Category: 8. Health

New research sheds light on the intricate connection between the gut microbiome and the brain, suggesting that a prebiotic diet might boost brain GABA (gamma-aminobutyric acid) levels, a key neurotransmitter associated with calming effects and neurological health.

GABA is an amino acid functioning as the principal inhibitory neurotransmitter that can act on the brain to slow or stop the reception of certain signals to the brain, leading to a calmer and more relaxed state.

Low GABA levels in the brain have been associated with neurological disorders and diseases like depression, Alzheimer’s, or epilepsy.

“Our study suggests that prebiotics have the ability to prevent or treat those brain diseases by increasing brain GABA levels via promoting gut GABA production through modulating gut microbiota,” details study corresponding author Thunatchaporn Kumrungsee, associate professor at Hiroshima University’s Graduate School of Integrated Sciences for Life.

Dietary interventions on GABA

Recently, there has been a push towards understanding more about the gut’s influence on mood, behavior, and mental health, as well as what foods might fuel or hinder a healthy mind.

The study authors aimed to assess how information directly passes between these two systems, asking: “Can an increase in gut-derived GABA directly cause an increase in the levels of brain GABA?”

The researchers set to work on determining whether brain GABA levels can be increased through dietary additions with the aim of modulating the gut bacteria present in an individual to bypass the blood-brain barrier — through which research has not yet proven GABA can pass.

Through trials on mice, researchers confirmed a “direct association” between gut GABA, brain GABA, and the gut microbiota.

There are still no solid results on whether or not gut microbiota-derived GABA can cross the blood-brain barrier and directly increase brain GABA. However, the research team claims further studies indicate a potential for other pathways to cause an increase in brain GABA elevation, such as stimulation through the vagus nerve or hormonal pathways.

Prebiotics that elevate GABA

In trials conducted on mice, researchers identified fructo-oligosaccharides (FOS), non-digestible oligosaccharides, and Aspergillus-derived enzymes, lipase and protease, as prebiotics effective in elevating brain GABA through their influence on the gut. FOS significantly increased brain GABA in the mice’s cortex and hippocampus — regions where GABA acts to reduce excitability and induce calmness.

Additionally, FOS and enzyme supplementation also raised homocarnosine levels in the hippocampus.

“Food factors such as prebiotics and fungi-derived enzymes with prebiotic-like effects have an ability to increase brain GABA and homocarnosine, a GABA-containing brain-specific peptide, which can possibly in turn enhance brain health through gut microbiota modulation,” adds Kumrungsee.

Homocarnosine has also been linked to certain brain diseases, with a previous study by Kumrungsee showing that homocarnosine-deficient mice were more prone to exhibiting depression-like behaviors and instances of hyperactivity.

Despite the lack of confirmed data on a direct increase in brain GABA derived from gut microbiota, the study provides strong indications that prebiotic consumption may indeed increase the brain’s GABA levels.

Future research will focus on unraveling the precise mechanisms by which the gut influences the brain and identifying the specific pathways involved. Once clarified, the next objective will be to determine if the prebiotic treatments used in this study can be further employed to treat GABA-related diseases, such as epilepsy or depression.

The study is published in npj Science of Food.

Gut-brain axis research highlights

As interest in the human gut-brain axis picks up, the US FDA formally acknowledged the safety of Lactobacillus plantarum DR7 — a patented probiotic strain developed by Kaneka Probiotics and AB-Biotics. One study found DR7 modulated enzymes linked to neurotransmitter production, suggesting a role in supporting healthy serotonin and dopamine levels, two key chemicals that influence mood, cognition, and emotional well-being.

In May, a clinical study suggested that a combined intervention of a synbiotic and “gut-directed” hypnotherapy significantly reduces gastrointestinal discomfort, irritability, and anxiety symptoms in autistic children.

Meanwhile, in botanical innovations, a recent clinical paper in Nutrients demonstrated that ZenGut, a natural microalgae extract developed by Microphyt, relieves digestive discomfort while enhancing mood and mental well-being in healthy adults.

Introduction

Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS), which induces the progressive depletion of CD4⁺ T cells in humans.¹ Infection with HIV can result in life-threatening opportunistic infections and may progress to AIDS. Since the start of the HIV epidemic, approximately 88.4 million people have been infected and 42.3 million people have died from AIDS-related illnesses.² The Joint United Nations Programme on HIV/AIDS estimates that, in 2023, there were 39.9 million people living with HIV (PLWH), 1.3 million new infections, and 630,000 deaths from AIDS-related illnesses globally.² Antiretroviral therapy (ART) is an effective therapeutic approach for preventing and treating HIV, thereby promoting a healthy lifespan.³ However, many individuals struggle with adherence to ART because of side effects, pill fatigue or aversion, and stigma.⁴ Therefore, it is necessary to find new drugs or treatments, such as traditional Chinese medicine (TCM).

TCM formulations have been used to treat AIDS in China since 2004. A national trial was conducted in the 19 major provinces to investigate the effects of TCM on AIDS, in addition to a series of research studies funded by the National Natural Science Foundation of China.⁵ Consequently, multiple TCM preparations have been developed to treat AIDS, many of which have demonstrated effectiveness.⁶ The Shuyu pill is a classic Chinese herbal formula for the treatment of Xu Lao for more 1800 years. Yiaikang(YAK) capsules, a modified form of Shuyu pills for the treatment of AIDS, which has antiviral and immune activities and improves the level of clinical application of symptoms and signs (eg, weakness, shortness of breath, spontaneous sweating, and diarrhea) in China.⁷ The main active components of YAK capsules, which are derived from the TCM prescription known as “Shuyu-Wan”, have been identified.⁸ Although YAK contains a total of 22 Chinese herbs, we summarize here the six key herbs and 11 bioactive ingredients that are used to treat AIDS, with a focus on their main target pathways, mechanisms, and therapeutic effects (Table 1). Among the active ingredients, Astragaloside IV combined Ginsenoside Rg1 have synergitic inhibition on autophagy injury.⁹

Table 1 Active Ingredients, Targets/Pathways, Mechanisms, and Therapeutic Effects of the Six Major Chinese Herbs of Yiaikang (YAK) Capsules Against Human Immunodeficiency Virus (HIV)/Acquired Immunodeficiency Syndrome (AIDS)

Mechanisms of YAK in HIV Treatment

According to recent research,^21–27 the functions and mechanisms of YAK include the following: blocking virus binding to its receptor, increasing the CD4⁺ T-cell count, regulating cytokine and chemokine responses, regulating the balance of T helper (Th)17 cells and regulatory T cells (Tregs), increasing the cytotoxic function of natural killer (NK) cells and maintaining the integrity of the intestinal mucosal barrier (Figure 1). According to previous studies, long-term application of Yiaikang is both safety and efficacy.²⁸ YAK combined with LPV/r can alleviate liver injury caused by LPV/r and combined with FTC+PMPA+RAL has no significant effect on the routine blood parameters in the treatment of SIVmac239 infected rhesus monkey AIDS model.²⁹ Studies shown that YAK combined with LPV/r in the treatment of SIVmac239 infected rhesus can maintain the stability of blood biochemical levels, reduce thrombocytopenia caused by ART, alleviate the side effects caused by LPV/r, and increase the efficacy of ART.³⁰

Figure 1 Therapeutic actions of Yiaikang (YAK) capsules. (A) YAK inhibits the viral Tat and Rev proteins as well as host intercellular adhesion molecule 1 (ICAM-1), assisting with the replication of HIV. YAK also increases CD4+ T-cell counts, possibly by restoring the expression of C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4), as well as T-cell activation levels. YAK significantly inhibits interleukin (IL)-13, increases the IL-2 and interferon (IFN)-γ cytokine response, and enhances host antiviral defense. (B) YAK significantly promotes the proliferation of natural killer (NK) cells and increases secretion of IFN-γ, enhancing immune function. (C) YAK regulates the imbalance in T helper 17 (Th17) cell/ regulatory T cell (Treg) imbalance by increasing RORγt expression and reducing FoxP3 expression, enhancing host antiviral defense. (D) YAK maintains the integrity of the intestinal mucosal barrier through inhibition of the expression of tight junction proteins ZO-1, Claudin-1, and Claudin-5, and chemokines. Created in BioRender. Xue, D. (2025) https://BioRender.com/l95e206.

Inhibition of Viral Replication

The HIV-1 envelope consists of two noncovalently associated fragments: gp120 and gp41. During infection, gp120 binds to the CD4 receptor on the surface of the host cell, interacting through its V3 ring with a coreceptor (eg, C-C chemokine receptor type 5 [CCR5] or C-X-C chemokine receptor type 4 [CXCR4]). This induces a conformational change that activates gp41, leading to the insertion of its viral fusion peptide to promote fusion of the viral and host cell membranes.³¹ The main target cells of HIV invasion and replication are CD4⁺ T lymphocytes,³² which are destroyed, thereby damaging the immune system.³³

The HIV genome contains two regulatory genes (trans-activator of transcription [tat] and rev), three structural genes (gag, pol, and env), and four accessory genes (nef, vpr, vpu, and vif). HIV-1 gene expression and replication largely depend on the Tat and Rev regulatory proteins.³⁴ Viral mRNA transcription is driven by the long terminal repeat, regulated by Tat and several host factors. Rev is transported to the infected host cell nucleus via its nuclear localization signal, where it binds to the Rev response element on viral RNA, accelerating viral mRNA transport outside of the nucleus.³⁵ Li et al³⁶ found that tat/rev expression and HIV-1 load in venous blood decreased significantly in the group that administration with YAK for 6 months compared with that in the control group, which suggesting that YAK may inhibit replication of HIV-1 by reducing the expression of Tat/Rev (mRNA level).

The levels of intercellular adhesion molecule 1 (ICAM-1), a glycoprotein that participates in immune responses, are abnormally increased on the surface of various cells following HIV infection, even under ART. ICAM-1 expression exhibits rapid upregulation in response to stimulation by cytokines, including interferon (IFN)-γ.³⁷ High levels of cell-surface ICAM-1 promote HIV production and virus spread,³⁸ and are positively correlated with HIV disease progression. ICAM-1 binds to the integrin lymphocyte function-associated antigen (LFA)-1, thereby stimulating HIV-infected dendritic cells and T cells and promoting viral spread.³⁹ Inhibition of ICAM-1/LFA-1 reduces HIV replication and transmission in vitro, suggesting the potential of ICAM-1 as a therapeutic target in HIV infection. Yue et al⁴⁰ observed an elevation in CD4⁺ T cells in YAK+ART group, with significantly decreased expression of ICAM-1/LFA-1 in CD4⁺ T cells following 6 months of YAK administration compared with ART controls. These results suggest that YAK directly affects the immune response in PLWH by decreasing the expression of ICAM-1 and LFA-1.

Improvement of Immune Response

CD4⁺ T-cell depletion is key to disease progression in PLWH. HIV-1 infection occurs when the virus binds to chemokine receptors and CD4 molecules on the surface of T cells. As the first recognition sites for HIV on the host cell surface, chemokine receptors CCR5 and CXCR4 are crucial in HIV infection.^41,42 Targeting these receptors is an attractive strategy for blocking HIV entry into host cells.⁴³

The active ingredients and other components of YAK regulate T cells through multiple targets and pathways, increasing the number of CD4⁺ T cells (Figure 1A). Liu et al⁴⁴ found that anticoagulant whole blood collected from PLWH at 6 months of YAK treatment exhibited restoration of CCR5 and CXCR4 expression, an increased number of CD4⁺ T cells, and a decreased HIV-1 load compared with that from healthy controls. Such findings suggest that YAK may restore coreceptor and T-cell activation levels to reverse the virus-induced immune damage in PLWH.

