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Introduction

Atopic dermatitis (AD) is the most prevalent chronic and pruritic inflammatory skin disease,¹ characterized by epidermal desquamation, intense pruritus, and lichenified lesions.² This disease affects 20% of children and 4% of adults globally, causing significant quality-of-life impairment due to chronic pruritus, sleep disturbance, and psychosocial burden.^3,4 Common antigens triggering AD include house dust mites, Staphylococcus aureus enterotoxins, food allergens (eg, egg, milk), and environmental pollens.⁵ AD is a continuous process from acute to chronic phase, accompanied by three interconnected pathological mechanisms, skin barrier dysfunction, immune dysregulation, and abnormal neural signaling.^6–8 Skin barrier dysfunction manifests as downregulated expression of barrier genes, such as loricrin (LOR), filaggrin (FLG), and elongation of very long-chain fatty acid (ELOVL).^9–11 Immune dysregulation mainly results from sustained inflammation mediated by type I, II, and III adaptive immune responses driven by differentiated CD4+ T cells (Th1/ Th2/ Th17).¹² Abnormal neural signaling manifests as sensory nerve hyperinnervation, upregulated pruritogens, such as interleukin (IL) −31 and thymic stromal lymphopoietin (TSLP), and neuroimmune crosstalk amplifying itch-scratch cycles.¹³ These three components reinforce each other.¹⁴ Environmental antigens first damage the skin barrier, which activates dendritic cells (DCs) and recruits Th cells. Activated Th cells release cytokines like IL-4, IL-13, and IL-31. These cytokines further break down barrier proteins and sensitize sensory nerves. Finally, neurogenic inflammation causes tissue damage which makes the immune system overactive, establishing a harmful cycle that worsens AD.¹⁵

For decades, the cornerstone of AD treatment has relied on topical corticosteroids and topical calcineurin inhibitors,¹⁶ whose adverse effects include skin atrophy, pigmentation changes, anaphylaxis, and potential complications (eg, folliculitis or tinea infection) after drug discontinuation.¹⁷ Recent advances in biological agents and small molecule inhibitors have introduced novel treatment options for AD, such as dupilumab (targeting IL-4Rα), tralokinumab/ lebrikizumab (targeting IL-13), CIM331/ nemolizumab (targeting IL-31R), crisaborole (targeting Phosphodiesterase-4), and tofacitinib (targeting Janus kinase 1/ 3).¹⁸ At the same time, because of potential safety problems, high recurrence rate and high economic burden of the AD, the use of these drugs is limited. In this context, traditional Chinese medicine (TCM) has attracted the attention of clinicians and AD patients as a complementary treatment for AD, especially in and around China, due to its abundance of natural anti-inflammatory compounds.¹⁹

Yin-Hua Li-Shi Decoction (YLD) is a TCM approved by medical institutions, which is composed of six herbs aimed at removing dampness. It has a long history of being used for the treatment of AD. The chlorogenic acid and luteoloside derived from honeysuckle in YLD has been shown to inhibit the secretion of pro-inflammatory cytokines such as IL-6 and TSLP that acts on multiple cell lineages, including macrophages, mast cells, neutrophils, DCs, and T cells, ultimately suppressing moderate to severe immune responses.^20,21 However, the specific mechanisms underlying YLD in the treatment of AD were still unclear and lacked systematic validation.

The present study was aimed at exploring the therapeutic effects and mechanisms of YLD in AD. We used MC903-induced AD-like mouse model to evaluate YLD therapeutic benefits for AD and to clarify the mechanisms by which it regulated immunity and restored the skin barrier.

Material and Methods

Drugs and Reagents

All herbs were purchased from WanShiCheng Pharmaceutical Co., Ltd. (Shanghai, China), and authenticated according to the Pharmacopoeia of the People’s Republic of China 2020 Edition by Professor Huijun Pan from Shanghai Skin Disease Hospital, School of Medicine, Tongji University. Voucher specimens of these herbal materials were deposited at the Shanghai Skin Disease Hospital with reference numbers YL1-6. Chlorogenic acid and specnuezhenide used as standard compounds were from Meilunbio (Shanghai, China). MC903 was purchased from Macklin Biochemical Co., Ltd. (#C833062, Shanghai, China). Antibodies used in the study were obtained from the following sources: anti-FLG (#GTX23137, GeneTex, Beijing, China), anti-LOR (#A21039, ABclonal, Wuhan, China), anti-ELOVL6 (#A21094, ABclonal, Wuhan, China), anti-TSLP (#ab188766, Abcam, Cambridge, U.K)., anti-β-Actin (#AC026, ABclonal, Wuhan, China), HRP-conjugated Goat anti-Rabbit IgG (H+L) (#AS014, ABclonal, Wuhan, China), anti-IL-4 (#25-7042-42, Invitrogen, California, USA), anti-IL-17A (#506904, Biolegend, California, USA), anti-IFN-γ (#563376, BD Biosciences, New Jersey, USA), anti-rabbit IgG (H+L) Ab HRP Affinity purified polyclonal (#95058–730, KPL, Maryland, USA), anti-CD4 (#ab183685, Abcam, Cambridge, U.K)., anti-CD4 (#555349, BD Biosciences, New Jersey, USA), anti-CD8 (#100733, Biolegend, California, USA), anti-CD86 and anti-CD80 (#561962 and #561955, BD Biosciences, New Jersey, USA). ELISA kits used for analysis were IL-4 (#EM3199M, WellBio, Shanghai, China), IL-13 (#EM3167M, WellBio, Shanghai, China), TNF-α (#RK00027, ABclonal, Wuhan, China), and IgE (#RK00170, ABclonal, Wuhan, China). Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Vazyme Biotechnology Co., Ltd (#A211-02, Nanjing, China). All other chemicals used in the experiments were of analytical grade.

Preparation of YLD

The YLD consists of six Chinese herbal medicines, including Jinyinhua (Honeysuckle), Shanyao (Yam, Siberian), Huangjing (Solomonseal rhizome), Digupi (Cortex lycii radices), Nvzhenzi (Fructus ligustri lucidi), Yiyiren (Coix seed), and the daily dose of adult clinical YLD is 54 g of crude drugs (Table 1). YLD extraction is the first step to adding 8 times amount of water in crude drugs (w/w), decocting 2 h after filtering, the rest of the student to join six times the amount of water decoction 1 h again. The above water extract was concentrated to 40 mL using a EYELA N-1300D-WB rotary evaporator, and the final YLD concentration had a crude drug equivalent of 6.00 g/mL. The concentration was appropriately diluted to crude drug equivalents of 3.00 g/mL and 1.50 g/mL to form medium and low doses, respectively. All prepared YLD samples were stored at 4 °C until subsequent use.

Table 1 The Herbal Composition of YLD

Quality Control of YLD

Fingerprint Identification

High-performance liquid chromatography (HPLC) was employed to construct a fingerprint profile and evaluate the quality of YLD and for quality control. Chlorogenic acid and specnuezhenide were dissolved in methanol to prepare a stock solution. The YLD reference solution (R) (200 μg/mL) was prepared by diluting the original 1.50 g/mL YLD solution and then filtering through a 0.45 μm membrane filter. The concentration of YLD extract (g crude herb/mL) represented a drug extract ratio, which was calculated by the quotient of total dry herb mass over final decoction volume.²² The YLD HPLC test solution (YLD sample) (150 μg/mL) was prepared by diluting the original 1.50 g/mL YLD solution and then filtering through a 0.45 μm membrane filter. The quality consistency validation and methodological parameters are detailed in Supplementary Information 1 Table S1. The content of the reference compounds in YLD was calculated based on the pre-constructed standard curves of chlorogenic acid and specnuezhenide. The fingerprint profile of YLD was identified by comparing the relative retention time and ultraviolet characteristics of the internal reference with the YLD test sample. The quality of YLD was controlled by comparing the similarity of the fingerprint profiles among 10 batches of YLD (S1-S10). To ensure batch consistency, all 10 tested batches were derived from the same cultivation batch of raw plants.

The liquid chromatography system used was an Waters Alliance HPLC (Waters, Massachusetts, USA), consisting of an E2695 separation module and a 2998 photodiode array detector with an autosampler. HPLC was performed on an ZORBAX Eclipse Plus C18 column (4.6 mm × 250 mm, 5 μm) (Agilent, California, USA). The mobile phase consisted of solvent A (acetonitrile) and solvent B (0.1% phosphoric acid water), and the gradient elution conditions were shown in Table 2. The UV absorption wavelength was set at 230 nm, column temperature at 25 °C, injection volume at 10 μL, and flow rate at 1.0 mL/min.

Table 2 HPLC Gradient Evaluation Conditions of YLD

Ingredient Identification

The components of YLD water extract were identified by liquid chromatography-tandem mass spectrometry (LC-MS). The YLD LC-MS test solution (600 μg/mL) was prepared by diluting the original 1.50 g/mL YLD solution and then filtering through a 0.45 μm membrane filter. The YLD water extract was analyzed using a LC-MS system composed of an ACQUITY UPLC I-Class HF ultra-high performance liquid chromatography coupled with a QE high-resolution mass spectrometer. The mobile phase consisted of solvent A (0.1% formic acid aqueous solution) and solvent B (acetonitrile). The sample was separated at a flow rate of 0.35 mL/min, and the gradient elution conditions are shown in Table 3.

Table 3 LC-MS Gradient Elution Conditions of YLD

LC-MS detection was performed using an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) (Waters, Massachusetts, USA). Mass spectrometry data acquisition was carried out in electrospray ionization (ESI) positive and negative modes, and the data-dependent acquisition (DDA) mode was used, with a mass range of m/z 90 to 1300. The capillary temperature was set to 320 °C, and the probe heating temperature was set to 350 °C.

