Category: 3. Business

Introduction

As a benign disease, keloids are featured by the overgrowth of dense fibrous tissue on the affected skin. Although benign, they often cause troublesome symptoms including itching, pain, and pruritus. Furthermore, their high recurrence rates make them particularly challenging in clinical practice.¹ Recommended treatments for keloids include compression therapy, intralesional injections, laser therapy, topical treatments, surgical excision, postoperative radiotherapy, and cryotherapy.² However, the nonspecific nature of these therapies increases the risk of damaging surrounding tissues. Intralesional corticosteroid injections, a standard first-line treatment, frequently result in side effects such as localized pain and changes in skin appearance.³ The combination of corticosteroids and 5-Fluorouracil has demonstrated potential in improving treatment outcomes, yet persistent side effects remain a concern.⁴ Furthermore, irregular post-operative radiotherapy, or intralesional injections, and high-risk sites have also been implicated in the recurrence of keloids.^5–7 Therefore, targeting the distinct genetic and protein expression profiles of keloids compared to normal skin necessitates the identification of novel therapeutic targets and corresponding ligands to advance keloid treatment.⁸

Previous studies on keloid therapy have primarily focused on the transforming growth factor-beta (TGF-β)/Smad signaling pathway,^9,10 which plays a crucial role in the prolonged stimulation of fibroblasts and myofibroblasts, leading to excessive collagen production in keloids.⁹ However, considering the involvement of TGF-β in multiple biological processes, therapies targeting this signaling may cause systemic cytotoxic effects, limiting the advancement of TGF-β-based treatments for fibrotic diseases.¹⁰ Therefore, further research is warranted to refine targeted therapies for keloids.

In our previous study, we identified several potential targets for keloid therapy through Mendelian randomization and single-cell sequencing analyses. However, these studies did not reveal any functional targets. Notably, proteins from the BAG family have attracted our interest due to their overexpression in both tumors and, to some extent, keloids, highlighting the tumor-like properties of these lesions.¹¹ Among them, BAG cochaperone 2 (BAG2) has emerged as a promising target for keloid therapy according to the single-cell sequencing analysis. Previous research has shown that the high BAG2 expression among tumor-associated fibroblasts correlated with the poor prognosis in breast cancer and its anti-apoptotic characteristics,^12,13 suggesting that BAG2 may play a similar role in keloid progression. Mechanistically, BAG2 negatively regulates the chaperone-associated ubiquitin ligase, C terminus of Hsc70-interacting protein (CHIP), which facilitates the ubiquitin-mediated degradation of misfolded proteins. This suggests that BAG2 may inhibit the degradation of specific proteins, including overexpressed collagen in keloids. Additionally, BAG2 interacts with the MAPK signaling pathway, influencing downstream cellular proliferation.^14,15 In keloids, BAG2 may inhibit collagen degradation and promote abnormal collagen accumulation, warranting further investigation.

This study aimed to validate the role of BAG2 in keloid progression and identify compounds targeting BAG2, employing high-throughput screening technology tailored for keloid patients. The experimental setup is illustrated in Figure S1. Additionally, we explored the intrinsic connection between BAG2 and the progression of keloids to provide insights for more precise keloid management.

Materials and Methods

Ethics Statement

Keloid biopsies (10 patients with 4 men and 6 women age ranging from 26 to 42 years old, with keloids from operative excision of previous abdominal incisionor chest incision, having the lesion of over one year without reduction, without previous surgical treatment or radiotherapy, diagnosed by 2 experienced clinical experts with lesions extending beyond the wound boundary into the normal skin and other clinical features) and normal skin samples (3 female patients, aged 38, 41 and 42, with skin excision from facial plastic surgery as brow lifting) were obtained from the Shanghai Jiao Tong University affiliated ninth people’s hospital in accordance with the institutional review board (SH9H-2024-TK561). All patients provided formal, informed and written consent to supply a biopsy for this study. The study complied with the Declaration of Helsinki.