Regulation of Cytokine and Chemokine Responses

HIV infection of the human body activates T cells, which rapidly proliferate and secrete high levels of two inflammatory markers: interleukin (IL)-6 and sCD14.⁴⁵ IL-6 and sCD14 predict disease progression and are associated with increased risks of HIV/AIDS and death.⁴⁶ Th1 cells produce IL-2, triggering IFN-γ expression to activate NK cells and leading to apoptosis of HIV-1-infected T cells, which may be critical for controlling HIV-1.^47,48 Cytokine IL-13 has received considerable attention as the regulator of CD4⁺ Th2 immunity,⁴⁹ with inhibition of IL-13 expression increasing the activity of CD8⁺ T cells and protecting against viral infection.⁵⁰

Li et al⁵¹ collected the venous blood of PLWH to screen for HIV-1 load and cytokines at 6 months and 12 months of YAK treatment. They found improvements in the levels of IL-2, IL-13, and IFN-γ, number of CD4⁺ T cells, and anti-HIV activity at 12 months, suggesting that YAK may improving the immune status of PLWH by increasing cytokines and inhibiting HIV-1 (Figure 1A).

Increased Proliferation of NK Cells

HIV infection changes the distribution and functions of the NK cell subpopulation, even after ART.⁵² NK cells are important in HIV-1 infection, inhibiting viral entry into CD4⁺ T cells and preventing HIV-1 transmission.⁵³ IFN-γ (also known as immune IFN) is produced by T cells and is involved in immune regulation, activating NK cells and increasing their cytotoxic capabilities.⁵⁴ Targeting NK cells to restore their residual functionality can bolster their antiviral effects.

YAK was shown to significantly increase the proliferation and cytotoxic function of the human NK cell line NK-92MI⁵⁵ (Figure 1B). Qian et al⁵⁵ cultured NK-92MI cells in blank control and YAK-containing serum (4%, 8%, 15%, 25%) in vitro. Compared with the control, NKG2A expression was downregulated and IFN-γ secretion was upregulated at 6 hours of culture (P < 0.05). Thus, YAK significantly increased the proliferation and cytotoxicity of NK-92MI by inhibiting NKG2A receptors and increasing secretion of IFN-γ.

Regulation of Th17/Treg Balance

An altered Th17/Treg ratio, indicative of rapid depletion of Th17 cells and increased frequency of Tregs, is a hallmark of HIV infection and a marker of disease progression. This imbalance contributes to immune dysfunction and microbial translocation, which leads to chronic immune activation/inflammation and disease progression.⁵⁶ Th17 cells and Tregs are important gatekeepers of mucosal interfaces, with transcriptional profiles that are controlled by the lineage transcription factors RORγt/RORC2 and FOXP3, respectively.⁵⁷ HIV-1 infection can modify the mRNA expression of these transcription factors, which may decrease the immune response of Tregs and Th17 cells.⁵⁸

YAK regulates Th17/Treg imbalance by increasing the level of Th17 cells and decreasing the level of Tregs (Figure 1C). Huang⁵⁹ found that, compared with healthy control, 6 months of YAK treatment led to increases in the proportion of Th17 cells and the expression of RORγt mRNA, alongside reduced levels of Tregs and mRNA expression of Foxp3, in peripheral blood mononuclear cells. Therefore, YAK appears to increase CD4⁺ T cell counts by regulating the Th17/Treg ratio.

Maintenance of the Intestinal Mucosal Barrier

HIV-1 infection disrupts gut-associated lymphatic tissue, leading to loss of intestinal integrity, translocation of pathological microorganisms across the compromised gastrointestinal barrier, and systemic immune activation, even after ART.⁶⁰ CD4⁺ memory T cells in the gut carry higher levels of HIV DNA compared with blood.⁶¹ Virus-induced changes in microbial translocation and damage to the intestinal barrier contribute to inflammation and immune activation, induces apoptosis of CD4+T cells, and aggravates immune failure.⁶² The homing of lymphocytes from the bloodstream to the intestine is a prerequisite for establishing the immune barrier of the intestinal mucosa. This occurs through binding of the homing receptors on the surface of lymphocytes to specific ligands in the intestinal mucosal tissue.⁶³ Maintaining the integrity of intestinal mucosa and the balance of intestinal flora is crucial in the pathogenesis of HIV infection.

YAK reduces the permeability of the intestinal mucosal barrier, maintaining its integrity, through inhibition of the expression of tight junction (TJ) proteins (Figure 1D). Li et al⁶⁴ found that two ingredients of astragalus polysaccharide and ginseng stem saponin in YAK, significantly upregulated expression of the chemokines CCL25 and CCL28, chemokine receptors CCR9 and CCR10, CD80, CD86, major histocompatibility complex II (MHC-II), Toll-like receptor 4 (TLR4), and nuclear factor (NF)-ĸB p65 in intestinal mucosal tissue. These changes promoted the homing of intestinal lymphocytes and stimulated the activation of other immune cells (T and B cells), thereby enhancing intestinal mucosal immunity.

To simulate the intestinal mucosal injury induced by HIV-1, Sang et al⁶⁵ stimulated monolayers of Caco-2 human epithelial cells with IFN-γ. They then examined changes in membrane electrical impedance, fluorescein sodium transmittance, and mRNA expression of genes encoding TJ proteins at different time points following treatment with YAK or blank control group. YAK reduced the permeability of the simulated intestinal mucosal barrier and maintained its integrity, which was found to be related to inhibition of the expression of proteins ZO-1, Claudin-1, and Claudin-5.

Traditional Chinese medicine has unique advantages in regulating the abundance of flora and restoring immune reconstitution. YAK can regulates the intestinal flora, improves intestinal homeostasis, promotes immune reconstitution, and enhances immune function. YAK combined with ART has a certain clinical effect on PLWH, which can improve the proportion of protective factors of intestinal mucosa bacteria (such as Streptococcus, Macromonas, Wesneria, Streptococcus lactis).⁶⁶ YAK decreased the abundance of Lachnoclostridium, Muribaculaceae, Lactobacillaceae, Alphaproteobacteria, Aeromonadales and Prevotella and increase the abundance of Fusobacteriota and Lachnospiraceae and enhance body immunity in HIV/AIDS patients with poor immune reconstitution.⁶⁷

Regulation of Abnormal Lipid Metabolism of HIV

HIV-1 infection, chronic inflammation, and ART therapy are all related to changes in lipid metabolism, posing risk factors for cardiovascular and cerebrovascular diseases among PLWH.^68,69

Shen et al⁷⁰ used the 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and fluorogenic quantitative PCR to examine the effects of a range of concentrations of YAK-containing serum and ritonavir at different time points on the proliferation of human hepatoma Hep G2 cells and the expression of genes related to lipid metabolism, respectively. Compared with the blank control, the ritonavir + YAK treatment group inhibited cell viability at 24 and 48 hours, while the YAK group upregulated the expression of CYP7A1 and downregulated that of HMG-CoA reductase and peroxisome proliferator-activated receptor (PPAR)α at 48 hours. The role of YAK in lipid lowering may involve inhibition of endogenous cholesterol, promotion of fatty acid transport, and removal of lipids from. Baicalin, a compound present in YAK, has metabolic effects exerted through increasing the activation of brown and white adipose tissue via the AMPK/PGC1α pathway.^71,72

Clinical Applications of YAK in AIDS

Impacts on Clinical Manifestations, Quality of Life (QoL), Survival Rate, Anemia, and Lung Infections

YAK can be used to improve the clinical symptoms of HIV/AIDS. Clinical trials showed that patients receiving YAK had significantly improved clinical manifestations of AIDS compared with combination therapy of YAK and ART has been shown to benefit AIDS patients. China National Knowledge Infrastructure, Wanfang, Chinese Biomedicine Literature Database, PubMed, Embase and Medline were searched for studies on the effect of YAK on HIV/AIDS published up to February 2025. Data was presented in the Table 2.

Table 2 Studies Showing the Benefits of Yiaikang (YAK) Capsules in Combination with Antiretroviral Therapy for the Treatment of Acquired Immunodeficiency Syndrome (AIDS)

The prevalence of anemia and lung infections among PLWH is high,^82,83 but can be prevented using YAK. In a cross-sectional analysis of 8632 PLWH, patients receiving YAK therapy had a lower prevalence of anemia than those who did not.⁸² In addition, a randomized placebo-controlled trial performed in Henan Province, China, found that 4.9% of PLWH in the treatment group experienced a lung infection, compared with 6.0% of PLWH in the control group.⁸⁴

Survival rate is an indicator of the effectiveness of AIDS management. Jin et al reported a retrospective cohort study that compared the survival rate of HIV infection in patients with and without YAK treatment. A total of 3229 HIV-infected patients were followed for 21,876 person-years, showing 8-year cumulative survival rates of 78.5% in the YAK group (n = 1442) and 74.0% in the non-YAK group (n = 1787). The follow-up studies also showed that YAK increased the survival rate and increased lifetime in HIV-infected patients.^85,86

QoL refers to awareness and satisfaction with social status and living conditions. PLWH often face both physical and psychological stress, and thus require greater attention to their QoL. In a cross-sectional study of 275 PLWH, mean QoL scores (excluding spirituality/personal beliefs) were significantly higher in the YAK + ART group than in the ART group (P < 0.05).⁸⁷

Adjuvant Drugs for Common AIDS-Related Conditions

As an adjuvant medicine, YAK can improve the efficacy of the primary treatment for many of the symptoms and illnesses common in PLWH. It has been widely used to treat abnormal lipid metabolism, diarrhea, anxiety/depression, ulcers, cough, and HIV/hepatitis C with hepatic fibrosis (Table 3).

Table 3 Illnesses and Symptoms Treated with Yiaikang (YAK) Capsule Therapy in Patients with Human Immunodeficiency Virus (HIV) Infection

Improvement in Clinical Symptoms of Abnormal Lipid Metabolism

Yu et al⁹⁵ performed a clinical trial with 40 PLWH who took YAK + ART. After 6 months of YAK treatment, patients experience alleviation of clinical symptoms (asthma, spontaneous sweating, chest tightness, fatigue, and palpitation), achieved through regulation of phosphatidylcholine, phosphatidylethanolamine, cholesterol ester, and sphingomyelin, along with improvements in lipid metabolism and decreased vascular endothelial injury.