Cell Culture

Murine macrophage cell line (RAW264.7) and human keratinocyte cell line (HaCaT) were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). RAW264.7 and HaCaT cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin-streptomycin (Beyotime, China). All cultures were maintained in a humidified atmosphere with 5% CO₂ at 37 °C.

Animals

Male BALB/c mice aged 8–9 weeks (weighing 22–25 g) were obtained from the SiPeiFu Biotechnology Co., Ltd. (Shanghai, China) and housed under SPF conditions. The mice were maintained at a room temperature of 26 °C with a relative humidity of 40% and a 12:12-hour light/dark cycle. They were provided with standard mouse maintenance feed and water ad libitum.

Apoptosis Assay

HaCaT cells were cultured in 6-well plates (30×10⁴ cells per well) for 24 h. The 6-well plate was then subjected to a 24-hour incubation in the following groups: the PBS group (0 µg/mL YLD) and the PBS+YLD group (150 µg/mL YLD). Apoptosis of HaCaT cells was assessed using propidium iodide (PI) and fluorescein isothiocyanate (FITC)-labeled Annexin V staining, followed by detection and analysis with Navios 6 COLORS/2 LASER flow cytometer and FlowJo Software.

Analysis of Macrophage Phenotype

To induce the inflammatory phenotype of macrophages, RAW264.7 cells were treated with 0.5 µg/mL LPS and 2 ng/mL IFN-γ. 1 h prior to LPS and IFN-γ stimulation, RAW264.7 cells were treated with YLD (150 µg/mL) to assess the impact of YLD on macrophages. After 18 h, cells were incubated with CD86 and CD80 antibodies for 30 minutes. The Navios 6 COLORS/2 LASER flow cytometer and FlowJo software were used to evaluate the differentiation of YLD in suppressing the inflammatory phenotype of macrophages.

Induction of AD-Like Mice Model and YLD Administration

Fifty male BALB/c mice were induced with AD by repeated topical application of MC903 on their ears for 14 days. The mice were randomly divided into five groups (n = 10/group): control mice treated with ethanol (Ethanol), control mice treated with MC903 alone (MC903), experimental mice treated with both MC903 and low-dose YLD (YLD-L), experimental mice treated with both MC903 and medium-dose YLD (YLD-M), and experimental mice treated with both MC903 and high-dose YLD (YLD-H). In brief, Ethanol group was topically applied with 20 μL of ethanol on the ears daily, while other groups were sensitized with 2 nmol of MC903 on the ears daily for 15 days (Day 0 to Day 14). From Day 1 to Day 14, Ethanol group received daily treatment with ethanol, and YLD-treated groups received oral administration after diluting YLD extract. Three different dosage levels were used: low, medium, and high, corresponding to final concentrations of 1.50 g/mL, 3.00 g/mL, and 6.00 g/mL of the YLD extract, respectively. The lose dose was calculated based on the clinical dose converted to mouse dose referring to the FDA human dose conversion table for animal doses. And each 20 g mouse received 0.15 mL decoction orally daily. The efficacy of the treatment was evaluated 12 h after the last administration (Day 15). Blood samples were collected by retro-orbital bleeding to obtain serum samples, and tissue samples were collected after cervical dislocation for subsequent experimental analysis.

AD-Related Evaluation

During the study, daily monitoring of mice included recording body weight using an electronic balance, measuring transepidermal water loss (TEWL) using an intelligent skin analyzer (#W-2100, Yizi-moqi, Guangzhou, China), assessing ear thickness using vernier calipers, and scoring AD (SCORAD). The SCORAD consisted of the following items: (i) pruritus/itching, (ii) erythema/hemorrhage, (iii) edema, (iv) excoriation/erosion, and (v) desquamation/dryness. Each symptom was graded as follows: 0 (no symptoms), 1 (mild), 2 (moderate), or 3 (severe). The total score for AD ranged from 0 to 15. Additionally, spleen index was measured on Day 15. The spleen index was calculated using the formula:

Spleen Index (mg/g) = Spleen weight (mg) /Mouse weight (g).

Histopathological Analysis

Tissue specimens from the inflamed area of the ear were fixed in formalin and embedded in paraffin. For hematoxylin and eosin (HE) staining, paraffin sections were stained with hematoxylin and eosin. Samples were examined and captured using an optical microscope (QT50GS, Yuehe, Shanghai, China), and the epidermal thickness was counted at five randomly selected sites under 40× and 100× magnification. For toluidine blue (TB) staining, paraffin sections were stained with TB. Samples were examined and captured using an optical microscope, and the number of mast cells was counted at five randomly selected sites under 40× magnification.

Enzyme-Linked Immunosorbent Assay (ELISA)

Mouse blood samples were centrifuged at 12,000 g and 4 °C for 20 minutes to separate the upper serum. ELISA was performed to measure the concentrations of IL-4, IL-13, TNF-α, and IgE in the serum. The measurements were carried out using commercially available ELISA kits following the manufacturer’s instructions.

Immunohistochemical Analysis

Paraffin-embedded tissue specimens were incubated overnight with anti-FLG (1:500), anti-TSLP (1:500), and anti-CD4 (1:500). After washing with PBS, Anti-Rabbit IgG (H+L) Ab HRP Affinity purified polyclonal (1:200) were added and incubated for 1 h, followed by DAB staining. The sections were counterstained with hematoxylin. Immunohistochemically stained slides were examined and captured using an optical microscope, and records were taken at five randomly selected sites under 40× magnification.

Western Blot Analysis

Tissue protein lysates were obtained in RIPA buffer (Beyotime, Shanghai, China) containing PMSF (Beyotime, Shanghai, China). Protein concentration was quantified using a BCA assay kit (Beyotime, Shanghai, China). Proteins were separated by 10% SDS-PAGE (Servicebio, Wuhan, China) and then transferred onto PVDF membranes. The membranes were incubated overnight at 4 °C with corresponding primary antibodies: anti-FLG, anti-LOR, anti-ELOVL6, anti-TSLP (1:1000), and anti-β-Actin (1:2000). Subsequently, the membranes were incubated with secondary HRP-conjugated (1:1000) for 1 h to detect antibody binding. β-Actin was used as an internal reference. The target protein signals were analyzed using Image J application.

Analysis of Spleen T Cells

Spleen tissue was dissociated and filtered through a 70 μm cell strainer. The cell suspension was centrifuged, and resuspended in PBS to prepare a single-cell suspension. Red blood cells in the splenocytes were removed using ACK lysis buffer (#A1049201, Thermo Fisher Scientific, Massachusetts, USA) and washed before staining. Cells were incubated at 4 °C in the dark for 30 minutes with 2.5 μL of CD4 antibody and CD8 antibody. Subsequently, a permeabilization wash buffer was added. Then, 2.5 μL of IL-4, IFN-γ, and IL-17A antibodies were added, and the cells were incubated at 4 °C for 30 minutes. The analysis was performed using a Beckman moflo Astrios EQ flow cytometer with FlowJo Software, and the proportions of Th1, Th2, and Th17 cells in CD4+ T cells were recorded.

Statistical Analysis

Data analysis and graph plotting were performed using GraphPad software (version 8.0). Normality of data distribution was verified using the Shapiro–Wilk test (α=0.05). Normally distributed data were analyzed by one-way ANOVA with Tukey’s post hoc test. Data were expressed as mean ± standard deviation (SD), and one-way ANOVA was used for comparisons among multiple groups. A P-value < 0.05 was considered statistically significant.

Results

Fingerprint and Component Identification of YLD

To ensure the stability of YLD, chlorogenic acid and specnuezhenide were selected as reference compounds and studied as specific indicators of YLD (Figure 1A). HPLC analysis was performed on the reference compounds, with a retention time of 12.7 minutes for chlorogenic acid and 26.3 minutes for specnuezhenide. The peak shape of both chlorogenic acid and specnuezhenide exhibited Gaussian distribution, with sharp and symmetrical peaks.

Figure 1 HPLC fingerprint of YLD. Chromatogram at 230 nm showing the reference compounds and the chromatogram (A), and fingerprints of ten different batches (B) of YLD. (C) Distribution chart of the top 10 components in terms of content in YLD. (D) BPC chromatograms of YLD in positive and negative ion modes.

The standard curves of chlorogenic acid and specnuezhenide reference compounds were provided in the Supplementary Information 1 Tables S2 and S3, Figure S1a and b). Based on the standard curves of these two reference compounds, the content of chlorogenic acid in YLD (clinical) was determined to be 2.697 mg/mL and the content of specnuezhenide was determined to be 8.405 mg/mL. We conducted fingerprint analysis of YLD from 10 batches (Figure 1B), and the highest similarity exceeded 0.9006 (Supplementary Information 1 Table S4). The above analysis data indicated that YLD was stable and of controllable quality.

The chemical components of the YLD extract included a total of 705 compound molecules, which were identified using LC-MS. These 705 compounds were classified chemically based on their quantity and content, as shown in Figure 1C. The Base Peak Chromatogram (BPC) in both positive and negative ion modes is presented in Figure 1D. The top 15 most abundant compounds among the 705 metabolites identified are Secologanic acid, Citric acid, Cryptochlorogenic acid, GL3, Specnuezhenide, Secoxyloganin, Mannoheptulose, Sucrose, Turanose, Swertiamarin, D-Galactose, Verbenalol, Dambose, 5-Hydroxymethylfurfural, 6α-dihydrocornic acid, and Salidroside (Table 4). The quantitative and qualitative identification results of the 705 metabolites in the YLD extract are provided in the Supplementary Information 2.