Keloid Fibroblasts (KFs) Isolation and Culture

Ten keloid samples from four men and six women were obtained after surgical excision. The sterile samples were rinsed in PBS, cut into pieces, and digested with 0.2% collagenase IV for 4 hours at 37 °C. After centrifugation, the sedimentary cells were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin, and incubated at 37°C in a 5% CO₂ incubator. The spindle keloid fibroblasts (KFs) from at least three patients at passages 2 to 3 were mixed for usage in further experiments.¹⁶

Single-Cell Data Analysis

To explore BAG2 gene expression in different cell groups, we downloaded the single-cell dataset GSE181297 of keloid patients from the Gene Expression Omnibus (GEO) database.¹⁷ This dataset was generated via the Illumina NovaSeq 6000 platform for scRNA-seq including keloid and normal skin samples from human subjects. The data processing included normalizing, filtering low-quality cells, de-batching, selecting highly variable genes for dimensionality reduction, and analyzing BAG2 expression in different cell clusters.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

qRT-PCR was performed as previously described.¹⁸ Briefly, total RNA was extracted from cells after treatment using an RNA isolation kit (Takara Bio, Shiga, Japan). RNA purity was assessed by measuring the A260/A280 ratio, with acceptable values ranging from 1.8 to 2.0. The primers used for gene amplification are listed in Table 1. The results from three independent reactions were used to determine relative gene expression, normalized against β-actin expression.

Table 1 Primer Sequence

Cell Transfection

Small interference RNA (si-RNA) targeting BAG2 for BAG2 knockdown and its negative control (si-NC) were synthesized by Sangon Biotech (Shanghai, China). The sequence of si-BAG2 was listed in Supplementary Table 1. The non-transfected KFs were set as controls. Cell transfection was conducted using RNATransMate transfection reagents (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol.

Lentiviral Production and Infection

DNA oligonucleotides encoding primers for obtaining the target gene BAG2 (sequences: BAG2-1, 5’-AGGTCGACTCTAGAGGATCCCGCCACCATGGCTCAGGCGAAGATCAACGCTAAAG-3’, BAG2-2, 5’-TCCTTGTAGTCCATGGATCCATTGAATCTGCTTTCAGCATTTTG-3’), or a negative control were inserted into the lentiviral vector GV703 (Genechem, Shanghai, China). Human mRNA-BAG2 cDNA (synthesized by Genechem, Shanghai, China) was inserted into the BamHI/Age I sites of the lentiviral vector GV703. For lentiviral production, 293T cells were transfected with the lentiviral vector along with packaging plasmids using Lipofectamine 2000 (Life Science, USA) according to the manufacturer’s instructions. At 48 h and 72 h after transfection, culture media was collected, pooled and filtered. Human Fibroblast cell line (HHF1) obtained from The Institute of Cell Biology, Chinese Academy of Sciences was infected with the indicated lentivirus, and BAG2 expression was determined by WB at 48 h after infection.

Ex-Vivo Explant Culture of Keloid Tissue

After harvesting aseptic keloid tissue, and removing epidermis, the remaining dermis of keloids was cut into about 3×2 × 2 mm pieces by a scalpel. The dermal fragments were divided into different groups, and cultured for 3 days in DMEM containing 10% FBS as previously described.¹⁹ After tissue attachment, the medium was replaced for the control, siRNA-treated, and compounds-treated groups, which were then incubated for an additional five and seven days. The explants were collected after treatment.

Cell Proliferation Assay

The KFs transfected with si-BAG2 and si-NC (2×10³ cells/well) were seeded into a 96-well plate. Following the manufacturer’s protocol, 10 μL of CCK-8 reagent (Beyotime, Shanghai, China) was added to each well and incubated for 2 hours at 37°C. Subsequently, cell proliferation was assessed daily from day 1 to day 5. The number of cells was quantified by measuring absorbance at 450 nm using a microplate reader. Each treatment group was evaluated in triplicate to ensure reliable results.