Conclusions

The therapeutic effects and mechanisms of action of YAK in the treatment of patients with AIDS have become a focus of TCM research.⁹⁶ By outlining these mechanisms and progress in clinical research on YAK in HIV/AIDS, this review has highlighted novel therapeutic targets and effective complementary approaches to ART. The key actions of YAK include the following: blocking virus–receptor binding, elevating CD4⁺ T-cell counts, regulating cytokine/chemokine responses, regulating Th17/Treg balance, enhancing NK cytotoxicity, and maintaining the integrity of the intestinal mucosal barrier. These effects are mediated through a number of pathological pathways involving NF-ĸB, PPAR, PD-1, transforming growth factor (TGF)-β/Smad, T-cell receptors (TCRs), and TLRs. Clinical studies have demonstrated the therapeutic efficacy of YAK in terms of improvements in clinical symptoms and QoL, longer survival times, and reduced mortality in patients with AIDS. As an herbal medicine, YAK is complementary to ART. However, there is limited clinical trial data on the effects of YAK in AIDS. Firstly, its pharmacology and therapeutic mechanism have not been extensively explored, necessitating further research to optimize its clinical application. Secondly, due to limited number and quality of clinical studies included in the analysis, further research is needed. Lately, we support large-scale clinical trials to evaluate the protective efficacy of YAK in AIDS, as well as single-cell and spatial multi-omics studies of its mechanisms of action. YAK shows great promise as a complementary treatment to ART and warrants further exploration.

Funding

This work was supported by the Zhengzhou Medical and Health Science and Technology Innovation Guidance Program (2024YLZDJH134), Henan Province Pilot Project of Treating AIDS with Traditional Chinese Medicine(No. 2004ZYA109), Henan Province Key Research and Development and Promotion Project (252102310488) and Traditional Chinese Medicine Research Project of Henan Province (2025ZKY016).

Disclosure

The authors report no conflicts of interest in this work.

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66. Yang Q. To explore the clinical efficacy of Yiaikang capsule in the intervention of HIV/AIDS spleen-centered deficiency syndrome and its influence on intestinal flora based on the theory of “spleen-centered protection”. Master. 2024.

67. Y-n LIU, Z-b LIU, Sang F, Liu Z, P-y LI, H-j GUO. Effects of yiaikang capsules on intestinal microflora and immune function in HIV/AIDS patients with poor immune reconstitution of lung and spleen qi deficiency syndrome. China J Tradition Chin Med Pharm. 2022;37(05):2729–2733.

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73. Wei S, Kang B, Liu K. Clinical observation of Yiaikang capsule treating AIDS for 8 years. Modern Dis Control Prevention. 2014;25(01):108–110. doi:10.13515/j.cnki.hnjpm.2014.01.039

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81. Liu P, Zhang Y, Song J. Summary of clinical efficacy of Yiaikang in treating 1792 cases of spleen and stomach symptom of AIDS. Acta Chin Med. 2009;24(05):9–10. doi:10.16368/j.issn.1674-8999.2009.05.009

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88. Fu L. Siwu Xiaofeng decoction mainly treated 30 cases of chronic AIDS rash. Traditional Chin Med Res. 2013;26(09):16–18.

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Mack Roach III, MD, professor of radiation oncology, medical oncology, and urology at the University of California, San Francisco, discusses the development and evaluation of a multimodal artificial intelligence (AI) algorithm designed to predict prostate cancer outcomes and its performance across racial subgroups.

According to Roach, the motivation behind the study stemmed from a longstanding controversy in prostate cancer: the role of race in determining outcomes. While prostate cancer incidence is 1.5 to 2 times higher among Black men compared with other racial groups, the question of whether biological differences contribute to disparities in outcomes has been widely debated.

“For many years, there have been different opinions about whether there is an inherent biologic factor or not, and so it is important for us to distinguish between the incidence of the disease and the biologic behavior of a disease once it is diagnosed,” he explains.

To address this, researchers turned to a high-quality data set derived from prospective phase 3 randomized clinical trials. These trials ensured uniform treatment protocols, patient stratification, and systematic follow-up. “The value of that resource,” Roach explains, “is that the quality of care and the eligibility are controlled in such a way that biases do not really enter into the quality of treatment.” Importantly, variables such as insurance coverage, treatment type, and dose were standardized, which allowed for more accurate analysis of outcomes by race.

This dataset provided a unique opportunity to test whether AI could not only enhance prognostic accuracy, but also maintain fairness across racial groups. The AI model analyzed digitized biopsy slides along with clinical features like prostate-specific antigen, tumor grade, and stage, uncovering subtle prognostic indicators not readily visible to human pathologists.

The study, published in JCO Clinical Cancer Informatics, demonstrated that the algorithm accurately predicted outcomes such as recurrence and metastasis and did so without introducing racial bias. The results support the broader application of AI in oncology while reinforcing the importance of diverse, well-controlled data in developing equitable predictive tools.

REFERENCE:

Roach M 3rd, Zhang J, Mohamad O, et al. Assessing algorithmic fairness with a multimodal artificial intelligence model in men of African and non-African origin on NRG oncology prostate cancer phase III trials. JCO Clin Cancer Inform. 2025;9:e2400284. doi:10.1200/CCI-24-00284

We know that processed meat isn’t particularly good for us, having already been linked to dementia, diabetes, and cancer, but how much of it counts as a ‘safe’ level of consumption? According to new research, there’s no such thing.

US researchers reviewed over 70 previous studies (involving several million participants in total), analyzing the relationships between ultra-processed food and three health issues: type 2 diabetes, ischemic heart disease, and colorectal cancer.

Associations for processed meat, sugar-sweetened beverages, and trans fatty acids were looked into, and it was the processed meat that came out with the worst results – even if the amount eaten is only small.

“The monotonic increases in health risk with increased consumption of processed meat suggest that there is not a ‘safe’ amount of processed meat consumption with respect to diabetes or colorectal cancer risk,” the team from the University of Washington in Seattle writes in their published paper.

Related: The Secret to Better Sleep Could Be As Simple As Eating More Fruit And Veggies

It’s important to put the research into context. The associations found are relatively weak, they don’t prove direct cause and effect, and the analyzed studies relied on self-reported dietary habits (which may not be completely accurate).

However, the study has several strengths too – it uses a Burden of Proof method, which is more conservative when assessing impacts on health. The results tend to be minimum values, which means they likely underestimate the true health risk.

What’s particularly notable here is that minimal increases in consumption still raised risk levels.

“Habitual consumption of even small amounts of processed meat, sugary drinks, and trans fatty acids is linked to increased risk of developing type 2 diabetes, ischemic heart disease and colorectal cancer,” University of Washington nutrition biologist Demewoz Haile told CNN.

For example, the equivalent of one hot dog a day was associated with at least an 11 percent greater risk of type 2 diabetes, and at least a 7 percent greater risk of colorectal cancer, compared to eating no processed meat at all.

For beverages, an extra can of sugar-sweetened pop a day was linked to a 8 greater risk of type 2 diabetes, and a 2 percent greater risk of ischemic heart disease, compared to not drinking anything sugary.

For trans fatty acids, a small daily amount was associated with a 3 percent increase in risk of ischemic heart disease, compared with zero consumption.

“This information provides critical data for public health specialists and policymakers responsible for dietary guidelines and potential initiatives that aim to reduce the consumption of these processed foods,” write the researchers.

While the study has limitations, its scale and conservative methodology make it worth taking note of.

This is backed up by a commentary in the same journal, which does note the role of ultra-processed foods in improving food accessibility and shelf life, particularly in areas with limited access to fresh food.

The message from the research team is that cutting out ultra-processed foods as much as possible is the best option for our health.

The research has been published in Nature Medicine.

According to a new study published in Frontiers in Nutrition, swapping out meat, eggs, and dairy for greens and beans may help reduce inflammation and support sustainable weight loss.

What Did the Study Find?

In a randomised cross-over trial, researchers studied 62 overweight adults who were randomly assigned to a Mediterranean diet or a low-fat vegan diet for 16 weeks. After a four-week cleansing period, each group followed the alternate diet for another 16 weeks, which means they acted as their own controls.

Researchers measured the participants’ dietary acid load, which is calculated using two scores: potential renal acid load (PRAL) and net endogenous acid production (NEAP). The former estimates how much acid the kidneys need to remove, and the latter estimates the total amount of acid your body produces, including from digestion and metabolism.

If that all sounds a bit complex, the main thing to know here is that increased dietary acid load is linked to chronic inflammation, which can disrupt metabolism and lead to increased body weight. So, essentially, the researchers were looking for lower dietary acid load scores.

The Mediterranean diet followed the PREDIMED protocol, which includes fruits, vegetables, legumes (eg, lentils, chickpeas, peas, beans or soy), nuts or seeds, fish or shellfish, and prioritises lean white meats over red meats. Participants were also asked to consume 50g of extra-virgin olive oil every day. Meanwhile, the vegan diet included vegetables, grains, fruits, and legumes.

Following a statistical analysis, the researchers found that both PRAL and NEAP scores decreased significantly on the vegan diet, with no significant change on the Mediterranean diet. This reduction in dietary acid load was associated with weight loss, which the researchers say remained significant even after they accounted for the higher calorie intake on the Mediterranean diet. Body weight was reduced by an average of six kilograms on the vegan diet, compared with no change on the Mediterranean diet.

What Does This Mean for Us?

Animal products like meat, fish, eggs, and cheese do cause the body to produce more acid, and the researchers say that the vegan diet’s alkalising effect, which increases the body’s pH level to make it less acidic, may be what helps promote weight loss.

Top alkalising foods include:

• Leafy greens
• Broccoli, beets
• Asparagus
• Garlic
• Carrots
• Cabbage
• Berries
• Apples
• Cherries
• Apricots
• Cantaloupe
• Lentils
• Chickpeas
• Peas
• Beans
• Soy
• Quinoa
• Millet

These results highlight the benefits of a plant-based diet for reducing inflammation. Because plant-based diets are more alkaline, the researchers say they are generally associated with weight loss, improved insulin sensitivity, and lower blood pressure.

That’s not to say you need to go entirely plant-based to reap those benefits, but the findings suggest that if your aim is to (sustainably) lose weight, a wholly plant-based approach might be optimum, seeing as the Mediterranean diet – which is also high in plants – didn’t produce the same results.

It’s important to remember that the study looked at overweight individuals, so while it suggests a temporary plant-based diet could be a good way to kickstart a sustainable weight loss journey, it’s probably not for you if you’re already quite active. We need ample fats and carbs to fuel regular training, and while a vegan diet can absolutely still work, it likely wouldn’t in conjunction with a low-fat approach.

However, when weight loss drugs (which can be a valid and helpful option for some people) are increasingly being promoted by unqualified sources as quick-fix solutions over nutrition and exercise, studies like this highlight the potential powers of nutritional interventions first.

The Bottom Line

Although the study had a rigorous design, it was relatively small and relied on self-reported dietary data from participants. Also, while the researchers say their statistical analysis accounts for the extra energy intake on the Mediterranean diet, some might argue that it could still have impacted outcomes.

With that in mind, more research is needed to confirm the results. However, for now, the study highlights the potentially powerful health benefits of including more plants in your diet – and how a temporary vegan diet could help you kickstart a sustainable weight loss strategy.

Hannah Bradfield is a Senior Nutrition Writer across Women’s Health UK and Men’s Health UK. An NCTJ-accredited journalist, Hannah graduated from Loughborough University with a BA in English and Sport Science and an MA in Media and Cultural Analysis. 

She has been covering sports, health and fitness for the last five years and has created content for outlets including BBC Sport, BBC Sounds, Runner’s World and Stylist. She especially enjoys interviewing those working within the community to improve access to sport, exercise and wellness. Hannah is a 2024 John Schofield Trust Fellow and was also named a 2022 Rising Star in Journalism by The Printing Charity. 