Table 4 Top 15 Most Abundant Compounds Among YLD Metabolites

YLD Did Not Induce Apoptosis in HaCaT

Flow cytometry, utilizing PI and membrane-associated protein V-FITC staining, was employed to assess the impact of YLD on apoptosis in HaCaT. After treatment with 150 μg/mL of YLD for 24 h, there was an increase in the percentage of apoptotic HaCaT but was no significant difference (Figure 2A). However, the viability of HaCaT remained above 80%. Generally, a cell survival rate greater than 80% post-drug treatment is considered indicative that the drug does not induce apoptosis.

Figure 2 In vitro pharmacological activity of YLD. (A) Apoptotic HaCaT treated with 150 μg/mL YLD for 24 h were detected by flow cytometry. (B) M1 macrophages were treated with YLD (150 μg/mL) for 24 h and then detected the M1-phenotype surface marker (CD86 and CD80) by flow cytometry. The data was collected from three independent experiments and was presented as a mean ± SD.

YLD Inhibited Macrophage to M1 Differentiation

Macrophages, essential phagocytic cells of the immune system, play a crucial role in coordinating innate immune responses.²³ It is noteworthy that macrophages also possess antigen-presenting capabilities, facilitating the presentation of antigen peptides to T cells, thereby initiating adaptive immune responses.²⁴ M1 macrophages represent classically activated macrophages that exhibit a pro-inflammatory phenotype, characterized by the production of high levels of cytokines such as IL-1β, IL-6, and TNF-α.²⁵

Stimulation of RAW264.7 with 0.5 µg/mL LPS and 2 ng/mL IFN-γ for 24 h induced the differentiation of macrophages into the M1 subtype. CD86 and CD80 are widely used as markers for M1 polarization, with their upregulation considered indicative of activated macrophages polarizing towards the M1 phenotype. Examination of M1 macrophages stimulated by LPS and IFN-γ revealed higher expression of the specific functional markers CD86 and CD80, suggesting the induction of M0 differentiation into M1. In comparison to the LPS + IFN-γ group, YLD treatment for 24 h significantly downregulated the positivity rates of CD86 and CD80, which indicated that YLD possessed the capability to inhibit the differentiation of macrophages into the inflammatory phenotype (Figure 2B).

YLD Alleviated Clinical Symptoms of MC903-Induced AD in Mice

The experimental design for the model construction in this study was shown in Figure 3A. MC903 is a vitamin D3 analog that has been widely used as an experimental drug for establishing AD animal models.²⁶ Under the stimulation of MC903, keratinocytes in mouse skin express and secrete TSLP, which induces the development of immature DCs to a mature phenotype by binding to TSLP receptors on DCs.^27,28 The activated DCs further initiate the differentiation of naive Th0 cells into Th2 subsets, thereby inducing Th2-mediated inflammatory response, down-regulating the expression of skin barrier related proteins, and promoting the production of allergen-specific IgE by B cells.²⁹ Thus, the mechanism by which MC903 induces AD-like skin lesions is similar to the pathogenesis of human AD.³⁰

Figure 3 Improvement of AD symptoms in mice by YLD. (A) Schematic representation of the construction of the AD animal model and treatment regimen: BALB/c mice aged 9 weeks were administered MC903 at a dose of 2 nmol/ear for 15 consecutive days, followed by treatment with 0.2 mL of YLD-L, YLD-M, or YLD-H for 14 days. (B) Visual images/representative phenotypic manifestations of the ears of mice from the Ethanol group, MC903 group, YLD-L group, YLD-M group, and YLD-H group on day 15 of AD induction. (C) Daily SCORAD during YLD treatment. (D) Daily percentage change in body weight of mice during YLD treatment. (E) The TEWL of AD-like mice during YLD treatment. (F) Percentage change in ear thickness of mice during YLD treatment measured using a micrometer. Data were expressed as mean ± SD (n = 10 for each group). ***P < 0.001.

Compared to the ethanol group, continuous stimulation with MC903 resulted in typical AD symptoms, significant ear epidermal swelling, erythema, and crust formation in mice (Figure 3B). The SCORAD, used to evaluate the severity of skin lesions in the MC903 group, reached 8.50 (Figure 3C). However, the YLD-M and YLD-H groups shown significantly reduced severity of skin damage compared to the MC903 group, with SCORAD of 6.08 and 6.62, respectively. Additionally, AD caused a decrease in body weight in the MC903 group, which was improved after oral administration of YLD. The YLD-M group exhibited the best control of body weight (Figure 3D). Moreover, while both the YLD-M and YLD-H groups shown therapeutic effects, it was observed that the YLD-H group caused adverse reactions in the gastrointestinal tract of AD-like mice.

Comparing the dynamic changes in TEWL in the lesional skin of mice in each group during YLD treatment, the results showed that the TEWL of mice in the MC903 group continued to increase under the action of MC903. There were significant differences between the MC903 group and other groups. After YLD treatment, TEWL in AD mice was significantly down-regulated, but there was no significant difference in TEWL between gradient YLD treatment groups (Figure 3E). AD-like mice’s ear thickness changes due to ear swelling during YLD treatment were statistically analyzed. Prolonged stimulation with MC903 resulted in significant ear swelling in mice, with an average change rate in ear thickness of 40.61% in the MC903 group at the end of AD induction. However, the average change rate in ear thickness was 25.24% in the YLD-L group, 23.17% in the YLD-M group, and 17.49% in the YLD-H group (Figure 3F), which was weaker than the counterpart in the MC903 group.

YLD Ameliorated Skin Lesions in AD Mice

HE staining was used to compare the epidermal lesions in the ears of AD-like mice and the YLD-treated groups. Compared to healthy ears, MC903-induced AD-like ears exhibited apparent hyperkeratosis (with a thickness of up to 95.36), incomplete keratinization, thickened spinous layers, increased granular layer, and infiltration of numerous inflammatory cells and eosinophils in the dermis (Figure 4A). The epidermal thickness in healthy mice was 36.31. In the YLD-L group, the epidermal thickness in mice could be reduced to 71.01. However, results shown that the mice in the YLD-M and YLD-H groups gradually reduced these skin lesion symptoms and decreased epidermal thickness (69.7 in the YLD-M group and 62.52 in the YLD-H group). From the perspective of epidermal thickness, the therapeutic effects of YLD-L, YLD-M, and YLD-H in AD-like mice demonstrated a dose-dependent pattern.

TB staining was used to process ear specimens for counting classical immune sentinel mast cells to compare the number of inflammatory cells in the ears of AD-like mice and the YLD-treated groups. Mast cells are considered classical immune cells implicated in itch sensation, and their excessive infiltration can directly contribute to itching behavior in AD-like mice. By comparing sections from the blank group and AD-like skin, it was observed that the infiltration of mast cells in the dermis and subcutaneous tissue of AD-like mice significantly increased, surpassing the levels in healthy mice. However, in AD-like mice administered YLD orally, the infiltration of mast cells in the dermis and subcutaneous tissue decreased, and this reduction exhibited a correlation with the dosage (Figure 4B).

Figure 4 Improvement of skin lesions in AD-like mice by YLD. (A) HE staining of longitudinal cross-sections of ears and quantification histogram of stratum corneum thickness within the field of view. A solid black line represented the stratum corneum. Scale bar represented 100 μm. (B) TB staining of longitudinal cross-sections of ears and quantification histogram of mast cell infiltration within the field of view. Red arrows indicated mast cells. Scale bar represented 100 μm. Data were expressed as mean ± SD (n = 10 for each group). ***P < 0.001.

YLD Suppressed Pro-Inflammatory Cytokines in Serum

To evaluate the inflammatory status of mice, the concentrations of pro-inflammatory cytokines and an antibody in collected serum samples were measured using ELISA. Treatment with MC903 increased levels of IL-4, IL-13, TNF-α cytokines, and IgE antibodies in the serum (Figure 5A). However, YLD reduced the serum levels of these pro-inflammatory factors in AD-like mice. Mainly, in the YLD-H group, the mice shown a decrease of 49% in IL-4, 38% in IL-13, 38% in TNF-α, and 35% in IgE.

Figure 5 YLD alleviated AD by maintaining barrier protein expression and inhibiting pro-inflammatory factors. (A) Serum ELISA results shown that YLD downregulated the levels of typical pro-inflammatory factors IL-4/13, TNF-α, and IgE antibodies in AD-like mice, exhibiting a dose-dependent relationship. (B) Western blot results of AD-like skin lesions demonstrated that compared to MC903, YLD maintained the expression of typical barrier proteins FLG, LOR, and ELOVL6 in the skin lesions of mice with AD-like symptoms, with a dose-dependent effect observed for LOR and ELOVL6. Furthermore, compared to MC903, YLD downregulated the expression of pro-inflammatory factor TSLP in the skin lesions of AD-like mice. (C) Immunohistochemistry results of AD-like skin lesions shown that YLD maintained the expression of barrier protein FLG and inhibited the expression of pro-inflammatory factor TSLP, with a dose-dependent effect observed for the inhibition of TSLP. Scale bar represented 100 μm. Data were expressed as mean ± SD (n = 10 for each group). **P < 0.01, ***P < 0.001.

YLD Upregulated Barrier Proteins and Downregulated Pro-Inflammatory Factors in Lesional Skin

Previous studies indicated that AD lesional skin exhibits defects or downregulation of FLG, LOR, and ELOVL barrier proteins. To investigate the role of YLD in improving the downregulation of barrier proteins in AD-affected skin, western blot analysis was performed to examine the expression levels of barrier proteins. As shown in Figure 5B, the expression of FLG, LOR, and ELOVL was downregulated in the skin of mice treated with MC903, consistent with impaired barrier function in AD skin. However, in mice treated with YLD, the expression of FLG, LOR, and ELOVL increased, with the most significant upregulation observed in the YLD-H group. YLD also exhibited a typical downregulation effect on the pro-inflammatory cytokine TSLP. These protein expression assessments suggested that the anti-AD results of YLD were achieved by upregulating barrier protein expression and downregulating inflammatory factors.