Cell Cycle Analysis

KFs were treated with si-BAG2, C16-PAF (Targetmol, #T21547, 10 nM), or si-BAG2 alone for 24 and 48 hours, along with the si-NC control. After treatment, the cells were rinsed once with PBS, and fixed with 70% ethanol overnight. The cell cycle analyses were performed according to the instructions of the Cell Cycle Kit (Qihai Biotechnology, Shanghai, China), and flow cytometric analyses were performed by a flow cytometer (Beckman Coulter) equipped with ModiFit LT v2.0 software.

Cell Migration Assay

To evaluate the effect of BAG2 knockdown on the KFs migration, a scratch assay was performed. KFs were seeded in multi-well plates and cultured to confluence. The culture medium was then removed, and the cell monolayers were scratched using a 200 µL pipette tip. The cells were rinsed with PBS and subsequently treated with either si-BAG2, si-NC, or control. Images of cell migration were captured at 0 and 24 hours using an inverted light microscope (TE2000 Nikon, Japan) at 40×magnification at 0 and 24 hours. These experiments were performed in triplicate, and migration areas were quantified using ImageJ analysis software (National Institutes of Health, Bethesda, MD).

Histological, Immunohistochemical and Immunofluorescence Analyses

Keloid tissue and explant samples were fixed overnight in 4% paraformaldehyde at 4°C, embedded in paraffin, and sectioned to 5 μm thickness. The sections were subsequently stained with hematoxylin and eosin (H&E) and Masson’s trichrome for histological examination. Furthermore, keloid tissue sections were also treated with antibodies specific to BAG2 and α-SMA at dilutions from 1:2000 to 1:100. Antibody binding was visualized using 3,3’-diaminobenzidine (DAB) chromogen (Dako, Glostrup, Denmark) and counterstained with hematoxylin for immunohistochemical analyses. For immunofluorescence staining, after overnight incubation with primary antibodies, specimens were incubated with secondary antibodies for 1 hour. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Finally, digital images were obtained using a V10-ASW 4.2 computerized image analysis system (Olympus, Tokyo, Japan). The antibodies used were: BAG2 antibody (Affinity, #DF2650, 1:100), Goat Anti-Rabbit IgG (H+L) HRP (Jackson, #111-035-045, 1:200), α-SMA antibody (Proteintech, #67735-1-Ig, 1:100), Goat Anti-Mouse IgG (H+L) Red (Jackson, #115-295-003, 1:200), and Goat Anti-Rabbit IgG (H+L) FITC (Jackson, #111-095-003, 1:200). The positive rate of BAG2 was defined as the proportion of BAG2-positive cells among all the cells in the field.

Western Blot

Western blot was performed on tissue samples or cultured cells as indicated. Total protein was extracted using RIPA lysis buffer, as previously described.¹⁸ The protein concentration of each lysate was determined using a BCA protein assay kit. Protein samples were separated by SDS-PAGE electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skimmed milk and immunoblotted with specific primary antibodies, including COL1, COL3, α-SMA, MEK, p-MEK and β-actin, diluted between 1:2000 and 1:1000 in TBST. After overnight incubation with the primary antibodies and three subsequent TBST washes, the membranes were incubated with the appropriate secondary antibodies for 1 hour at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Chalfont St. Giles, UK). The intensity of each protein band was normalized against the β-actin band for comparison. The primary antibodies used in this study were: COL3 (Abcam, #Ab184993, 1:1000), COL1 (Huabio, #HA722517, 1:1000), α-SMA (Affinity, #AF1032, 1:1000), BAG2 (Affinity, #DF2650, 1:1000), MEK (Affinity, #AF3385, 1:1000), p-MEK (Affinity, #AF6385, 1:1000), and β-actin (Affinity, #AF7018, 1:2000).