A keen runner, Hannah was firmly a sprinter growing up (also dabbling in long jump) but has since transitioned to longer-distance running. While 10K is her favoured race distance, she loves running or volunteering at parkrun every Saturday, followed, of course, by pastries. She’s always looking for fun new runs and races to do and brunch spots to try.

“Forever chemicals” or per- and polyfluoroalkyl substances (PFAS) have been widely used in consumer and industrial products for the better part of a century, but do not break down in the natural environment. One PFAS, perfluorohexanoic acid or PFHxA, is made up of a shorter chain of molecules and is thought to have less of an impact on human health. New research from the Del Monte Institute for Neuroscience at the University of Rochester suggests otherwise, finding that early life exposure to PFHxA may increase anxiety-related behaviors and memory deficits in male mice.

“Although these effects were mild, finding behavioral effects only in males was reminiscent of the many neurodevelopmental disorders that are male-biased,” said Ania Majewska, PhD, professor of Neuroscience and senior author of the study out today in the European Journal of Neuroscience. Research has shown, males are more often diagnosed with neurodevelopmental disorders such as autism and ADHD. “This finding suggests that the male brain might be more vulnerable to environmental insults during neurodevelopment.”

Researchers exposed mice to PFHxA through a mealworm treat given to the mother during gestation and lactation. They found that the male mice exposed to higher doses of PFHxA in utero and through the mother’s breastmilk showed mild developmental changes, including a decrease in activity levels, increased anxiety-like behaviors, and memory deficits. They did not find any behavioral effects in females that were exposed to PFHxA in the same way.

Finding that developmental exposure to PFHxA has long-term behavioral consequences in a mammalian model is concerning when considering short-chain PFAS are thought to be safer alternatives to the legacy PFAS that have been phased-out of production. Understanding the impacts of PFHxA on the developing brain is critical when proposing regulations around this chemical. Hopefully, this is the first of many studies evaluating the neurotoxicity of PFHxA.”

 Elizabeth Plunk, PhD (’25), an alumna of the Toxicology graduate program at the University of Rochester School of Medicine and Dentistry and first author of the study

Researchers followed these mice into adulthood and found that in the male mice PFHxA exposure affects behavior long after exposure stops, suggesting that PFHxA exposure could have effects on the developing brain that have long-term consequences.

“This work points to the need for more research in short-chain PFAS. To our knowledge, PFHxA has not been evaluated for developmental neurobehavioral toxicity in a rodent model,” said Majewska. “Future studies should evaluate the cellular and molecular effects of PFHxA, including cell-type specific effects, in regions associated with motor, emotional/fear, and memory domains to elucidate mechanistic underpinnings.”

Despite its shorter chain, PFHxA has been found to be persistent in water and was restricted by the European Union in 2024. This follows years of restrictions on longer chain PFAS. Last year, the Environmental Protection Agency set its first-ever national drinking water standard for PFAS, which will reduce PFAS exposure for millions of people. PFAS are man-made chemicals that have the unique ability to repel stains, oil, and water have been found in food, water, animals, and people. They are linked to a range of health issues, including developmental issues in babies and kidney cancer.

Additional authors include Marissa Sobolewski, PhD, of the University of Rochester Medical Center, Katherine Manz, PhD, of the University of Michigan, and Andre Gomes, and Kurt Pennel, PhD, of Brown University. The research was supported by the National Institutes of Health, the University of Rochester Intellectual and Developmental Disabilities Research Center, and the University of Rochester Environmental Health Services Center.

Introduction

Tetralogy of Fallot (ToF) is a congenital cyanotic heart defect, first described in detail by Étienne-Louis Fallot in 1888. It is characterized by a combination of four distinct anatomical abnormalities: right ventricular (RV) hypertrophy, a ventricular septal defect (VSD), obstruction of the right ventricular outflow tract (RVOT), and an overriding aorta. These structural anomalies result in altered hemodynamics, reducing pulmonary blood flow and systemic cyanosis.¹ Early primary repair of Tetralogy of Fallot (ToF) has been advocated since the 1970s and is now routinely performed with excellent outcomes.² This approach has become the standard of care, as it promptly addresses the anatomical abnormalities and associated hemodynamic consequences, reducing long-term morbidity and mortality. However, despite the widespread adoption of early surgical intervention, occasional cases of patients who have not undergone repair survive into adulthood.^3,4 However, delaying the diagnosis and late intervention are highly associated with poor outcomes.⁵ According to large observational series, 24% of individuals with an uncorrected TOF die before the age of 10, and only 4% survive beyond their thirties.^6,7. We present a unique case of an individual with uncorrected Tetralogy of Fallot (TOF) who has survived into adulthood with a relatively preserved quality of life despite the absence of surgical intervention. The lack of corrective surgery was primarily due to financial constraints and limited access to specialized cardiovascular care. The patient resides in Somalia, a low-income country where healthcare infrastructure remains underdeveloped, and access to advanced medical and surgical interventions is severely restricted. This case underscores the significant challenges faced by individuals with congenital heart disease (CHD) in resource-limited settings. It highlights the urgent need for improved access to pediatric and adult congenital cardiac care in such environments.

Case Presentation

A 43-year-old male presented with a history of recurrent syncope, reporting three episodes over the past six months. The most recent episode occurred at approximately 3:00 AM, during which the patient was found lying unresponsive on his bed by his family and was subsequently rushed to the hospital. The patient has a longstanding history of exertional dyspnea dating back to childhood, which was particularly pronounced during activities such as climbing hills. Due to his family’s nomadic lifestyle, he was often transported on camels during travels to accommodate his physical limitations. Additionally, the patient reported episodes of epistaxis and hemoptysis, further complicating his clinical picture.

The patient’s past medical history includes a diagnosis of congenital heart disease of unknown type in 1997. Despite being referred to a cardiac center in Djibouti, a neighboring country, for further management, financial constraints prevented him from accessing specialized care. Over the years, the patient has experienced progressive exertional dyspnea, which has significantly impacted his quality of life and occupational capacity. Initially, he worked in charcoal production and later as a painter, but he was forced to retire due to worsening symptoms. He is the father of six children and has been the primary provider for his family.

Physical Examination

General Appearance

The patient appeared to be in no acute distress and exhibited a normal overall appearance without signs of cachexia.

Vital Signs Were

Blood pressure 110/60 mmHg, heart rate 80 beats/minute, respiratory rate 18 breaths/minute, temperature 35.8 °C, and oxygen saturation 85% on room air.

General Examination

• Clubbing: Grade 3 clubbing was observed see Figure 5.
• Cyanosis: No cyanosis was noted.
• Edema: No peripheral edema was present.

Cardiovascular System (CVS)

• S1 and s2 are audible with a pansystolic murmur, graded 3/6 in intensity, was auscultated. The murmur was most prominent in the tricuspid and mitral areas but was not associated with a palpable thrill.

Abdomen

• The abdomen was soft and non-tender on palpation.
• No abdominal swelling or organomegaly was detected.

Peripheral Examination

• No ankle edema or other signs of peripheral vascular compromise were observed.

Nervous Examination

• A brief neurological assessment showed the patient to be alert and fully oriented to person, place, and time. There were no obvious motor or sensory impairments. Examination of cranial nerves II through XII revealed no gross abnormalities. Additionally, there were no clinical signs suggestive of meningeal irritation.

Investigations

Complete Blood Count (CBC)

• Polycythemia: Hemoglobin (Hb) level was elevated at 21 mg/dL, indicative of secondary polycythemia, likely due to chronic hypoxemia associated with congenital heart disease.
• Thrombocytopenia: A Low platelet count was noted, possibly related to chronic disease or other underlying factors.

Echocardiographic evaluation of the patient revealed a large malalignment subaortic ventricular septal defect (VSD)with an overriding aorta of less than 50%, indicative of abnormal conal septal development. Right ventricular hypertrophy (RVH) was observed, consistent with chronic pressure overload. Infundibular pulmonary stenosis was noted, contributing to right ventricular outflow obstruction. Additionally, an interatrial septal (IAS) aneurysm was identified, suggesting a structural abnormality that may have clinical significance (Figures 1 and 2).

Figure 1 Is a long-axis view demonstrating the VSD with color Doppler imaging, showing flow across the defect penetrating the interventricular septum.

Figure 2 Is an apical four-chamber view providing additional visualization of the VSD.

Assessment

• Tetralogy of Fallot (ToF): Known congenital heart defect with concerns for arrhythmias due to recent syncope.
• Possible Old Inferior Myocardial Infarction (MI): ECG shows pathological Q waves in inferior leads, possibly due to chronic hypoxemia or coronary artery disease.
• Secondary Polycythemia: Elevated hemoglobin (21 mg/dL) as a compensatory response to chronic hypoxemia, increasing thromboembolic risk.
• Thrombocytopenia: Low platelet count, possibly secondary to chronic disease or polycythemia.

Plan

• Medical Management:

  • Aspirin (81 mg daily): To reduce thromboembolic risk from severe polycythemia and possible atherosclerosis indicated by ECG.
  • Atorvastatin (20 mg daily): Secondary prevention for suspected old inferior MI despite unknown lipid levels.
  • Hydroxyurea (500 mg daily): To control symptomatic secondary polycythemia by lowering erythropoiesis and blood viscosity.
  • Home Oxygen Therapy (as needed): To relieve chronic hypoxemia symptoms, especially exertional dyspnea.

• Phlebotomy: Weekly 250 mL blood transfusion to manage polycythemia, with caution due to thrombocytopenia.
• Further Investigation: Holter monitoring for arrhythmias.
• Referral for Cardiac Surgery: Consideration for pulmonary valve replacement or complete repair.
• Lifestyle & Follow-up: Avoid strenuous activity, and regularly monitor hemoglobin, platelets, and cardiac function.

Electrocardiogram (ECG)

• Rate: 75 beats per minute.
• Rhythm: Sinus rhythm.
• Axis: Extreme right axis deviation.
• P Wave: P-pulmonale (tall, peaked P waves in leads II, III, and aVF) suggests right atrial enlargement.
• Q Wave: Pathological Q waves were observed in the inferior leads (II, III, and aVF), suggesting a possible old inferior wall myocardial infarction.
• R Wave:

  1. Prominent R wave in V1 and aVR.
  2. Deep S wave in V5 and V6.
  3. Poor R-wave progression across the precordial leads (Figure 3).
     

     Figure 3 ECG showing right ventricular hypertrophy, right atrial enlargement (P-pulmonale), and pathological Q waves in inferior leads suggest an old inferior wall myocardial infarction.

• Findings:

  • Right Ventricular Hypertrophy (RVH): Suggested by the prominent R wave in V1 and deep S wave in V5/V6.
  • P-pulmonale: Indicative of right atrial enlargement, likely secondary to pulmonary hypertension or chronic right heart strain.
  • Old Infarction: Pathological Q waves in the inferior leads raise the possibility of a prior inferior wall myocardial infarction.

Chest X-Ray

• The chest X-ray reveals characteristics of Tetralogy of Fallot, notably a boot-shaped heart caused by enlargement of the right ventricle (Figure 4).
  

  Figure 4 Chest X-ray showing features of tetralogy of Fallot, including a boot-shaped heart due to right ventricular hypertrophy.

Physical Appearance

• Hand of the patient showing evidence of digital clubbing (Figure 5).
  