Furthermore, the expression of FLG and TSLP in the skin was visually observed using immunohistochemistry (Figure 5C). Under continuous stimulation by MC903, the expression level of FLG in mice significantly decreased while TSLP expression increased. After YLD treatment, the expression level of FLG in AD lesional tissues recovered, and the highly expressed level of TSLP was controlled. These results were consistent with the findings from the western blot. However, in immunohistochemistry, dose-dependency was primarily reflected in FLG, and no obvious dose-dependency was observed for TSLP.

YLD Inhibited Splenic Atopic Immune Responses

To elucidate the mechanisms by which YLD regulated immune responses, immunohistochemistry analysis was used to analyze the infiltration of CD4+ T cells in lesional tissues and flow cytometry was used to analyze the quantity and proportion of Th1/ Th2/ Th17 immune cells in the spleens of mice from different treatment groups, aiming to characterize the regulation of YLD on CD4+ T cell subsets in AD-like mice. Compared to the skin tissue of healthy mice, CD4+ T cell infiltration in the epidermis of AD lesional skin significantly increased, indicating severe adaptive immune responses induced by MC903 (Figure 6A). After YLD-L, YLD-M, and YLD-H treatments, a gradient reduction in infiltrating CD4+ T cells in the epidermis and dermis was observed, showing a correlation with the dosage. In the YLD-H treatment, the infiltration of CD4+ T cells in lesional areas approached levels seen in normal skin. As a typical immune organ, the spleen exhibited morphological changes indicative of AD-like immune responses, such as volume reduction. The number of immune cells in the spleen can represent the degree of immune responses. Morphologically, the spleen shown different degrees of shrinkage after treatment with MC903. However, YLD treatment attenuated the degree of spleen shrinkage (Figure 6B). Significant differences were observed in the spleens of the YLD-L and YLD-H groups compared to the MC903 group, which was also reflected in the spleen index.

Figure 6 YLD exerted therapeutic effects in AD by inhibiting the activation of immune cells in the lesional skin. (A) Immunohistochemistry results shown a significant reduction in CD4+ T cell infiltrates in mice epidermis and dermis of the lesional skin after YLD treatment, compared to MC903. A slight dose-dependent relationship was observed among the three doses of YLD treatment. Red arrows indicated positive signal CD4+ T cells. (B) YLD inhibited splenic atopic immune responses, improved the typical atopic symptom of spleen shrinkage in AD-like mice, and restored the spleen index. Among the different doses of YLD treatment, YLD-H exhibited the most optimal inhibition of splenic atopic immune responses. Data were expressed as mean ± SD (n = 10 for each group). ***P < 0.001.

Flow cytometry results shown that the proportions of CD4+ T, CD8+ T cells, and Th1, Th2, and Th17 cells in the spleens of AD-like mice significantly increased (Figure 7). Compared to healthy mice, the proportions of Th1, Th2, and Th17 cells in AD-like mice approximately doubled, representing the occurrence of type I, II, and III adaptive immune responses. Oral administration of YLD at all three dosages reduced the proportions of CD4+ T and CD8+ T cells in the spleen, indicating that YLD slowed down excessive immune responses. Moreover, YLD exerted different degrees of regulatory effects on type I, II, and III adaptive immune responses, as evidenced by the downregulation of the proportions of Th1, Th2, and Th17 cells.

Figure 7 YLD exerted therapeutic effects in AD by modulating the balance of Th1/ Th2/ Th17 cells. YLD downregulated the CD4+/ CD8+ ratio and effectively controls the differentiation of Th1/ Th2/ Th17 cells induced by MC903 in CD4+ T cells after YLD treatment, relieving type I, type II, and type III immune responses. Furthermore, the inhibitory effect on Th cells differentiation correlated with the dose of YLD, with YLD-H exhibiting the best inhibitory effect on Th cells differentiation.

Discussion

The predominant mechanisms underlying AD pathogenesis were the adaptive immune response mediated by Th cells and the downregulation of barrier genes such as LOR, FLG, and ELOVL6, leading to epidermal barrier impairment.³¹ In non-lesional phase of AD, allergen stimulation of the skin leaded to excessive scratching, initially compromising the skin barrier. This response activated epidermal langerhans cells (LCs) and dermal DCs,³² resulting in infiltration and low-level activation of various Th cell subsets (Th1/ Th2/ Th17/ Th22).^33,34 During the acute phase of AD, Th2/ Th22 cells significantly increased and released multiple inflammatory mediators,³⁵ such as Th2-associated cytokines IL-4, IL-5, IL-13, IL-31, C-C motif chemokine ligand 18 (CCL18), and Th22-associated cytokines IL-22, S100A proteins, leading to acute skin inflammation.³⁶ The immunomodulatory cytokines IL-4 and IL-13 released by Th2 cells induce significantly reduced expression of FLG,³⁷ LOR,³⁸ and ELOVLs³⁹ in differentiated keratinocytes, thereby inhibiting the production of antimicrobial peptides and promoting colonization by Staphylococcus aureus.⁴⁰ These consequences further worsened skin barrier impairment. In the chronic phase of AD, apart from Th2/ Th22 cells, Th1/ Th17 cells contributed to epidermal remodeling and hyperplasia.⁴¹

The safety and efficacy of TCM in alleviating AD have been well established, and they were commonly used as an adjunctive therapy in clinical AD treatment.⁴² This type of TCM formula contains plants with anti-inflammatory effects, which can exert synergistic effects through different mechanisms and then produce stable therapeutic effects.⁴³ This analysis revealed abundant contents of chlorogenic acid, luteoloside, and specnuezhenide in YLD. Chlorogenic acid accounted for 13.06% and specnuezhenide accounted for 8.83% of YLD, respectively. In previous studies, these components have demonstrated anti-inflammatory activity through mechanisms including inhibition of the MAPK/ERK/JNK pathway, the NF-κB pathway, and the JAK2/STAT3 pathway.^44–46 From the compositional point of view, YLD seems to have a therapeutic effect on AD. However, the pharmacological actions of YLD in the treatment of AD remain unclear and lack systematic validation.

Flow cytometry analysis showed that YLD treatment did not cause keratinocyte apoptosis, suggesting that it was not cytotoxic at commonly used doses. After specifying the concentration at which YLD had no effect on cell growth, we examined the effect of YLD on the regulation of antigen-presenting cells (APCs). T cell activation is mediated by APCs such as dendritic cells and M1-type macrophages.⁴⁷ Costimulatory molecules such as CD80 and CD86 expressed by APCs activate specific immunity by interacting with CD28 on the surface of T cells.^48,49 We confirmed in vitro that YLD inhibited the differentiation of M0-type macrophages into M1, suggesting that YLD may reduce T cell activation through this process.

In vivo study, we used the vitamin D3 analog MC903 to induce AD-like skin lesions in mice. Following the continuous application of MC903 to mice, AD-like symptoms such as ear swelling, redness, and dryness were observed. Still, these symptoms were alleviated to varying degrees by oral administration of YLD. YLD was found to reduce the thickness of the stratum corneum, and alleviated epidermal edema, thereby reducing SCORAD in mice in a dose-dependent manner, with higher doses of YLD being more effective. YLD at all doses reduced mast cell infiltration in the epidermis and dermis and attenuated MC903-induced weight loss. The above results validate the therapeutic effect of YLD on AD.

We found by western blot and immunohistochemistry that YLD reduced the elevated levels of the pro-inflammatory cytokines IL-4, IL-13, TNF-α, and TSLP, and IgE antibodies in AD-like mice, which were generally elevated in patients with moderate to severe AD. Notably, YLD may suppress pathogenic IgE production by downregulating IL-4 and IL-13, thereby inhibiting STAT6-mediated IgE class-switching in B cells.⁵⁰ Additionally, YLD promoted the expression of essential proteins involved in maintaining epidermal barrier integrity, including FLG, LOR, and ELOVL, whose expression was down-regulated in MC903-treated mouse skin. As expected, we found that YLD reduced the proportion of CD4+T cells and CD8+ T cells, and downregulated the proportion of Th1/ Th2/ Th17 cells in splenic lymphocytes. These results suggest that the mechanisms of YLD in treating AD include regulating T cell differentiation and regulating type I, II and III immune responses. The ability of YLD to inhibit the differentiation of CD4+ T cells was dose-dependent. Among the three doses of YLD, YLD-H exhibited a potent inhibitory effect on immune responses, while YLD-M had a more suitable immunomodulatory effect with the number and proportion of Th cells approaching those of normal mice. YLD-M is the equivalent dose for human clinical use, and its appropriate immunosuppressive effect meets the clinical safety requirements.

Although this work confirmed YLD efficacy in modulating Th responses and barrier repair, it remained unclear which component contributed to its anti-AD upstream signaling mechanisms. Future research will integrate existing LC-MS phytochemical data with network pharmacology approaches to identify its critical pharmacodynamic material basis and molecular targets, and clarify the synergistic mechanisms of its multicomponent system.

Conclusions

This study demonstrated that YLD can alleviate AD skin lesions, improve the histopathological characteristics of skin tissue, downregulate the levels of pro-inflammatory cytokines in serum and tissues, upregulate the expression of barrier genes, and inhibit T cell differentiation. These findings supported the therapeutic potential of YLD in AD by maintaining skin barrier function and suppressing adaptive immune responses, while also suggesting its potential for treating other inflammatory diseases. In summary, this study provided systematic validation of the therapeutic efficacy of YLD in AD, elucidated the mechanisms underlying its action in AD treatment, and provided a basis for applying YLD in AD.

Data Sharing Statement

The datasets generated and analyzed during this study are available from the primary corresponding author, Professor Zhongjian Chen, upon reasonable request.