Surface Plasmon Resonance (SPR) for Affinity Screening and Affinity Determination

SPR experiments were performed in at 25 °C on a BIAcore T200 with CM5 sensor chips, and data were analyzed with BIAcore T200 Evaluation software (GE Healthcare), following the manufacturer’s instruction. BIAcore T200 optical biosensor was used to screen for BAG2 affinity and to measure equilibrium dissociation constant (K_D) values for protein–ligand interactions. A cell on the CM5 sensor chip was activated using a mixture of 200 μM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 50 μM N-hydroxysuccinimide at a flow rate of 10 μL/min for 420 seconds. Subsequently, BAG2 protein (50 μL) was mixed with 180 μL of 10 mM sodium acetate solution at pH 5.0 and immobilized on the cell surface at the same flow rate and duration for two cycles as previously described.²⁰ The cell was then blocked using 1 M ethanolamine. A neighboring channel, used as a reference, underwent the same activation and blocking steps, except immobilization was carried out with PBS adjusted to pH 5.0. Both channels were equilibrated with PBS afterward. Grouped SPR single concentration screening: Fifty compounds were grouped, with each compound contributing 1 μL, combined in an EP tube to total 50 μL. EP tubes were labeled from 1 to 54, and 950 μL of PBS was added to achieve a screening concentration of 10 μM for each compound. The DMSO content was maintained at 5% within the correction range. Each run flowed at 10 μL/min for 150 seconds. After each run, the chip was regenerated for 5 minutes with 10 mM glycine-HCl (pH 2.0), repeating until all groups (54 in total) were tested. Sequential SPR single concentration screening: The two groups with the highest RU values from the first screening round (100 compounds total) were selected. Each compound was taken as 2 μL from the plate and mixed with 198 μL of PBS to achieve a screening concentration of 100 μM. Each run flowed at 10 μL/min for 150 seconds. At the end of each run, the chip was regenerated for 5 minutes with 10 mM glycine-HCl (pH 2.0) solution, repeating this process until all compounds (100 in total) were completed. Data were collected from the sample cell using BIAcore T200 Control software (v. 2.0, GE Healthcare) and normalized against the reference cell. Association and dissociation constants were calculated using global fitting to a 1:1 Langmuir binding model with BIAcore T200 Evaluation software (v. 2.0, GE Healthcare). Final figures were generated using Origin 7 software (v. 7.0552, OriginLab).

To determine the K_D value for protein-ligand interactions, each compound was diluted 6 times from 12 μM to 15.625 nM. The compounds were flowed over a chip with immobilized BAG2 protein from lowest to highest concentration at 10 μL/min for 150 seconds. After each concentration, the chip was regenerated with 10 mM glycine-HCl (pH 2.0) for 5 minutes at the same flow rate. Data were recorded in real time, with molecular weight adjustment and solvent correction applied to address non-specific binding and signal drift molecular effects. Data analysis was conducted using BIAcore T200 software (GE Healthcare) following the manufacturer’s guidelines.

Statistical Analysis

All data are presented as means ± SD and analyzed using GraphPad Prism version 8 software (GraphPad Software Incorporation, La Jolla, California, United States). The cell migration area data were analyzed using Image J software. Sample sizes were determined based on practical and experimental considerations, without predetermined statistical methods. Differences among multiple groups were analyzed using analysis of variance (ANOVA), followed by Tukey’s post hoc test for comparisons between groups (α = 0.05). Differences among two groups were analyzed using t-test. Statistically significant differences were indicated by asterisks, with a two-tailed p-value of less than 0.05 considered significant.