  Figure 5 Hand of the patient showing evidence of digital clubbing.

Discussion

Tetralogy of Fallot (TOF) is a congenital cardiac anomaly characterized by a ventricular septal defect, right ventricular outflow tract obstruction, overriding of the aortic root, and right ventricular hypertrophy. It occurs in approximately 3 per 10000 live births, accounting for 7–10% of all congenital heart defects. Clinical presentation typically occurs in the neonatal period, with cyanosis varying in severity based on the degree of right ventricular outflow obstruction. The etiology is multifactorial, with genetic and environmental factors playing a role. Maternal conditions such as diabetes and phenylketonuria, as well as chromosomal anomalies like trisomies 21, 18, and 13, have been associated with TOF, though recent evidence suggests a stronger link with 22q11.2 microdeletion. The recurrence risk in affected families is approximately 3%.⁸

In diagnosing congenital heart diseases like Tetralogy of Fallot (ToF), readily available tools such as electrocardiography (ECG) and echocardiography are indispensable, offering a non-invasive approach. An ECG can reveal right axis deviation and right ventricular hypertrophy, indicative of the strain on the heart caused by ToF. Furthermore, echocardiography (2D ECHO) is crucial for confirming the diagnosis of ToF, as it can visualize the key anatomical defects, including ventricular septal defect (VSD), overriding aorta, and right ventricular outflow tract (RVOT) obstruction.⁹

While ECG and standard echocardiography are essential for the initial diagnosis and monitoring of Tetralogy of Fallot (ToF), particularly in resource-limited settings due to their accessibility, advanced imaging modalities are often required for comprehensive anatomical and hemodynamic evaluation. Cardiac MRI (cMRI) is central in assessing ventricular function, pulmonary valve pathology, and right ventricular outflow tract (RVOT) morphology. Emerging techniques such as 4D-flow MRI enhance diagnostic precision by enabling dynamic flow analysis, offering insights into adverse hemodynamic patterns, and assisting with risk stratification and intervention planning.¹⁰ While cardiac catheterization remains the gold standard for direct hemodynamic measurements and detailed visualization of pulmonary arteries or complex vascular anomalies like Major Aortopulmonary Collateral Arteries (MAPCAs), its use is often limited in low-resource environments. Therefore, ECG and echocardiography remain indispensable tools for initial evaluation and follow-up in such settings.⁹ In our patient, ECG and echocardiography were indispensable for diagnosing Tof.

Tetralogy of Fallot (TOF) is a cyanotic congenital heart disease associated with high mortality rates among unrepaired patients. Survival beyond 20 years is limited to approximately 10%, with only 3% reaching 40. In contrast, over 90% of patients undergoing surgical repair survive into adulthood. In developed countries, most individuals with TOF receive timely surgical intervention, which alleviates right ventricular outflow tract (RVOT) obstruction and significantly reduces mortality. However, in low-resource settings, access to surgical repair remains limited, particularly among patients from low socioeconomic backgrounds. This disparity contributes to poorer long-term outcomes and increased mortality in these populations.¹¹

In some cases, patients may not receive an accurate diagnosis during childhood, as was observed in our case. The first clinical suspicion of Tetralogy of Fallot (TOF) only arose when the patient reached adulthood. Confirming the diagnosis required advanced imaging studies, which were often financially inaccessible and frequently unavailable in his setting, both during initial assessment and routine follow-up. The lack of continuous monitoring and access to diagnostic tools contributed significantly to the delayed diagnosis and management of his condition. This case highlights the critical importance of making bedside echocardiography available, even in resource-limited environments. Point-of-care ultrasound can enable earlier detection and timely intervention in congenital heart diseases, ultimately improving patient outcomes.

In the electrocardiogram (ECG) of our patient, the presence of QS waves in the inferior leads raised suspicion of an inferior myocardial infarction (MI). The relationship between myocardial infarction and Tetralogy of Fallot (TOF) is reported in the literature. A case from Turkey described a patient with TOF who experienced an MI, providing valuable insights into this uncommon association. In that case, the absence of resting cyanosis suggested a minor right-to-left shunt, potentially explaining the patient’s prolonged survival. However, following the myocardial infarction, both ventricles rapidly deteriorated, leading to severe heart failure and early mortality.¹².

The patient, a 43-year-old man, was admitted with chest pain, dyspnea, and diaphoresis. His medical history revealed dyspnea, exertional cyanosis, and palpitations since childhood. He had been diagnosed with TOF 13 years earlier, at which time cardiac catheterization was performed. However, he declined TOF corrective surgery, which may have contributed to his later cardiovascular complications.¹² This case underscores the complex interplay between congenital heart defects and ischemic heart disease, highlighting the need for careful cardiovascular risk assessment in patients with uncorrected TOF. In the literature, several cases also describe similar situations, where the coexistence of TOF and MI significantly worsens the prognosis.¹³

In our patient, syncope raised suspicion of an underlying arrhythmia. Syncope is a concerning symptom in individuals with repaired Tetralogy of Fallot (TOF). It is often associated with arrhythmias and conduction abnormalities, common long-term sequelae of surgical repair. While advancements in surgical techniques have significantly improved survival rates, residual cardiac abnormalities, scarring from patch material, atriotomy, and ventriculotomy contribute to the development of rhythm disturbances.¹⁴ However, cases of arrhythmias have also been reported in patients with unrepaired TOF.¹⁵ Similar pathophysiological mechanisms could potentially account for the syncopal episode observed in our patient.

Ideally, ambulatory ECG monitoring—such as a Holter monitor—would have been indicated to assess for transient arrhythmias that might not be captured on routine ECG. Contemporary diagnostic strategies often rely on a spectrum of prolonged monitoring modalities, including traditional 24–48-hour Holter monitors, external loop recorders (ELRs), wearable patch devices, and implantable cardiac monitors (ICMs) for prolonged surveillance in select patients. These tools are critical for establishing symptom–rhythm correlation, especially when symptoms are infrequent or unpredictable.¹⁶ As Carrington et al (2022) emphasize, the choice of monitoring modality is primarily dictated by the frequency and nature of symptoms, with longer-duration or patient-activated monitors being particularly advantageous for episodic events. Unfortunately, such diagnostic tools were unavailable due to limitations inherent in our resource-constrained setting. Consequently, only a standard one-minute 12-lead ECG was performed, which showed no evidence of acute ischemia or arrhythmic disturbances at the recording time.

Conclusion

Tetralogy of Fallot (TOF) is a congenital heart defect with significant long-term risks, particularly in uncorrected cases. Our patient’s ECG suggested an inferior myocardial infarction, while syncope raised suspicion of arrhythmias, though Holter monitoring was unavailable. This case underscores the critical need for early diagnosis, timely surgical intervention, and continuous cardiac surveillance. In resource-limited settings, improving access to echocardiography and ambulatory ECG monitoring is essential for mitigating complications and improving outcomes in patients with uncorrected TOF.

Authors’ Information

• Dr Abdirahman A Warfaa: Cardiology Specialist at Darussalam Health Care and Cigaal Interventional Cardiology Center; also teaches at Amoud University.• Dr. Abdirahman Ibrahim Said: Internal Medicine Specialist at Borama Regional Hospital and Alaaleh Hospital; Clinical Coordinator for undergraduate programs at Amoud University College of Health Sciences.• Dr. Mohamoud Abdulahi: Orthopedics Specialist at Al-Hayat Hospital, Dar es Salaam Polyclinic, and Alaaleh Hospital; Dean of the School of Medicine, Amoud University.• Mohamed Said Hassan: Public Health Researcher; Head of the Medical School Research Committee.

Manuscript Submission

We confirm that this manuscript is original and has not been submitted elsewhere for publication.

Ethical Approval

The Ethical Committee of Amoud University granted ethical approval for this study, including permission for publication (Reference: 0100-AU-REC-2025).

Consent for Publication

The patient provided written informed consent after receiving a detailed explanation of the study’s purpose, procedures, and potential for publication. This consent included permission to publish all clinical details and associated images in this report. Patient anonymity has been preserved.

Acknowledgments

We extend our gratitude to the healthcare team at Darussalam Health Care for the care provided to the patient and the follow-up they gave.

Author Contributions

All authors contributed significantly to developing this case report, including the conception and interpretation of clinical findings. They participated in drafting, revising, or critically reviewing the manuscript; approved the final version to be published; agreed on the journal to which the case report was submitted; and took full responsibility for all aspects of the work.

Funding

This research received no financial support from external sources.

Disclosure

The authors declare that there are no conflicts of interest regarding the content or publication of this manuscript.

References

1. Boyer R, Kim HJ, Krishnan R. Management of unoperated tetralogy of Fallot in a 59-year-old patient. J Investig Med High Impact Case Reports. 2020;8. doi:10.1177/2324709620926908

2. Van Arsdell GS, Maharaj GS, Tom J, et al. What is the optimal age for repair of tetralogy of Fallot? Circulation. 2000;102(19). doi:10.1161/circ.102.suppl_3.iii-123

3. Dockery D. 1993. The New England journal of medicine was downloaded from nejm.org at Uniwersytet Jagiellonski Collegium Medicum on February 9, 2012. For personal use only. No other uses without permission. Copyright © 1993 Massachusetts medical society. All rights reserved. New Engl.

4. Bertranou EG, Blackstone EH, Hazelrig JB, Turner ME, Kirklin JW. Life expectancy without surgery in tetralogy of Fallot. Am J Cardiol. 1978;42(3):458–466. doi:10.1016/0002-9149(78)90941-4

5. Bernier PL, Stefanescu A, Samoukovic G, Tchervenkov CI. The challenge of congenital heart disease worldwide: epidemiologic and demographic facts. Semin Thorac Cardiovasc Surg Pediatr Card Surg Ann. 2010;13(1):26–34. doi:10.1053/j.pcsu.2010.02.005

6. Campbell M. Natural history of cyanotic malformations and comparison of all common cardiac malformations. Br Heart J. 1972;34(1):3–8. doi:10.1136/hrt.34.1.3

7. Roman S, Castellarin M. A uniquely compensated boot-shaped heart: a case of unrepaired tetralogy of Fallot with delayed symptom onset in adulthood. Chest. 2020;158(4):A280. doi:10.1016/j.chest.2020.08.281

8. Bailliard F, Anderson RH. Tetralogy of Fallot. Orphanet J Rare Dis. 2009;4(1). doi:10.1186/1750-1172-4-2

9. Kaur KS, Gupta ML, Rajput HS, Sajan C. Tetralogy of Fallot in adult – uncorrected and rare presentation: a case report. J Young Pharm. 2023;15(1):189–192. doi:10.5530/097515050444

10. Schäfer M, Mawad W. Advanced imaging technologies for assessing tetralogy of Fallot: insights into flow dynamics. CJC Pediatr Congenit Hear Dis. 2023;2(6):380–392. doi:10.1016/j.cjcpc.2023.09.011

11. Bhattarai P, Karki M, Purewal JK, Devarakonda Kumar. Unrepaired tetralogy of Fallot: a tale of delayed presentation and limited access to care. Chest. 2023;164(4):A386–A387. doi:10.1016/j.chest.2023.07.316

12. Kudat H, Ahmet BS, Vakur A, Ozcan M. A case of Fallot tetralogy admitted for acute myocardial. Case Rep. 2020;4(1):4–5.