Ethics Approval

The animal study was carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Shanghai Skin Disease Hospital (Tongji University, Shanghai, China) (grant number: 2021-107).

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 work was supported by the National Natural Science Foundation of China [grant numbers 82305231] and the Science and Technology Commission of Shanghai Municipality [grant numbers 21S21900900 and 22S21902700].

Disclosure

The authors report no conflicts of interest in this work.

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Introduction

Palmitic acid (PA) is a 16-carbon long-chain saturated fatty acid (SFA),^1,2 which is widely found in animals and plants.³ It is an essential constituent acid of adipose tissue and the most abundant SFAs in the body,⁴ accounting for approximately 44–52% of the body’s total fat content⁵ and 28–32% of the total serum fatty acid (FA).⁶

Cardiovascular diseases (CVD) is one of the deadliest diseases worldwide.⁷ CVD mainly includes coronary heart disease, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, and pulmonary embolism.⁸ In addition, atrial fibrillation is very closely linked to atherosclerosis (AS) and has largely the same pathophysiological basis as other CVD: endothelial dysfunction and inflammation, coronary artery disease is an important and clinically relevant risk factor of atrial fibrillation.⁹ According to the World Health Organization, 17.3 million people died from CVD in 2016, accounting for 31.5% of all deaths. This number is expected to increase to 23.6 million by 2030.⁸ The mortality rate of CVD has exceeded that of cancer, infectious diseases, maternal diseases, and neonatal diseases.¹⁰ Hyperlipidaemia (elevated total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and reduced high-density lipoprotein cholesterol (HDL-C)), Systemic inflammation, and oxidative stress play a crucial role in the development of CVD.^11–14

Emerging evidence indicates that elevated circulating FA levels correlate with CVD incidence, and free fatty acids show diagnostic potential as early biomarkers for AS.^15–20 In vivo and in vitro experiments evidence suggests potential mechanisms linking PA intake with CVD pathogenesis.^21,22 Epidemiological studies also indicate that high dietary PA exposure associates with increased CVD risk across diverse populations.^23,24 Elevated serum PA concentrations have been proposed to heighten atrial fibrillation risk primarily through PA’s impact on endothelial dysfunction and inflammation.²⁵ However, critical gaps persist in current research: the analysis of PA’s biosynthetic pathways remains incomplete, with insufficient mechanistic delineation specific to individual pathologies, particularly AS, ischemic heart disease (IHD), and ischemic stroke (IS); in addition, lack of translational research frameworks connecting PA-related molecular mechanisms to therapeutic strategies; finally, while substantial evidence supports PA’s detrimental cardiovascular effects, several studies report context-dependent outcomes (Table 1). To address these gaps, this review: Systematically synthesizes PA’s anabolic pathways and pathological mechanisms in AS, IHD, and IS; Identifies novel targetable nodes for CVD prevention/treatment by pinpointing therapeutically exploitable sites within key biological pathways.

Table 1 Palmitic Acid Associations with Traditional Cardiovascular Risk Factors

Methods

A systematic search was performed across four electronic databases (PubMed, Scopus, Web of Science, and Google Scholar) to comprehensively identify literature examining the association between palmitic acid and specific cardiovascular diseases, namely atherosclerosis, ischemic heart disease, and ischemic stroke. Search results were merged and deduplicated. Initial study inclusion/exclusion was determined by screening titles and abstracts. The review encompassed literature published through December 2024.

The Anabolic Pathways of Palmitic Acid

Endogenous Synthesis and Exogenous Uptake of Palmitic Acid

Palmitic acid is mainly synthesized in the liver. In the de novo synthesis, glucose and glutamine produce pyruvate by glycolysis, which undergoes the tricarboxylic acid cycle in the mitochondria to produce citrate. And then the citrate is cleaved in the cytoplasm by ATP-citrate lyase (ACLY) to Acetyl-CoA and oxaloacetate. Acetyl-CoA is then carboxylated to malonyl-CoA by acetyl-CoA carboxylase (ACC) and condensed by fatty acid synthase (FASN) in a repeated reactions to generate PA.^30,31 The endogenous synthesis of PA is controlled precisely under normal circumstances. However, when carbohydrate intake is excessive, the carbohydrate response element-binding protein (ChREBP) is activated, upregulation of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) and resulting in insulin production, which subsequently increases PA production^32–34 (Figure 1).

Figure 1 Endogenous synthesis and exogenous uptake of palmitic acid. In the de novo synthesis, glucose and glutamine are enzymatically catalyzed to produce citrate, which is cleaved to acetyl-CoA and oxaloacetate. Acetyl-CoA is carboxylated to malonyl-CoA, which is condensed by the repeated actions of FASN to produce PA. In the process of exogenous uptake, dietary fat is digested into free fatty acids and monoglycerides through the emulsification of bile acids in the duodenum and upper jejunum, which are then absorbed and converted into TG by intestinal epithelial cells, and then combined with apolipoproteins to form chylous particles, which enter the lymphatic system and the ultimately the bloodstream.

Exogenous Uptake Pathway of Palmitic Acid

Palmitic acid is found in plant oils including palm oil, peanut oil, and coconut oil, as well as in animal fats like butter and cream. Therefore, the human body can also obtain PA through exogenous dietary intake.^35,36 The primary sites for digestion and absorption of fats in the human body are the duodenum and the upper jejunum. When the body consumes fats containing PA from the diet, they are emulsified by bile acids to form hydrophobic fat globules, which are then further broken down into smaller droplets. These droplets are subsequently hydrolyzed by pancreatic lipase into free fatty acids and monoacylglycerol, which are absorbed by the intestinal epithelial cells. In the endoplasmic reticulum (ER) of the epithelial cells, free fatty acids are converted into TG, which then combine with apolipoproteins. These TG, together with apolipoproteins, are transported through chylomicrons to the lymphatic system and eventually enter the bloodstream^37,38 (Figure 1).

Metabolism of Palmitic Acid

The distribution and metabolism of PA in tissues is strictly controlled by the organism, which normally regulates the de novo synthesis pathway according to the amount of exogenous PA consumed.^39,40 First, PA as a kind of FA, have the capability of providing the body with energy through the process of oxidative catabolism.^41,42 PA combines with carnitine to produce an acylcarnitine molecular, and then the acylcarnitine molecular is transported across the mitochondrial membrane to the mitochondrial matrix to generate a molecule of nicotinamide adenine dinucleotide (NADH), a molecule of flavin adenine dinucleotide, reduced (FADH₂), and an acetyl-CoA, which is eventually consumed as energy for the body.⁴³ Secondly, PA is elongated or desaturated for conversion to other FA or compounds,⁴⁴ which are produced in the presence of FA elongases (elongation of very long-chain fatty acids 1–7 (ELOVL1-7)) to produce longer chain FA (eg, stearic acid (SA) and arachidonic acid).⁴⁵ Moreover, PA synthesized endogenously in adipocytes is converted to other FA or compounds through elongation and desaturation in preference to exogenous PA, thus ensuring that the concentration of PA in tissues is within the normal range to maintain cell membrane fluidity and insulin sensitivity.^46,47 Finally, PA itself can be transformed into an important component of biofilms (phospholipids), which plays an important role in biological processes (eg, cellular proliferation, reproductive processes, and intracellular transport). PA was found to generate phosphatidylcholine and phosphatidylethanolamine (PE) by deacylation in rat hepatocyte, the final synthesis of membrane phospholipids.⁴⁸ This process is regulated by membrane-binding transcription factors and can further regulate lipid synthesis.⁴⁹

In obese subjects, the activity of stearoyl coenzyme a desaturase 1 (SCD1) was increased, and SCD1 was associated with insulin sensitivity.⁴⁶ However, under pathological conditions including insulin resistance and chronic nutritional imbalance, this regulatory mechanism can be disrupted, leading to excessive PA deposition in the liver and eventually to a series of CVD.^50,51 Several studies have measured plasma PA concentrations in healthy subjects, indicating a range of 100~409 µM. Nevertheless, patients with diabetes, hypertriglyceridemia, and CVD have elevated plasma PA levels (Table 2).

Table 2 Plasma Palmitic Acid Levels

Palmitic Acid and Cardiovascular Diseases

Palmitic Acid and Atherosclerosis

Atherosclerosis is the basis of most CVD and causes of death, for example, coronary heart disease and stroke.⁵⁸ It is characterized by the endothelial dysfunction and inflammation, form cells formation from macrophage, atherosclerotic plaque formation in the intima of arteries and apoptosis,^59–61 which may result in acute cardiovascular events due to plaque rupture and thrombosis.⁶² Studies have demonstrated that the high concentrations of PA in blood are involved in the formation of AS through a variety of biological processes, including hyperlipidaemia,^63,64 inflammation,⁶⁵ vascular endothelial damage,⁶⁶ form cells formation,⁶⁷ and downregulation of apolipoprotein M (APOM).⁶⁸

Palmitic Acid Induces Hyperlipidemia

There is an increased risk of CVD associated with high levels of TC, LDL-C, and lower levels of HDL-C.^69,70 PA can induce AS by altering blood cholesterol levels, particularly through elevating LDL-C levels.^63,64 PA inhibits the expression of low density lipoprotein (LDL) receptors and accelerates the secretion of very low-density lipoprotein (VLDL) from the liver.⁵¹ Genes related to lipid transport, adipogenesis, lipid droplet formation, and glucose and FA metabolism were found to be upregulated after incubation with PA in human hepatocytes cultured in vitro, similar effects were observed in primary cultures of human pancreatic islets.^71,72 Specifically, PA promoted lipid accumulation by upregulating the CCN1/integrin α5β1 pathway.⁷³ Lipid accumulation and apoptosis were also observed in PA-treated human kidney-2 (HK2).⁷⁴ Increased dietary levels of 18:2(n-6) FA lead to lower total and LDL-C levels, while at low dietary levels of 18:2(n-6) FA, increased PA content leads to a significant increase in total and LDL-C levels.⁷⁵ Meanwhile, in a controlled metabolic feeding study, PA intake promotes elevated blood cholesterol levels, consistent with previous studies.^76–78 In addition, PA also induces insulin resistance, leading to impaired lipid metabolism. Prolonged exposure of cultured human, rat or mouse islets to PA leads to reduced insulin transcription, impairment of glucose-induced insulin secretion, and finally to β-cell apoptosis.^79–81 PA promotes β-cell apoptosis via mTOR-mediated downregulation of protein kinase B (AKT).⁸² In human umbilical cord endothelial cells, PA induces insulin resistance by upregulating human regulator of G protein signaling 2 (RGS2) expression, which inhibit insulin-mediated AKT phosphorylation^83,84 (Figure 2).