Results

Higher BAG2 Expression in KF Clusters Compared to Normal Skin and Higher BAG2 Protein Expression in Keloid Tissue

After quality control of the GSE181297 dataset, we conducted dimensionality reduction and clustering analysis. Unique molecular identifiers between 200 and 6000 were selected. Cell clusters were categorized based on original molecular markers. These clusters included endothelial cell (EC), fibroblast (FB), proliferating myofibroblast (MFB), keratinocyte (KC), immune cells (lymphocytes, macrophages, and mast cells), lymphatic endothelial cell (LEC), and neural cell (Figure S2A and B). Besides, elevated BAG2 gene expression in KFs was observed in differential expression analysis (Figure S2C), with no significant difference in BAG2 expression between ECs and LECs in keloid and normal skin samples. Immunohistochemical staining confirmed BAG2 expression around fibrous nodules (Figure 1A). Additionally, immunofluorescent staining showed colocalization of BAG2 with α-SMA-positive cells, while BAG2 expression was also observed in α-SMA-negative cells, consistent with single-cell sequencing results (Figure 1B). Western blot analysis further demonstrated that BAG2 protein levels were significantly higher in keloid tissue than in normal skin samples (Figure 1C). The images of individual channel of immunofluorescence staining of keloid samples were showed in Figure S3A. The negative controls of immunohistochemical staining for keloid and normal skin, and the Hematoxylin and Eosin Staining of the keloid tissue was showed in Figure S3B and Figure S4.

Figure 1 The expression of BAG2 in keloid and normal skin samples. (A) Immunohistochemical analysis reveals enhanced positive staining for BAG2 in and around the fibrous nodules of keloid tissues compared to normal skin samples. (Scale bar = 100 μm at 200× magnification; Scale bar = 50 μm at 400× magnification); (B) The statistical analysis of BAG2 protein expression in keloid tissues and normal skin samples, as detected by immunohistochemical staining; (C) Immunofluorescence staining of a keloid sample slide shows nuclear staining with DAPI (blue) and colocalization of BAG2 (green) with α-SMA (red) positive cells; BAG2 expression is also visible in α-SMA negative cells. (Scale bar = 100 μm); (D) BAG2 protein levels in keloid tissue were significantly elevated compared to normal skin samples. (K: Keloid; (N) Normal Skin). ***P<0.001.

Inhibition of BAG2 Reduced Collagen Synthesis and Deposition of KFs, and in an Ex-Vivo Model

The relative gene expression of collagen type I (COL1), which is associated with collagen synthesis and excessive deposition in keloids,²¹ and Tissue inhibitor of metalloproteinase 1 (TIMP1), which inhibits matrix metalloproteinases and subsequently promotes COL1 deposition in keloids,¹⁹ was significantly decreased following BAG2 inhibition (Figure 2A). Additionally, Western Blot analysis showed reduced protein expression of BAG2, COL1, collagen type III (COL3), and α-SMA in si-BAG2-treated KFs, highlighting the role of BAG2 in abnormal collagen deposition (Figure 2B). Consistent with these findings, collagen structures in si-BAG2-treated ex-vivo keloid explants appeared thinner and more degraded compared to the control group (Figure 2C). Furthermore, COL1 and COL3 protein levels were significantly reduced in ex-vivo explants treated with si-BAG2 (Figure 2D).

Figure 2 Inhibition of BAG2 reduced keloid collagen synthesis and deposition. (A) The relative gene expression of COL1A1 and TIMP1 in si-BAG2-treated keloid fibroblasts compared to control groups. Gene expression related to collagen synthesis and the inhibition of collagen degradation was significantly reduced following BAG2 inhibition; (B) Western blot analysis showing reduced protein expression of BAG2, COL1, COL3, and α-SMA in si-BAG2-treated keloid fibroblasts; (C) Masson’s trichrome staining of keloid explants from different groups. Collagen structures in the si-BAG2-treated groups appeared thinner compared to control groups; (D) Western blot analysis demonstrates decreasing protein expression of COL1 and COL3 in si-BAG2-treated ex-vivo keloid explants. The reduction in COL1 and COL3 protein expression was statistically significant. Statistical analysis of relative protein expression is provided (n=3). **P<0.01, **** P<0.0001.