13. Shteerman E, Singh V, Nero T, Lee M, Wilentz J, Menon V. Acute myocardial infarction in uncorrected tetralogy of Fallot. Circulation. 2002;106(4):1–2. doi:10.1161/01.cir.0000023883.39017.f4

14. Ghazaryan N, Adamyan M, Khachatryan L, Hovakimyan T. Syncope in a pregnant woman with repaired Tetralogy of Fallot: a case report. Eur Heart J Case Reports. 2022;6(6):1–5. doi:10.1093/ehjcr/ytac209

15. Gorla R, Macchi A, Franzoni I, et al. Unrepaired tetralogy of Fallot in an 85-year-old man. Congenit Heart Dis. 2012;7(5):1–4. doi:10.1111/j.1747-0803.2012.00642.x

16. Carrington M, Providência R, Chahal AAC, et al. Monitoring and diagnosing intermittent arrhythmias: evidence-based guidance and role of novel monitoring strategies. Eur Heart J Open. 2022;2(6):1–10. doi:10.1093/ehjopen/oeac072

Spending time outside can boost your mood, promote better sleep and support your immune system (plus, it’s free!). The only drawback is that outdoor time also exposes you to the sun’s skin-damaging UV rays. Over time, that could set the stage for skin cancer, the most commonly diagnosed cancer in the United States. “By far, the top risk factor for developing skin cancer is unprotected UV exposure, followed by genetic predisposition,” says dermatologist Geeta Yadav, M.D. 

There is good news, though. According to the Centers for Disease Control and Prevention,  many cases of skin cancer are largely preventable. Adopting safe sun habits like applying a broad-spectrum sunscreen, wearing a hat, sunglasses and clothes that cover your arms and legs, and staying in the shade can all lower your UV exposure and significantly reduce your risk. So can avoiding tanning beds, which also emit large amounts of UV light. 

You can also bolster your skin’s defenses from the inside out by eating more antioxidants. While diet plays a smaller role in skin cancer prevention, research reveals that antioxidants can provide additional protection to safeguard your skin from this all-too-common cancer.

How Antioxidants May Protect Against Skin Cancer

Skin cancer occurs when abnormal skin cells develop in the skin’s outermost layer, called the epidermis. What causes those abnormal cells to develop and grow? The most common cause is DNA damage from exposure to UV rays, either from the sun or tanning beds. However, there are other risk factors too, like getting older or having a family history of skin cancer. You may also be more likely to develop skin cancer if you have blue or green eyes, red or blond hair, or have skin that’s fair or burns or freckles easily. 

Of course, most of these risk factors are beyond your control. But there is one helpful step you can take, and that’s eating an antioxidant-rich diet. In fact, research has found that dietary antioxidants can help counteract some of the damage caused by UV exposure before it turns into cancer. And the list is long: selenium, zinc, copper, carotenoids, polyphenols and vitamins A, C and E may all be protective, according to research. 

They Combat Oxidative Stress

Exposure to UV light sets off a chain reaction that creates a storm of skin-damaging compounds called free radicals. That’s where antioxidants step in. “Antioxidants combat free radicals, unstable molecules that can damage cells and their DNA, proteins and lipids,” says Yadav. “When there are too many free radicals in the body to the point that antioxidants cannot help neutralize them, oxidative stress occurs, leading to cellular dysfunction. This dysfunction could manifest as early signs of aging, but it could also manifest as cancer.” Regularly consuming antioxidant-rich foods equips your body with the defenders needed to neutralize those free radicals before they cause long-term harm. 

They May Prevent the Spread of Cancerous Cells

Not all DNA damage leads to cancer. In fact, our bodies have a natural defense mechanism to kill off DNA-damaged cells before they turn cancerous and start to spread. However, it’s not foolproof, and some damage can fall through the cracks. Fortunately, research reveals that antioxidants called anthocyanins may help speed the process. While anthocyanins are found in lots of fruits and vegetables, one of the best sources for skin protection is berries. So, load up on these juicy fruits for an extra dose of prevention. 

They Help Boost Internal Sun Protection

Sunburns aren’t just painful. This inflammatory reaction in your skin can cause long-lasting damage.  Enter antioxidant-rich foods. Research has found that they help absorb some of the sun’s harmful UV rays and reduce inflammation to decrease the development of sunburn., For instance, one study found that carotenoids, antioxidants found in yellow, orange and red fruits and vegetables, could provide the equivalent sun protection to SPF 4 sunscreen. For the biggest bang, think tomatoes. They’re filled with a carotenoid called lycopene that’s been shown to guard against sun damage from the inside out. 

Tips to Enjoy More Antioxidants

If you’re gearing up to spend more time outdoors, these tips can help you provide your skin with an extra layer of antioxidant protection. 

• Eat the Rainbow: An easy rule of thumb for adding more antioxidants to your diet is to add more color to your plate. Fruits and vegetables with bright, deep hues are often the richest source of these beneficial compounds. 
• Brew a Cup of Green Tea: There’s a reason green tea is added to face creams, masks and serums. It’s rich in antioxidants called catechins that have been shown to calm UV-related skin inflammation. 
• Savor Some Dark Chocolate: While chocolate may not prevent skin cancer, it contains inflammation-taming antioxidants called polyphenols that may improve skin hydration and circulation. Since dark chocolate contains the most polyphenols, the darker the chocolate, the better!

Our Expert Take

Getting regular skin checks and practicing safe sun habits like applying sunscreen, wearing a hat and protective clothing, and staying in the shade may all help reduce your risk of skin cancer. While diet plays a much smaller role, research has found that antioxidants may offer additional protection. Antioxidants are believed to combat cancer-causing oxidative stress, slow the spread of cancer cells and boost your body’s internal defenses against inflammation and sunburn. And the best way to get more of them isn’t a pill or powder. It’s a diet rich in colorful fruits and vegetables. So, before you hit the beach, park or pool, head to the produce aisle!

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a widespread chronic liver condition, affecting an estimated global prevalence of 37.8%, which has significantly increased from 25.5% around 2005.¹ The terminology for this condition has evolved to metabolic dysfunction-associated steatotic liver disease (MASLD), which more accurately reflects its metabolic basis.² However, we continue to use NAFLD in this manuscript for consistency with historical GWAS datasets. NAFLD encompasses a spectrum of liver disorders, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which can potentially progress to advanced stages like fibrosis, cirrhosis, and hepatocellular carcinoma.³ The disease is linked to a high risk of liver-related morbidity and metabolic syndromes, imposing a substantial burden on healthcare systems.³ Despite advancements in its treatments, the exact causes of NAFLD are not completely understood and are likely influenced by a complex interplay of genetic and environmental factors, such as lifestyle choices, dietary habits, and exposure to certain medications or toxins.⁴

Cell senescence is a state of irreversible cell-cycle arrest that occurs in response to various stressors, such as DNA damage, oxidative stress, and telomere shortening.⁵ It is characterized by a distinct secretory phenotype known as the senescence-associated secretory phenotype (SASP), which involves the secretion of pro-inflammatory cytokines, chemokines, and matrix metalloproteinases.⁶ In the context of NAFLD, cellular senescence is thought to play a role in the transition from simple steatosis to NASH, and potentially to more advanced stages such as fibrosis and cirrhosis.⁷ The SASP can create a pro-inflammatory and profibrotic microenvironment, which may contribute to the progression of liver disease.⁸ Additionally, senescent hepatocytes and hepatic stellate cells may directly influence the development of liver cancer through the secretion of factors that promote cell proliferation and invasion.^9,10 However, whether senescence is a marker or a potential mediator of NAFLD progression remains unclear. Therefore, a comprehensive analysis of senescence-related genes in NAFLD using a robust method is necessary to determine whether senescence is a cause or consequence of NAFLD.

Mendelian randomization (MR) offers an alternative to conduct causality assumptions that cannot be readily obtained from conventional observational studies.¹¹ By utilizing randomly allocated genetic variants as instrumental variables (IVs), MR investigates the causal connections between two factors, thereby mitigating confounding bias and reverse causality.^12,13 Summary-data-based Mendelian randomization (SMR) utilizes independent genome-wide association study (GWAS) summary statistics and quantitative trait locus (QTL) data to identify causal genes from GWAS results.¹⁴ Unlike traditional MR analysis, SMR combines multi-omics data including genetic, epigenetic, proteomic evidence to improve the accuracy and reliability of causal inference. Using this approach, potential causal associations between senescence-related genes and NAFLD were identified, followed by a heterogeneity in independent instruments (HEIDI) test.¹⁵

Here, an SMR analysis was executed to investigate the potential associations of senescence-related genes methylation, expression, and protein abundance with the risk of NAFLD.

Methods

Study Design

Figure 1 summarized the overall study design. The current SMR analysis was based on publicly available datasets obtained from previous studies and the FinnGen. In this study, IVs for senescence-related genes extracted at the methylation, gene expression and protein abundance levels. Subsequent SMR analysis was conducted for NAFLD, NASH or liver cirrhosis at these levels. To strengthen the causal inference, colocalization analysis was conducted. Through the integration of results obtained from SMR analysis at these levels, we identified causal candidate genes or proteins. The reporting of MR analysis adhered to the Strengthening the Reporting of Observational Studies in Epidemiology using Mendelian Randomization (STROBE-MR) guidelines.¹⁶

Figure 1 Overall study design of the MR analysis. A flow chart depicts how the SMR analysis was conducted in this study.

Data Sources

GWAS summary statistics for NAFLD was obtained from publicly available databases. The primary discovery dataset (GCST90275041), which comprised 6,623 cases and 26,318 controls of the European ancestry,¹⁷ was supplemented with validation from three independent cohorts: NAFLD (2,568 cases and 409,613 controls) and NASH (175 cases and 412,006 controls) cohorts from FinnGen, and cohort of liver cirrhosis in NAFLD (1,106 cases and 8,571 controls).¹⁸ The definition of diseases is based on the International Classification of Diseases, 9th and 10th Revision (ICD-9 and ICD-10).The detailed information for each phenotypic outcome data was provided in Supplementary Table 1. There is no overlap in samples between the discovery and validation cohorts. This study utilized summary statistics from public GWAS studies, for which ethic approvement has been obtained. Consequently, no further ethical approval was necessary.

949 senescence-related genes were extracted from the CellAge (https://genomics.senescence.info/cells/) database (Build 3) using the keyword “cell senescence”. QTLs can uncover the relationships between SNPs and variations in DNA methylation, gene expression, and protein abundance. Blood eQTL summary statistics were obtained from eQTLGen, encompassing genetic data of blood gene expression in 31,684 individuals from 37 datasets.¹⁸ Blood mQTL summary data were generated from a meta-analysis of two European cohorts: the Brisbane Systems Genetics Study (n = 614) and the Lothian Birth Cohorts (n = 1366).¹⁵ Data on genetic associations with circulating protein levels were sourced from a protein quantitative trait loci (pQTL) investigation involving 54219 individuals.¹⁷

Summary-Data-Based MR Analysis

SMR was employed to assess the association of senescence-related genes methylation, expression, and protein abundance with the risk of NAFLD. Leveraging top associated cis-QTLs, SMR achieved enhanced statistical power compared to conventional MR analysis, particularly in scenarios with large sample sizes and independent datasets for exposure and outcome. Cis-QTLs were selected based on a ±1000 kb window around the gene of interest and a significance threshold of 5.0×10⁻⁸.¹⁹ SNPs with allele frequency differences exceeding 0.2 between datasets were excluded. Thresholds for pQTL, mQTL, and eQTL were set at 0.05. The original version of SMR only uses the lead cis-QTL variant as IV, and it has since been extended to SMR-multi to accommodate the potential presence of multiple cis-xQTL causal variants.¹⁵

In addition to exploring the causal associations between QTLs and NAFLD, the study further investigated the causal relationships between mQTL as the exposure and eQTL as the outcome. The key findings linking mQTL and eQTL with NAFLD are highlighted as signals of particular interest between mQTL and eQTL. Additionally, this study extends to the causal connections between eQTL and pQTL, with a focus on key genes from the mQTL-eQTL association and significant findings from NAFLD GWAS analysis associated with pQTL.