Figure 2 Overview of the mechanisms by which palmitic acid promotes atherosclerosis. PA promotes the progression of by inducing hyperlipidemia, vascular endothelial cell injury, foam cell formation, downregulation of APOM, and proinflammatory effects. Its proinflammatory effect is by activating TLR2 and TLR4, enhanced LPS production and synergistic interactions with LPS, promoting FABP4 expression, amplification of proinflammatory T-cell responses, and induction of ER stress and oxidative stress (↑: increase/activation; ↓: decrease/inhibition).

Palmitic Acid Mediates Inflammation

Palmitic Acid Promotes the Production of Inflammatory Factors

PA has been shown to directly increase levels of interleukin-6 (IL-6) in vivo and in vitro.^85–87 PA upregulates the expression of C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs), thereby triggering an inflammatory response in cardiac fibroblasts and inducing apoptosis in VSMCs.⁸⁸ PA increases the level of the cysteine-rich angiogenic inducer 61 (CYR61) in endothelial cells, thereby stimulating the production of pro-inflammatory cytokines and pro-apoptotic factors.⁸⁹ PA also induces the secretion of interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and TNF-α by peritoneal macrophages, which activated the inflammatory process in LDLr KO mice and ultimately induced AS formation.⁹⁰ In microvascular endothelial cells (EOMA lineage), palmitate stimulates the activation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome.⁸⁸ Further studies showed that PA treatment of mouse primary macrophages induced the formation of crystals within the macrophages, which activated the NLRP3 inflammasome, resulting in lysosomal dysfunction and increased IL-1β release⁹¹ (Figure 2).

Palmitic Acid Activates Toll-Like Receptor 4 (TLR4) to Promote Inflammation

During the inflammatory response, toll-like receptors (TLR) serve as receptors for lipopolysaccharide (LPS).^92–94 Several studies have demonstrated that PA is a TLR agonist that activates TLR4 and TLR2, and induces dimerization among TLR2 and TLR1, TLR2 and TLR6, or TLR4 and TLR6.^95,96 TLR4 translocates into lipid rafts after activation and recruits its downstream adapter molecules (MyD88 and TRIF) to the rafts. After dimerizing with MyD88 or TRIF, initiates pro-inflammatory cytokine and type I interferon production.⁸⁴ In addition, activated TLR4 forms a complex with myeloid differentiation protein 2 (MD2), which triggers downstream signaling. However, it is uncertain whether PA is a direct agonist of TLR4-MD2.⁹⁷ During the activation of TLR4, atypical protein kinase Czeta (PKCζ) is triggered by RhoA, next PKCζ activates transforming growth factor β-activated kinase 1 (TAK1), which then participates in the activation of NF-κB,⁹⁸ which results in the production of inflammatory cytokines (eg, TNF-α and IL-6).⁹⁰ PA promoted the TLR4/phosphorylated-NF-κB signaling pathway by inhibiting Krüppel-like factor 4 (KLF4), upregulated Galectin-3 expression, and improved insulin resistance in macrophage⁹⁹ (Figure 2).

Palmitic Acid Activates the Proinflammatory Function of T Cells

T cells are an instrumental component of adaptive immunity and account for 10% of all cells in atherosclerotic plaques.^100,101 Using single-cell sequencing techniques, T cells were found to account for approximately 30–65% of white blood cells in atherosclerotic plaques in humans and mice.^102–104 CD4⁺ T cells are the predominant T cell subtype in AS and exacerbate atherogenesis in immunodeficient Apoe-/- mice.¹⁰⁵ Researchers found that both CD4⁺ T cells and CD8⁺ T cells were increased at atherosclerotic lesion sites associated with acute coronary syndrome.¹⁰⁶ PA activates the proinflammatory function of T cells in four ways: metabolism, activation, proliferation, and polarization.¹⁰⁷ There is evidence that PA increases insulin receptors (IR), insulin-like growth factors 1 (IGF-1), glucose transporter type 4 (GLUT4), and insulin receptor substrate 1 (IRS1) on the surface of T cells, resulting in T cell activation. PA also stimulates the proliferation of T cells and induces the polarization of T cells into proinflammatory subpopulations (Th1 cells and Th17 cells), which then induce an inflammatory response.¹⁰⁷ The addition of 1 mM PA to peripheral blood mononuclear cells activated with anti-CD3 and anti-CD28 increased the proportion of Th1 and Th17 cells, while decreasing that of TH 2 and Treg cells. After in vitro exposure to PA, CD4⁺ T cells or CD8⁺ T cells isolated from five healthy, non-diabetic, and glucose-tolerant individuals were found to be activated in a time and concentration-dependent manner¹⁰⁸ (Figure 2).

Palmitic Acid Promotes Inflammation in Synergy with LPS

A high-fat diet increases the levels of short-chain FA by altering the gut microbiome, which leads to elevated levels of LPS and enhanced activation of TLR4.¹⁰⁹ PA also increases ceramide production through de initio synthesis and sphingolipid hydrolysis, thereby enhancing IL-6 expression and TNF-α stimulation induced by LPS.¹¹⁰ Researchers fed mice both LPS and a high-fat diet rich in PA, which accelerated thoracic aortic atherosclerosis.¹¹¹ In human aortic endothelial cells (HAECs) and cardiac microvascular endothelial cells (MICECs), co-treatment with LPS and PA increased IL-6 expression at 36 hours¹¹¹ (Figure 2).

Palmitic Acid Promotes the Expression of Fatty Acid‑binding Protein 4 (FABP4)

As a cytoplasmic FA carrier protein, FABP4 regulates lipid transport and responses in cells, and is associated with metabolic and inflammatory pathways.^112–115 FABP4 bind a long-chain FA, including PA, SA, oleic acid (OA), linoleic acid (LA), and facilitates the translocation of FAs to specific organelles in the cell (eg mitochondria, peroxisomes, ER, and nucleus), regulates enzymatic activity, and stores excess FAs as lipid droplets.¹¹⁶ The FABP4 protein has a high affinity for free monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) in cells under normal conditions, however, under oxidative stress conditions, the conformation of FABP4 changes, losing its affinity for most FA (except PA), and triggers an inflammatory response.¹¹⁶ PA increases FABP4 protein expression in macrophages via ER stress.^117,118 The genetic ablation of FABP4 in macrophages showed inhibition of inflammatory signaling, reduced NF-κB pathway activation, and reduced ER stress, protecting mice from AS and dyslipidemia.^119,120 In C2C12 skeletal muscle cells, overexpression of FABP4 protein decreases expression the expression of Sirtuin 3, uncoupling protein 2 (UCP2), and Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), ultimately leading to increased ROS production in mitochondria and inflammation^121–123 (Figure 2).

Palmitic Acid Activates ER Stress

The ER is involved in the biosynthesis of cholesterol, steroids, and other lipids. A high concentration of free fatty acids (eg PA) may disrupt lipid metabolism, which triggers stress in the ER. When PA is transformed into phospholipids and diacylglycerol (DAG), it accumulates in the ER, causing disruptions in the structure of the ER and activation of the stress sensors.^124–126 The extracellular signal-regulated kinase (ERK) pathway mediates translation of CCAAT/enhancer binding protein (C/EBP) homologous proteins and genes involved in autophagy that are dependent on activating transcription factor 4 (ATF4). Inositol-requiring enzyme 1α (IRE1-α) mediates the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulated kinase 1 (ASK1)/C-jun N-terminal kinase (JNK). They contribute to the ability of stress cells to maintain autophagy, which ultimately triggers ER oxidative and inflammatory signaling pathways leading to apoptosis.^127–131 Phosphorylated ERK, IRE1α, and JNK activation are elevated in both adipose tissue and liver of high fat diet fed mice, which triggers ER stress, eventually leads to apoptosis.^132–135 By upregulating ATF4 and C/EBP homologous protein (CHOP) expression, decreasing cytoplasmic NAD+/NADH, and reducing Sirt1 activity, PA induced ER stress in H9c2 myogblasts.¹³⁶ Heart-specific sirt1 knockout mice fed a high palmitate diet were found to express higher levels of CHOP and ATF4.¹³⁶ In obese individuals and type 2 diabetes mellitus (T2DM) patients, chronic exposure of β-cells to FA results in ER stress and lipotoxicity¹³⁷ (Figure 2).

Palmitic Acid Induces Oxidative Stress

Increased reactive oxygen species (ROS) are the primary cause of palmitate-induced oxidative stress. PA enhances ROS production by promoting lipid uptake in podocytes, and the activity calcium/protein kinase Cα/NADH oxidase 4 (NOX4) pathway in endothelial cells, inhibited mitochondrial respiratory chain complex I and complex III. And the activity of adenine nucleotide carrier protein (ADP/ATP carrier protein).^138–141 Normal mouse hepatocytes AML12 treated with PA. Lipid accumulation, expression of total ROS, mitochondrial ROS, NOX4, inflammasomes, and IL-1β were detected in hepatocytes after 24 h¹⁴² (Figure 2).