Inhibition of BAG2 Reduced Migration and Proliferation of KFs, Correlating with the MEK Pathway

BAG2 inhibition significantly reduced the migration and proliferation of KFs (Figure 3A and B). Additionally, the relative gene expression of TGF-β, an indicator of the proliferation and migration of KFs, was significantly decreased in si-BAG2-treated KFs (Figure 3C).²² In addition, Western Blot further demonstrated decreased levels of phosphorylated MEK (p-MEK) following si-BAG2 treatment (Figure 3D). Furthermore, si-BAG2 treatment significantly increased KFs’ percentage within the G0/G1 phase, suggesting cell cycle arrest. This effect was reversed by C16-PAF, an MEK activator, which restored G0/G1 phase percentages in si-BAG2-treated KFs (Figure 3E).

Figure 3 Inhibition of BAG2 reduced KF migration and proliferation via the MEK signaling pathway. (A) Representative images and bar graph depict the migration of KFs treated with or without si-BAG2 at 0 and 24 hours after scratching (n=3). Scale bar = 500 μm; (B) CCK-8 assays were performed on control and si-BAG2-treated KFs, showing a significant reduction in the proliferation of si-BAG2-treated cells (n=3); (C) Relative gene expression of TGF-β in si-BAG2-treated and control KFs, with significantly decreased TGF-β expression observed following si-BAG2 treatment (n=3); (D) Western blot analysis revealing decreased p-MEK in si-BAG2-treated cells; (E) Representative cell cycle profiles of KFs from various groups, assessed by flow cytometry, accompanied by statistical analysis (n=3). *P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001. KF, keloid fibroblast.

Screening of Compounds with High‑affinity to BAG2 via Surface Plasmon Resonance (SPR) and Their Cell Inhibition Rates

BAG2 protein was found to play a significant role in collagen deposition in keloid tissues and the proliferation of KFs, making it a potential target for keloid treatment. To explore compounds with high-affinity to BAG2, the structure of BAG2 predicted by AlphaFold3 is shown in Figure S5. SPR was employed as an affinity screening technology to identify potential high-affinity compounds for BAG2.

A total of 2,732 compounds from the FDA and anti-fibrosis libraries were screened (Supplementary Table 2). In the first round, 2 groups of compounds were identified, and 6 compounds were identified as putative BAG2 ligands in the second round (Figure 4A and B). The primary cell inhibition rates of these 6 compounds on KFs were determined via CCK8 assays on Day 3 (Supplementary Table 3). Three compounds (Saikosaponin B1, Bazedoxifene acetate, and Ponesimod) showed significant inhibition of KFs and were selected for further investigation. Subsequently, the affinity index (K_D values) of these compounds with BAG2 was determined using SPR, and their inhibitory effects on KFs were evaluated at varying concentrations. The results indicated that Saikosaponin B1, Bazedoxifene acetate, and Ponesimod had K_D values of 6.35E−7 M, 2.84E−6 M, and 5.87E−6 M, respectively. Additionally, the fitted curves demonstrating the effects of different compound concentrations on the relative cell viability of KFs revealed IC50 values of 9.32 μM for Bazedoxifene acetate and 24.96 μM for Ponesimod, indicating that both drugs exhibit strong binding affinity and inhibitory effects (Figure 4C and D). Although Saikosaponin B1 exhibited relatively high binding affinity to BAG2, its inhibitory effect on KFs was below 40% at a concentration of 50 μM (Figure S6), suggesting it is not an optimal candidate for BAG2 targeting. The association rate constants (Ka) and dissociation rate constants (Kd) of these compounds are listed in Supplementary Table 4. Overexpressing BAG2 in human fibroblasts, and adding Bazedoxifene (10μM) acetate or Ponesimod (25μM) for 48h, it was found that these two compounds could inhibit BAG2-mediated cell cycle transition and cell proliferation (Figure 4E). The overexpression of BAG2 was validated by Western blot (Figure S7).