To differentiate between pleiotropy and linkage, we employed the HEIDI test, with P-HEIDI <0.05 indicating potential pleiotropy and leading to exclusion from the analysis. Associations meeting the criteria (p SMR < 0.05, multi-SNP-based P-value < 0.05 and P-HEIDI > 0.05) were considered for colocalization analysis in mQTL, eQTL and pQTL datasets.

Colocalization Analysis

We conducted colocalization analyses using the R package “coloc” to identify shared causal variants between NAFLD and the mQTLs, eQTLs, or pQTLs of senescence-related genes. In these analyses, five different posterior probabilities are reported, corresponding to the following hypotheses: H0 (no causal variants for either trait), H1 (a causal variant for gene expression only), H2 (a causal variant for disease risk only), H3 (distinct causal variants for two traits), and H4 (the same shared causal variant for both traits).²⁰ When GWAS signals and QTLs are found to colocalize, it suggests that the GWAS locus may influence the complex trait or disease phenotype by modulating gene expression or splicing.^21,22 For colocalization analysis, all SNPs within 1000 kb upstream and downstream of each top cis-QTL were retrieved to determine the posterior probability of H4 (PPH4). A PPH4 > 0.5 was used as the cut-off, indicating strong evidence of colocalization between GWAS and QTL associations.²³

Cell Culture and Treatments

The human liver-7702 (HL-7702) cell line was obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The complete culture medium for the HL-7702 cells consisted of DMEM/F-12 (1:1) (Gibco, 11330–032) with 89 mL ITS liquid medium (Sigma, I3146), 1 mL dexamethasone (Sigma, D4902-100mg), and 10 mL FBS (Gibco). The cells were cultured at 37°C in a 5% CO2 incubator. Once the cells reached 60–70% confluence, they were divided into two groups (n=3): (1) Control group (treated with normal saline for 24 hours) and (2) NAFLD group (treated with 1 mM oleic acid (OA; Sigma, USA) for 24 hours). Cell conditions were assessed using Oil Red O staining.

Creation of NAFLD Mouse Model and Histological Process

Six 8-week-old male, C57BL/6 WT mice, were utilized in this experiment. In the experimental group, male C57BL/6 mice were given a diet high in fat, sugar, and cholesterol, along with a high-sugar solution (23.1g/Ld fructose and 18.9g/Ld glucose) and a weekly low dose (0.2 ul /g) of carbon tetrachloride (dissolved in olive oil) administered intraperitoneally. After 16 weeks, NAFLD/NASH mouse models were established. In the control group, male C57 BL/6 mice were given a standard maintenance diet and a weekly intraperitoneal injection of the same dose of olive oil as the experimental group.

After 16 weeks, all mice were euthanized, and blood was drawn from the inferior vena cava using a 1 mL syringe and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected to obtain mouse plasma. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using an automatic biochemical analyzer (ANTECH Diagnostics, Los Angeles, CA, USA). Liver tissue samples were also collected from the mice. A portion of each liver sample was immediately frozen in liquid nitrogen in an EP tube. The remaining tissue was fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (HE). A section of the freshly frozen liver tissue, 8 μm thick, was stained with Masson. All animal experiments received approval from the Institutional Animal Care and Use Committee of Guilin Medical University (GLMC-IACUC-20241090). All animal experiments strictly adhered to the National Standards for Laboratory Animal Welfare issued by the Chinese government (GB/T 35892–2018) and the Guide for the Care and Use of Laboratory Animals (National Research Council, 8th Edition, 2011).

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

The frozen liver tissue was weighed, lysed, homogenized, and mixed with anhydrous ethanol. RNA was extracted from both mouse liver tissues and the HL-7702 cell line using TRIzol reagent (VAZYME, China). After extraction and elution through an RNA binding column, purified total RNA samples were obtained. cDNA was synthesized using the first strand cDNA synthesis kit. SYBR Green qRT-PCR premix was used for quantitative PCR, with gene expression levels normalized to GAPDH. RNA reverse transcription was performed with the PrimeScript™ RT Reagent Kit (VAZYME, China), and qRT-PCR was conducted using an FX Connect system (VAZYME, China) and SYBR^® Green Supermix (VAZYME, China). qRT-PCR was performed in triplicate, with primer details provided in Supplementary Table 2.

Statistical Analysis

All statistical analyses were performed using R (v4.3.0). The R package “ggplot2” and “ggrepel” was used for Manhattan plot generation, and “forestplot” for forest plot generation. The code for SMRLocusPlot and SMREffectPlot was sourced from Zhu et al.¹⁴

Results

Senescence-Related Genes Methylation and NAFLD

Results for causal effects of senescence-related genes methylation on NAFLD were visualized in Figure 2A (See full results in Supplementary Table 3). A total of 143 methylation loci (58 genes) passed the screening criteria (P-SMR < 0.05, multi-SNP-based P-value < 0.05 and P-HEIDI > 0.05). Of the identified signals, 40 near 13 unique genes were found to have strong colocalization evidence support (PPH4 >0.5) including ENDOG (cg13630871), S100A6 (cg24155129, cg01910639) and TP5313 (cg14273083). Specifically, ENDOG methylation at cg13630871 (OR = 1.02, 95% CI = 1–1.04) was linked to an increased risk of NAFLD. Conversely, certain methylation loci exhibited divergent association with NAFLD, such as S100A6, with cg24155129 (OR = 0.94, 95% CI = 0.9–0.98) linked to a decreased incidence of NAFLD and cg01910639 demonstrating the opposite (OR = 1.03, 95% CI = 1.01–1.06). The colocalization for representative methylation loci and NAFLD was visualized in Figure 2B. Among these identified CpG sites, the association for CD34 (cg15031826), PPARG (cg04632671), FOXP1 (cg06175008), TACC3 (cg10756475), FGFR3 (cg07041428, cg25342568, cg01464969, cg14661159, cg14101193, cg07458712) were replicated in the NAFLD replication cohort (FinnGen). The detailed associations in the NAFLD, NASH and liver cirrhosis replication cohorts were provided in Supplementary Tables 4–6.

Figure 2 SMR analyses of the causal effects of senescence-related genes mQTL on NAFLD. (A). Forest plot depicting the association between representative gene methylation and NAFLD. *Indicated causal associations supported by colocalization evidence. (B) Locus comparison plots between a representative gene (TP53I3) methylation loci and NAFLD. The scatter plot compares -log10(p) values from GWAS (x-axis) and mQTL (y-axis) analyses. Each point represents a SNP, with color indicating linkage disequilibrium with the lead SNP (highlighted in purple).

Senescence-Related Genes Expression and NAFLD

Causal effects of senescence-related genes expression on NAFLD were presented in Figure 3A (See full results in Supplementary Table 7). A total of 16 genes were found to be associated with NAFLD (P-SMR < 0.05, multi-SNP-based P-value < 0.05 and P-HEIDI > 0.05), in which S100A6, DTL, DNMT3A, ATG7, THRB, EGR2, FOXO1 and CHEK2 were positively associated with NAFLD incidence. Specifically, S100A6 (OR = 1.11, 95% CI = 1.04–1.19) was a potential risk factor for NAFLD and ENDOG (OR = 0.99, 95% CI = 0.97–1) exhibited the opposite. Among the loci corresponding to these genes, colocalization between representative genes and NAFLD was visualized (PPH4 > 0.5) (Figure 3B and C). Among the identified genes, none of them were replicated in the NAFLD cohort, NASH cohort and liver cirrhosis cohort (Supplementary Tables 8–10).

Figure 3 SMR analyses of the causal effects of senescence-related genes eQTL on NAFLD. (A) Forest plot depicting the association between representative gene expressions and NAFLD. *Indicated causal associations supported by colocalization evidence. Locus comparison plots between (B) ENDOG and (C) TP53I3 expression and NAFLD. The scatter plot compares -log10(p) values from GWAS (x-axis) and eQTL (y-axis) analyses. Each point represents a SNP, with color indicating linkage disequilibrium with the lead SNP (highlighted in purple).

Senescence-Related Protein Abundance and NAFLD

Causal effects of senescence-related protein abundance on NAFLD were presented in Figure 4A (See full results in Supplementary Table 11). In total, 6 proteins were found to be associated with NAFLD at the criteria (P-SMR < 0.05, multi-SNP-based P-value < 0.05 and P-HEIDI > 0.01), in which EIF2AK3, TIGAR and ING1 were positively associated with NAFLD incidence. Specifically, ING1 (OR = 1.16, 95% CI = 1.02–1.31) was a potential risk factor for NAFLD. Colocalization analysis between representative proteins and NAFLD were visualized (PPH4 > 0.5) Figure 4B and C. Among the identified proteins, only TIGAR was associated with NAFLD in the replication cohort (FinnGen) (Supplementary Tables 12–14).

Figure 4 SMR analyses of the causal effects of senescence-related protein abundance on NAFLD. (A) Forest plot depicting the association between representative protein abundance and NAFLD. *Indicated causal associations supported by colocalization evidence. Locus comparison plots between the level of (B) ING1 and (C) TIGAR and NAFLD. The scatter plot compares -log10(p) values from GWAS (x-axis) and pQTL (y-axis) analyses. Each point represents a SNP, with color indicating linkage disequilibrium with the lead SNP (highlighted in purple).

Tissue-Specific Validation

We further explored the causal associations between gene expression and NAFLD in the liver tissues. The expression of ENDOG in the liver tissues was negatively associated with NAFLD (OR = 0.98, 95% CI = 0.97–1), which was consistent with the protective role suggested in the SMR analysis. The detailed information regarding the association between identified genes with NAFLD in the liver tissues was provided in Supplementary Table 15.

Multi-Omics Data Integration

By integrating blood mQTL and eQTL data, we performed SMR with the methylation loci of the common genes in mQTL-GWAS and eQTL-GWAS results as the exposure and the expressions of these genes as the outcome. At a stringent criteria (P-SMR < 0.05, multi-SNP-based P-value < 0.05 and P-HEIDI > 0.05), S100A6 methylation at cg24155129 (OR = 0.6, 95% CI = 0.49–0.73) and cg01910639 (OR = 1.35, 95% CI = 1.24–1.47) were associated with a decreased and increased expression of S100A6 respectively (Table 1). The detailed integrated associations were provided in Supplementary Table 16.

Table 1 Causal Effects of the Senescence-Related Gene Methylation on Gene Expression

We did not identify common proteins between intersecting genes between mQTL and eQTL, and pQTL-GWAS results. Therefore, no SMR analysis was performed with the eQTL as the exposure and the pQTL as the outcome.