Palmitic Acid Induces Vascular Endothelial Injury

Vascular endothelial injury is an important pathological process in the process of AS. Endothelial dysfunction, characterized by impaired vasodilation, inflammation, and thrombosis, triggers future CVD.¹⁴³ Reduced endothelial progenitor cells are independent predictors of CVD morbidity and mortality.¹⁴⁴ Lipotoxicity of PA decreases immune surveillance protein DDX58/Rig-1 expression and activity, leading to impaired autophagy and apoptosis;¹⁴⁵ apoptosis in vascular endothelial cells induces endothelial injury and promotes AS progression.^146,147 A member of the angiopoietin-like protein family involved in lipid metabolism promotes endothelial cell proliferation and inhibits PA-induced endothelial cell injury by increasing autophagy, which may inhibit AS.⁶⁶ Also, activation of the interferon regulator 3 (IRF3) pathway causes endothelial inflammation.¹⁴⁸ Nitric oxide (NO) from enzymatic NO synthases (NOS) system importantly contributes to vascular homeostasis, in addition to the classical NOS system, NO can also be generated via the nitrate-nitrite-NO pathway.¹⁴⁹ The addition of PA to HAECs resulted in decreased cell viability, reduced intracellular NO production, increased migratory capacity of HAECs, and cellular oxidative stress, ultimately leading to endothelial-to-mesenchymal transition.¹⁵⁰ In endothelial cells, PA upregulated the expression of phosphorylated p38, JNK, and caspase-3, thereby increasing endothelial apoptosis dose- and time-dependently.^151,152 Patients with coronary artery disease showed significantly higher levels of phosphorylation of p38 and mitogen-activated protein kinase (MAPK) in endothelial progenitor cells than healthy individuals.¹⁵³ Inhibition or knockout of p38 and MAPK significantly increases the number of circulating endothelial progenitor cells¹⁵⁴ (Figure 2).

Palmitic Acid Promotes Foam Cells Formation

Form cells is one of the major causes of AS, which is due to the accumulation of oxidized LDL (oxLDL) in the arterial intima. Macrophages absorb accumulated oxLDL and form cells. The presence of high levels of PA in the blood enhances the ability of macrophages to take up oxLDL and produce more form cells. OxLDL is a dysfunctional lipid metabolite that is a major promoter of the prothrombotic state in both animal models and human patients.^67,155 In macrophages, PA enhances lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression, promotes oxLDL uptake, a process mediated mainly through the ROS-p38 MAPK pathway.¹⁵⁶ 5-hydroxytryptamine (5-HT) takes part in platelet aggregation, vasoconstriction, proliferation of VSMCs, ER function, and macrophage foam cell formation, play a key role in the development of AS.^157,158 In vitro experiments, exposure of macrophages and human umbilical vein endothelial Cells (HUVECs) to oxLDL or PA demonstrated that activation of 5-HT_2A receptor regulates TG synthesis and oxLDL uptake by activating PKCε, resulting in the formation of lipid droplets and even foam cells.¹⁵⁹ PA increases CD146 expression in macrophages, promoting foam cell formation and disrupting migration-related signaling,¹⁶⁰ by activating JNK signaling and inhibiting STAT3 activation, CD146 (Gp130) promotes proinflammatory polarization of M1-like adipose tissue macrophages (ATMs)¹⁶¹ (Figure 2).

Palmitic Acid Induces Apolipoprotein M Downregulation

Palmitic acid can downregulate the expression of human APOM, promote the accumulation of cholesterol in the blood and induce the development of AS. APOM facilitates HDL metabolism and stabilization, which can reduce blood cholesterol levels, with anti-AS, anti-inflammatory and antioxidant effects.¹⁶² Generally, it is found in hepatocytes and renal tubular epithelial cells, and is weakly expressed in colorectal tissues.^163,164 APOM has been shown to be a possible HDL-carrying receptor for sphingosine 1-phosphate, which enhances HDL-mediated antioxidant effects.^165,166 APOM plays a role in the formation of preb-HDL,^167,168 PA significantly inhibited APOM gene expression in HepG2 cells, and the peroxisome proliferator-activated receptor β/δ (PPAR β/δ) antagonist GSK3787 completely reversed PA-induced downregulation of APOM expression, indicating that PA-induced downregulation of APOM expression is mediated through the PPAR β/δ pathway.⁶⁸ A key regulator of lipid metabolism, peroxisome proliferator-activated receptor (PPAR), is expressed in platelets. This receptor upregulates the transcription of lipid metabolizing enzymes, including carnitine palmitoyl coenzyme A transferase-I (GPT-I) and acyl-CoA oxidase, both of which are important to thrombosis and hemostasis^169,170 (Figure 2).

Palmitic Acid and Ischemic Heart Disease

Ischemic heart disease is heart disease caused by narrowing/occlusion of the coronary arteries or by ischemia, hypoxia, or necrosis of the heart muscle due to spasm of the coronary arteries. Approximately 40–80% of the heart’s energy comes from FA, several cohort studies have revealed, compared with healthy young subjects, patients with chronic heart failure, myocardial ischemia, T2DM, and obese individuals elevated levels of free fatty acids (include PA) in the blood.^171–177 Additionally, there are studies that indicate that PA levels in adipose tissue are related to IHD incidence. Insull et al found that SA (18:0), lauric acid (12:0), palmitoleic acid (16:1), myristic acid (14:0), and LA (18:2) acids were associated with coronary artery disease, and PA (16:0) content in adipose tissue was associated with plasma cholesterol levels.¹⁷⁸ A study by Lee et al compared the FA composition of adipose tissue in two races with different prevalences of coronary heart disease and found significant differences in PA, palmitoleic, and OA (18:1).¹⁷⁹ Thus, high concentrations of PA, both circulation and adipose tissue, are associated with the incidence of IHD. There was a significant increase in FA uptake and FA oxidation in the heart when the supply of free FA was increased, according to Lopaschuk GD.¹⁸⁰ Replacing saturated FA (FA and SA) with plant-based proteins may reduce the risk of myocardial infarction.¹⁸¹

Palmitic Acid Induces Apoptosis in Cardiomyocytes

Palmitic acid induces cardiomyocyte apoptosis by promoting autophagy. Studies have shown that after treating rat cardiomyocytes with PA (0.25 and 0.5 mM) for 18 hours, the number of apoptotic cells and biochemical markers (caspase activation, DNA fragmentation), significantly increased.¹⁸² In cardiomyocytes, PA induces apoptosis by promoting the generation of ceramide and activating the mitochondrial apoptosis pathway, leading to the myofibril disintegration.¹⁸³ In a cohort study involving 4249 participants, the correlation between plasma ceramide (Cer) and sphingomyelin (SM) levels and the risk of sudden heart failure was investigated. The results showed that high levels of PA were associated with a higher risk of heart failure during a median follow-up of 9.4 years.¹⁸⁴ Ischemic events are believed to increase the flow of free fatty acids to cardiomyocytes, thereby increasing oxidative stress and causing cardiomyocyte damage.^185–188 When the heart is exposed to excessive energy (eg, glucose, free fatty acids, and TG) and growth factors (eg, insulin and leptin) over a long period, it accelerates the development of cardiomyopathy, leading to cardiac hypertrophy and failure. These processes are driven by oxidative stress induced by glucolipotoxicity and become the main drivers of cell apoptosis¹⁸⁹ (Figure 3).

Figure 3 Overview of the mechanism by which palmitic acid promotes ischemic heart disease. PA accelerates progression of induces oxidative stress and autophagic dysregulation, and further triggers cardiomyocyte apoptosis. Additionally, PA promotes cardiomyocyte ferroptosis by reducing the protein expression of Heat Shock Factor 1 and Glutathione Peroxidase 4. These mechanisms collectively drive pathogenesis the onset and development of IHD (↑: increase/activation; ↓: decrease/inhibition).

Palmitic Acid Promotes Cardiomyocyte Ferroptosis

Ferroptosis is an iron-dependent form of programmed cell death.¹⁹⁰ The primary mechanism of ferroptosis is the induction of cell death through the action of divalent iron or lipoxygenases. Additionally, the expression of the antioxidant systems glutathione and glutathione peroxidase 4 (GPX4) is also involved in the process.¹⁹¹ A large body of evidence has shown that ferroptosis is associated with CVD, particularly with ischemia-reperfusion injury and myocardial infarction.¹⁹² Using different ferroptosis inhibitors significantly reduced PA-induced death in both H9c2s and primary neonatal rat cardiomyocytes. Specifically, PA promotes ferroptosis by reducing the protein expression of heat shock factor 1 (HSF1) and GPX4, while overexpression of HSF1 and GPX4 effectively prevents PA-induced ferroptosis⁴ (Figure 3).