Figure 4 SPR screening of high-affinity compounds with BAG2 and validation in vitro. (A) Initial screening of 54 compound groups via SPR identified two groups demonstrating high affinity for BAG2 (shown in blue), compared to the positive control, BAG2 rabbit monoclonal antibody (mAb) from ABclonal Technology (A8775) (shown in red); (B) A second screening via SPR of two groups comprising 100 compounds revealed six compounds with high affinity for BAG2 (shown in blue), using the same BAG2 rabbit mAb as a positive control (shown in red); (C-D) SPR assays were used to analyze the binding affinities of Bazedoxifene acetate and Ponesimod to human BAG2 protein. Additionally, dose-response curves displaying the response of KFs to Bazedoxifene acetate and Ponesimod treatment over 72 hours are presented (n=6); (E) Representative cell cycle profiles of human fibroblasts from control, BAG2 overexpressed (oe-BAG2), oe-BAG2 treated with Bazedoxifene acetate or Ponesimod groups, assessed by flow cytometry, accompanied by statistical analysis (n=3). **** P<0.0001. KF, keloid fibroblast.

Compounds with High-Affinity to BAG2 Exhibited Inhibition of Collagen Deposition in Keloid Tissue

To further evaluate the therapeutic effects of the identified compounds on keloids, the effects of Bazedoxifene acetate and Ponesimod were assessed through protein expression analysis of COL1 and COL3, as well as histological evaluation of ex-vivo keloid explants. The experiments revealed that treatment with Bazedoxifene acetate and Ponesimod for five days or longer significantly reduced both the protein expression of collagen and the histological deposition of collagen in the ex-vivo explants (Figure 5). Additionally, Bazedoxifene acetate showed a superior inhibitory capacity for the expression of COL1 and COL3. These findings confirmed that compounds with high-affinity to BAG2 could markedly inhibit collagen deposition in ex-vivo keloid explants, indicating their clinical potential as therapeutic agents for keloids.

Figure 5 Masson’s trichrome of keloid explants in different groups. (A) In ex-vivo keloid explants treated with Bazedoxifene acetate and Ponesimod, the collagen structures appeared thinner compared to control groups; (B) Western blot analysis showed reduced protein expression of COL1 and COL3 in ex-vivo keloid explants treated with Bazedoxifene acetate and Ponesimod. Notably, Bazedoxifene acetate demonstrated a superior inhibitory effect on collagen deposition; (C) Statistical analysis of the relative protein expression is included (n=3). *P<0.05, **P<0.01.

Discussion

Keloids, a benign fibrogenic skin disease, share numerous characteristics with tumors, including the absence of spontaneous regression, excessive proliferation, and high recurrence rates.²³ Consequently, it is critical to explore therapeutic agents that target specific mechanisms involved in keloid formation. In this study, we identified BAG2 as a novel therapeutic target and screened thousands of compounds to find high-affinity BAG2 ligands. Our findings include the identification of Ponesimod and Bazedoxifene acetate as high-affinity ligands to BAG2, whose inhibitory effects were evaluated on keloid explants.