Integrating the multi-omics level evidence, we found that S100A6 may be causally associated with NAFLD. In particular, the methylation site cg01910639 showed a positive correlation with NAFLD risk and positively regulated S100A6 gene expression, which was positively associated with NAFLD risk. Additionally, cg24155129, which was also negatively correlated with NAFLD risk, negatively regulated S100A6 expression. Therefore, we propose that the higher methylation levels at cg20552903 and lower methylation levels at cg24155129 upregulates S100A6 gene expression, leading to an increased risk of NAFLD.

To visualize the results of our SMR analysis, we created locus plots for S100A6 methylation, expression and NAFLD (Figure 5A and B). Furthermore, we also provided the effect plots confirming the effects between S100A6 methylation and expression and NAFLD (Figure 6).

Figure 5 Locus plots showing (A) S100A6 methylation and (B) S100A6, their locations within the chromosome (lower panel). The Y-axis indicated the negative log of the p-values instrumental in deeming this locus significant in the SMR analysis.

Figure 6 SMR effect plots for (A) S100A6, (B) methylation site cg01910639 and cg24155129, and their associations with NAFLD. cis-QTLs were marked by blue dots, while top cis-QTLs were highlighted in red triangles.

Validation of Candidate Genes in Mouse and Cell Models of NAFLD

To validate the findings from the analysis above, we conducted experiments using both mouse and cell models of NAFLD. We assessed the expression levels of S100A6, ENDOG and TP53I3 in cell cultures (normal and steatotic). Oil Red O staining revealed substantial lipid accumulation in the NAFLD group cells, marked by an increased number of fat droplets (Figure 7A). qRT-PCR analysis of mRNA levels showed a significant rise in the expression of S100A6 and TP53I3, and lower expression of ENDOG in the NAFLD group compared to the control group (Figures 7B).

Figure 7 Expression of the Key Genes in a Cell and Mouse NAFLD Model. The NAFLD mouse model was generated in C57BL/6J mice. Pair-fed mice were used as controls. Serum and liver tissues were collected on the 16 weeks for further analysis. (A) Oil Red O staining. (B) The relative mRNA expression of S100A6, ENDOG and TP53I3 in cell NAFLD model was verified by qRT‒PCR. (C) HE and Masson staining. (D) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. (E) The relative mRNA expression of the S100A6 in mouse NAFLD model was verified by qRT‒PCR. N = 4 in mouse model and N = 3 in Cell model, *p < 0.05, **p < 0.01, ***p < 0.001.

In the animal model, we specifically focused on S100A6 due to multi-omics evidence suggesting that methylation at cg20552903 and/or cg24155129 might regulate its expression. H&E and Masson staining suggested hepatic steatosis in the NAFLD group (Figure 7C). AST and ALT levels were significantly higher in the NAFLD group than in the control group (Figures 7D), indicating successful establishment of the NAFLD model. qRT-PCR measurements revealed that the expression levels of S100A6 and ENDOG were significantly different in the NAFLD group compared to the control group (Figures 7E), suggesting their potential regulatory role in NAFLD development.

Discussion

In this study, we systematically investigated the causal relationships between the methylation, gene expression and protein abundance of senscence-related genes and NAFLD using a multi-omics approach and SMR analysis. We chose to use the term NAFLD, given the ongoing transition to MASLD terminology, to maintain consistency with historical datasets and clinical contexts. Integrated multi-omics evidence from blood mQTL and eQTL SMR analysis revealed 3 genes (S100A6, ENDOG and TP53I3) as potential causal genes associated with NAFLD. And we further confirmed these findings by validation in mouse and cell models of NAFLD.

At the mQTL and eQTL levels, S100A6 was found to be a potential risk factor for NAFLD. S100A6, also referred to as calcyclin, encodes a protein belonging to the S100 family and is integral to the regulation of cellular senescence. This gene has been shown to have an inhibitory effect on senescence-like changes in various cell types.²⁴ Its deficiency has been shown to induce morphological and biochemical features that are characteristic of cellular senescence.²⁵ Recently, there has been an ongoing research into the role of S100A6 in NAFLD. A recent study has identified a significant relationship between the liver-derived protein S100A6 and the progression of NAFLD.²⁶ Elevated serum levels of S100A6 were observed in both human patients with NAFLD and in a high-fat diet-induced mouse model, correlating negatively with β-cell insulin secretory capacity. Depletion of hepatic S100A6 in mice improved glycemia, suggesting a contributory role of S100A6 in the pathophysiology of diabetes associated with NAFLD. Additionally, a review by Delangre et al highlighted that the aberrant activity of S100 isoforms, including S100A6, contributes to the dysregulation of lipid metabolism leading to hepatic steatosis and insulin resistance (IR), which are hallmarks of NAFLD.²⁷ While the exact mechanisms are not fully elucidated, it was suggested that S100 proteins may influence cell proliferation, apoptosis, migration, and inflammation, which are all relevant to the pathophysiology of NAFLD. In our study, we discovered that higher levels of S100A6 might be associated with an increased risk of developing NAFLD, possibly by the dysregulation of lipid metabolism and promotion of hepatic steatosis. Furthermore, our findings propose a novel avenue for therapeutic intervention, where modulating S100A6 expression or its regulatory pathways could be explored as a strategy to slow or halt disease progression in NAFLD patients. Additional research is required to fully understand the complex role of S100A6 in hepatic health and disease, and to determine whether diminishing its effects could offer a viable treatment approach for those at risk of NAFLD.

In addition to S100A6, ENDOG was demonstrated to be a protective factor for NAFLD. ENDOG is a gene that encodes the mitochondrial protein Endonuclease G, a crucial enzyme involved in various cellular processes, particularly apoptosis and DNA metabolism. In the context of NAFLD, research has uncovered that ENDOG promotes NAFLD development via regulating the expression of lipid synthesis-associated genes like ACC1, ACC2, and FAS.²⁸ Loss of ENDOG was found to repress high-fat diet-induced liver lipid accumulation.²⁸ Therefore, targeting ENDOG could be a potential therapeutic approach for NAFLD. However, our study proposed the opposite, in which ENODG expression was negatively associated with NAFLD incidence. The controversy between ENDOG and NAFLD could be due to the multifactorial and dynamic nature of ENDOG in NAFLD pathogenesis. Additionally, the role of ENDOG might be context-dependent, with its expression and activity influenced by various environmental and genetic factors that could alter its function from protective to pathogenic, underscoring the complexity of its involvement in NAFLD.

TP53I3, also known as tumor protein p53 inducible protein 3, functions as a quinone oxidoreductase, which is involved in cellular redox reactions. Due to its role in apoptosis and stress responses, TP53I3 has been implicated in cancer research.²⁹ However, no direct evidence about TP53I3 in NAFLD has been presented. In this study, we demonstrated that TP53I3 expression was negatively associated with the incidence of NAFLD, suggesting it as a potential protective factor. We could postulate that TP53I3 is involved in the generation of ROS and participates in p53-mediated cell death pathways associated with NAFLD progression.

By integrating multi-omics analysis of mQTL and eQTL, we uncovered a potential regulatory axis in NAFLD pathogenesis: DNA methylation at specific loci suppresses S100A6 gene expression, reducing S100A6 protein levels and decreasing the susceptibility to NAFLD. This opens up new avenues for therapeutic intervention in NAFLD, such as targeting this regulatory axis to modulate gene expression. Potential interventions might include the use of methylating agents or therapies to reduce S100A6 expression. Additionally, the S100A6 methylation-S100A6 axis could serve as a biomarker for early detection, prognosis, and monitoring of therapeutic responses in NAFLD patients, thereby enhancing personalized clinical care.

This study represents the first evaluation of the associations between senescence-related genes and NAFLD using SMR and colocalization. The main strength of this study is its use of SMR, allowing simultaneous assessment of the associations between methylation, expression, and protein abundance of senescence-related genes and NAFLD in independent European populations. Additionally, colocalization approaches effectively eliminate potential bias caused by linkage disequilibrium. Additionally, GWAS datasets with large sample sizes increased the statistical power of our study. Nonetheless, some limitations have to be addressed. First, due to the limited number of senescence-related proteins in the pQTL dataset, the current study did not fully explore the causal relationship between senescence protein abundance and the risk of NAFLD. Second, the exclusive use of cis-QTLs in SMR analysis may limit the comprehensiveness of the identified genetic associations and overlook long-range regulatory effects relevant to NAFLD pathogenesis. Third, SMR also has limited ability to exclude horizontal pleiotropy, where a gene affects disease through pathways independent of expression. Fourth, the tissue-specific nature of eQTL/mQTL associations means that the relevance of the selected QTL tissues to the disease-affected tissues directly impacts the reliability of the findings. Fifth, conclusions should be treated with caution when extending to other populations, as this study was based solely on European ancestry. Lastly, the findings from SMR analysis, while valuable for identifying potential causal associations, may not fully reflect clinical observations. SMR relies on genetic data and statistical models, which may not capture the full complexity of biological pathways or the influence of environmental factors on NAFLD. Additionally, SMR reflects the lifelong exposure effects associated with genetic variants, which may differ from the short-term effects of interventions or environmental exposures. Therefore, the results need to be contextualized with observational or clinical studies to better understand their relevance and applicability in clinical settings.

Conclusions

Our findings suggest potential causal relationships between senescence-related gene methylation, expression, and protein abundance and NAFLD, with S100A6, ENDOG and TP53I3 emerging as notable candidates in NAFLD pathogenesis. These findings provide a foundation for future research endeavors and clinical applications, but further investigations are needed to confirm these associations and their therapeutic implications.

Abbreviations

GWAS, genome-wide association study; HEIDI, heterogeneity independent instruments; HEIDI, heterogeneity in the dependent instrument; HL-7702, Human Liver-7702; HE, hematoxylin and eosin; IVs, instrumental variables; MR, Mendelian randomization; NAFLD, Nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PPH4, posterior probability of H4; QTL, quantitative trait locus; qRT-PCR, Quantitative reverse transcription-polymerase chain reaction; SMR, summary-data Mendelian randomization; SASP, senescence-associated secretory phenotype.

Data Sharing Statement

The GWAS summary statistics for NAFLD can be accessed via the FinnGen and GWAS Catalog under the search term of GCST90275041 and GCST008469. The QTLs data for senescence-related genes can be obtained via CellAge.

Ethics Approval and Consent to Participate

According to Item 1 and 2 of Article 32 of “the Measures for Ethical Review of Life Science and Medical Research Involving Human Subjects”, this study is exempt from ethical review and approval, as it utilized summary statistics from public GWAS studies. All animal experiments received approval from the Institutional Animal Care and Use Committee of Guilin Medical University (GLMC-IACUC-20241090). All animal experiments strictly adhered to the National Standards for Laboratory Animal Welfare issued by the Chinese government (GB/T 35892-2018) and the Guide for the Care and Use of Laboratory Animals (National Research Council, 8th Edition, 2011).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study was funded by the First Affiliated Hospital of Guilin Medical University, PhD start-up fund, and The Project for Improving the Research Foundation Competence of Young and Middle-aged Teachers in Guangxi Universities (2025KY0526). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper.

Disclosure

The authors declare that they have no competing interests.

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