Palmitic Acid and Ischemic Stroke

Ischemic stroke has become a major cause of global disease burden due to its high incidence, prevalence, mortality, and disability rates.¹⁹³ In 2013, an estimated 6.9 million new IS cases occurred globally, with only 18.25 million surviving in good health, 3.32 million deaths, and 65.54 million disabilities.¹⁹⁴ Plasma levels of docosahexaenoic acid, LA, arachidonic acid, and PA were measured by gas chromatography in 943 participants from the Framingham Heart Study and 1406 participants from three cities of the Bordeaux Study. The results showed that PA is a risk factor for stroke.¹⁹⁵ In a study conducted at the Minneapolis Community Atherosclerosis Risk Center, 3870 white men and women aged 45–64 years (1987–1989) were assessed for plasma cholesterol esters and phospholipid FA, revealing a significant positive correlation between plasma SFAs (particularly PA) and IS.¹⁹⁶

Palmitic Acid Promotes Neuroinflammation

Palmitic acid can induce chronic inflammation in both peripheral tissues and the central nervous system, for example, hypothalamic neurons.^197–200 In in vitro experiments, PA was found to induce dysfunction in human adipose tissue and soft meningeal artery endothelial cells.²⁰¹ Researchers found that when Medin (a common amyloid protein) was combined with PA, there was upregulation of IL-6, IL-8, and PAI-1 gene expression in HUVECs, suggesting combined proinflammatory and prothrombotic effects in IS pathogenesis.^201,202 Mechanistically, PA promotes TLR4 recruitment to lipid rafts in SH-SY5Y neuroblastoma cells, facilitating TLR4/MYD88/TIRAP complex formation a process potentiated by heme-dependent TLR4 activation.⁹⁴ PA promoted the upregulation of IL-6 and TNF-α in primary hypothalamic cultures from rats.²⁰³ Further studies confirmed that mice fed a high PA diet showed increased hypothalamic cytokine levels, proinflammatory signaling, neuronal death, and impaired leptin and insulin signaling.^198,204 Direct intraventricular injection of PA also led to hypothalamic inflammation and insulin resistance.²⁰³ PA induces the expression of proinflammatory cytokines in cultured hypothalamic neurons (N42) by increasing ceramide accumulation and lipotoxicity.⁹² Additionally, PA interacts with LPS to activate microglial cells, upregulating the expression of proinflammatory cytokines via MAPK, NF-κB, and AP-1 signaling pathways, inducing neuroinflammation in HMC3 cells²⁰⁵ (Figure 4).

Figure 4 Overview of the mechanism by which palmitic acid promotes ischemic stroke. PA exacerbates IS through multi-target mechanisms: (1) Atherogenesis: Accelerates plaque formation via ceramide overproduction and proinflammatory cytokine induction. (2) Neuroinflammation: Triggers CNS inflammatory cascades through microglial TLR/NLRP3 activation and astrocytic metabolic reprogramming. (3) Neuronal Apoptosis: Induces ER stress-autophagy axis dysregulation in neurons. (4) Glial Activation: Directly stimulates microglial inflammatory signaling and astrocytic lipotoxicity. These interconnected pathways collectively drive neurovascular unit dysfunction, culminating in IS progression (↑: increase/activation).

Palmitic Acid Promotes Apoptosis of Neuronal

The lipotoxicity of PA triggers ER stress and autophagic impairment, leading to an increase in apoptosis and the regulation of neuronal plasticity. High concentrations of PA have been shown to induce ER stress in SH-SY5Y cells and mouse brain cells.²⁰⁶ In SH-SY5Y cells and human glioblastoma cells, PA-induced neurotoxicity and glial cell toxicity, as well as increased oxidative stress in neurons and astrocytes, further promoted cell apoptosis.²⁰⁷ Mechanistic studies reveal that PA upregulates fatty acid transport protein 1 (FATP1) expression, which enhances prefrontal cortical autophagy dysregulation and ER stress while downregulating neuroplasticity markers including synaptophysin (SYN), brain-derived neurotrophic factor (BDNF), and acetylcholine receptors (AChRs).²⁰⁸ High-fat diets containing PA activate the MST1/JNK/Caspase-3 signaling pathway in hippocampal HT22 cells, leading to neuronal apoptosis.^209,210 In in vitro experiments, PA significantly increased the autophagic flux in hypothalamic neurons. After PA exposure, the autophagic flux in hypothalamic neurons was suppressed, leading to impaired neuronal autophagy. This autophagic dysfunction was accompanied by changes in lysosomal dynamics, increased Rab7 GTPase activity, ERK phosphorylation, elevated expression of NADPH oxidase 4, and higher levels of inflammation, oxidative stress, and apoptosis in DRG neurons²¹¹ (Figure 4).

Palmitic Acid Activates Glial Cells

Glial cells, primarily composed of microglia and astrocytes, PA can activate glial cells. Microglia are the principal FA sensors in the hypothalamus related to neuronal stress and inflammation and are key mediators of the inflammatory response after stroke and brain injury.²¹² PA promotes inflammation by activating TLR receptors distributed in microglia, and also activates NLRP3 inflammasome by increasing TLR4/MyD88/NF-κB p65 signaling, Long-term activation of hypothalamic microglia inhibits neurogenesis in the medial basal hypothalamus (MBH), and the occurrence of IS further activates microglia and exacerbates disease progression.^213,214 Astrocytes are the primary cells responsible for FA oxidation in the brain and play an important role in chronic inflammatory responses associated with obesity and the development of secondary metabolic disorders.²¹⁵ Although the brain’s energy is primarily provided by glucose PA accumulation in astrocytes activates mitochondrial β-oxidation pathways, generating ATP while inducing proinflammatory activation²¹⁶ (Figure 4).

Conclusion and Future Directions

Cardiovascular impact of dietary fatty acids exhibits fundamental dichotomy: Saturated fatty acids, particularly PA, promote cardiovascular pathogenesis through pro-inflammatory, dyslipidemic, and endothelial dysfunction pathways. SA as one of the metabolic products of PA, that exhibits neutral metabolic effects. While monounsaturated (eg, oleic acid) and polyunsaturated fatty acids confer cardioprotection. As the most abundant endogenous and dietary SFA, PA serves as a pathophysiological pivot in atherosclerosis development and cerebrovascular complications. Translation of these mechanistic insights into balanced nutritional interventions represents an actionable strategy for global CVD burden reduction.

However, current limitations must be addressed: current evidence exhibits heterogeneity in PA exposure quantification across studies; different organizations, races, and diseases should adopt specific quantitative standards, rather than simply using the same standard for measurement; moreover, most interventional data derive from preclinical models requiring human validation.

To advance this field, future research should prioritize: establish specific quantitative standards for different organizations, races and diseases; elucidate tissue-specific signaling mechanisms (eg, endothelial vs glial PA sensing); develop targeted therapies disrupting PA-induced inflammatory cascades (eg, RGS2 inhibitors); conduct randomized trials testing precision and personalized nutrition approaches for high-risk populations; establish clinical biomarkers quantifying PA’s pathogenic contributions.

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this work, the authors used [deep seek] in order to [improve language and readability]. After using this tool, the authors reviewed and edited the content as needed and takes full responsibility for the content of the publication.

Abbreviations

PA, palmitic acid; SFAs, saturated fatty acids; CVD, cardiovascular diseases; AS, atherosclerosis; IHD, ischemic heart disease; IS, ischemic stroke; FA, fatty acid; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; ACLY, ATP-citrate lyase; ACC, acetyl-CoA carboxylase; FASN, fatty acid synthase; ChREBP, carbohydrate response element-binding protein; SREBP-1c, sterol regulatory element binding protein-1c; ER, endoplasmic reticulum; NADH, nicotinamide adenine dinucleotide; FADH₂, flavin adenine dinucleotide, reduced; ELOVL1-7, elongation of very long-chain fatty acids 1-7; SA, stearic acid; PE, phosphatidylethanolamine; SCD1, stearoyl coenzyme a desaturase 1; APOM, apolipoprotein M; LDL, low density lipoprotein; VLDL, very low-density lipoprotein; HK2, human kidney-2; AKT, protein kinase B; RGS2, human regulator of G protein signaling 2; IL-6,interleukin-6; CRP, C-reactive protein; TNF-α, tumor necrosis factor-α; Inos, nitric oxide synthase; VSMCs, vascular smooth muscle cells; CYR61, cysteine-rich angiogenic inducer 61; IL-1β, interleukin-1β; MCP-1, monocyte chemoattractant protein-1; NLRP3, NACHT, LRR and PYD domains-containing protein 3; TLR4, Toll-like receptor 4; TLR, toll-like receptors; LPS, lipopolysaccharide; MD2, myeloid differentiation protein 2; pkcζ, atypical protein kinase Czeta; TAK1, transforming growth factor β-activated kinase 1; KLF4, Krüppel-like factor 4; IR, insulin receptors; IGF-1, insulin-like growth factors 1; GLUT4, glucose transporter type 4; IRS1, insulin receptor substrate 1; HAECs, human aortic endothelial cells; MICECs, cardiac microvascular endothelial cells; FABP4, fatty acid‑binding protein 4; OA, oleic acid; LA, linoleic acid; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator 1α; UCP2, uncoupling protein 2; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; C/EBP, CCAAT/enhancer binding protein; ATF4, activating transcription factor 4; IRE1-α, inositol-requiring enzyme 1α; TRAF2, tumor necrosis factor receptor-associated factor 2; ASK1, apoptosis signal-regulated kinase 1; JNK, C-jun N-terminal kinase; CHOP, C/EBP homologous protein; T2DM, type 2 diabetes mellitus; ROS, reactive oxygen species; NOX4, calcium/protein kinase Cα/NADH oxidase 4; IRF3, interferon regulator 3; NO, nitric oxide; MAPK, mitogen-activated protein kinase; oxLDL, oxidized LDL; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; 5-HT, 5-hydroxytryptamine; HUVECs, human umbilical vein endothelial Cells; ATMs, M1-like adipose tissue macrophages; PPAR, eroxisome proliferator-activated receptor; GPT-I, carnitine palmitoyl coenzyme A transferase-I; Cer, ceramide; SM, sphingomyelin; GPX4, glutathione peroxidase 4; HSF1, heat shock factor 1; FATP1, fatty acid transport protein 1; SYN, synaptophysin; BDNF, brain-derived neurotrophic factor, AChRs, acetylcholine receptors; MBH, medial basal hypothalamus.

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 work was supported by the National Natural Science Foundation of China (32460138); Priority Union Foundation of Yunnan Provincial Science and Technology Department and Kunming Medical University (202101AC070461), and Basic Research Program of Yunnan Province Science and Technology Department (202301AT070083).

Disclosure

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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