The present study observed an upregulation of BAG2 in keloid tissues, meanwhile, decreasing BAG2 with siBAG2 inhibited the proliferation of KFs, which correlated with MEK signaling. Consistent with the alterations of TGF-β following BAG2 inhibition, TGF-β modulates cell proliferation through the MEK pathway via a Smad-independent signaling mechanism (Derynck et al, 2003). This is consistent with prior findings linking MEK pathway activation to the proliferation of KFs,²⁴ and to the proliferative and migratory behaviors of cancer cells.^25,26 As evidenced by our findings, si-BAG2 suppressed KFs’ progression at the G0/G1 phase, a process that could be reversed by MEK activation. This indicates that BAG2 facilitates KF proliferation, which is correlated with the MEK signaling pathway, as an early indicator of a long-term process involving ECM remodeling. In accordance with previous studies, inhibiting the MEK pathway could induce G0/G1 phase arrest.^27,28 Additionally, the initial phosphorylation site Ser20, located within the curled helical domain near the amino terminus of BAG2, is phosphorylated by MAPKAPK-2. The p38 MAPK-MAPKAPK-2-BAG2 phosphorylation cascade, activated in response to extracellular stress, influences cell proliferation activities,²⁹ and may explain BAG2’s role in activating the MEK pathway. The BAG2-Heat shock protein (HSP) complex maintains basement membrane integrity and regulates ECM and cell junctions in glial cells, suggesting a similar role in keloids.³⁰ BAG2 also has anti-apoptotic effects that promote cell survival and, in some cases, enhances TIMP synthesis and activity by boosting intracellular anti-apoptotic factors. Since TIMPs inhibit MMP activity to control ECM degradation, BAG2-mediated upregulation of TIMPs may reduce MMP-driven ECM breakdown, preventing excessive degradation.¹³

While the proliferation and migration of KFs are well-documented, accelerated collagen accumulation also plays a significant role in keloid pathology.¹⁹ Additionally, mechanical stress at keloid sites has been recognized as a risk factor for the initiation and progression of this condition.³¹ The stiffness of the extracellular matrix (ECM) in keloids, largely due to collagen accumulation, augments the effects of mechanical stress, which, in turn increases the production of ECM by KFs.³² Therefore, halting the collagen deposition cycle is vital for effective keloid therapy.³³ Traditional therapies for keloids fail to adequately target the essential pathological processes of collagen synthesis and degradation.³⁴ In this study, altering BAG2 activity reduced both collagen synthesis and deposition in vitro and ex vivo. Additionally, our findings demonstrate that drugs with high-affinity to BAG2 can significantly reduce collagen accumulation, highlighting their potential as novel targeted therapies for keloids that inhibit collagen deposition and reduce the viability of KFs. In pulmonary fibrosis, TGF-β treatment promotes α-SMA expression through acetylated CCAAT/enhancer binding protein β (C/EBP-β), and a similar mechanism may exist in keloids, which warrants further investigations.³⁵

Several skin diseases, including psoriasis, vitiligo, dermatitis, and skin cancers, have been successfully treated with targeted therapies.^36–39 Drugs such as vemurafenib and trametinib, which target the BRAF mutation and MEK respectively, have significantly advanced the field of targeted therapy, although many such drugs are still in the preclinical research stage. Specifically, drugs targeting keloids, due to their localized presence on the body surface, could offer more precise treatment options. Nevertheless, there are currently no specific inhibitors targeting BAG2, a keloid-associated marker identified through this study. In this study, several high-affinity BAG2 ligands with were identified, including Saikosaponin B1, Ponesimod, and Bazedoxifene acetate, and verified their capability to inhibit collagen expression and deposition in ex-vivo keloid tissue. Saikosaponin B1, a bioactive molecule found in Radix Bupleuri, has been shown to exhibit anticancer properties⁴⁰ and antifibrotic activity in liver fibrosis.⁴¹ Ponesimod, a Sphingosine 1-phosphate (S1P) modulator, has been investigated as a treatment for Multiple Sclerosis, focusing on its ability to regulate S1P activity in lymphocytes.⁴² Bazedoxifene acetate, a selective Estrogen Receptor modulator, was examined for its role as a BAG2 ligand and its capacity to inhibit the viability of KFs in this study.⁴³ Interestingly, tamoxifen, another Estrogen Receptor modulator, has also been shown to reduce keloid collagen fibers.⁴⁴ Like bazedoxifene acetate, tamoxifen’s impact on keloids appears to extend beyond simple Estrogen Receptor modulation. Bazedoxifene acetate appears to influence keloid pathology by correlating with the inhibition of BAG2. The mechanisms through which bazedoxifene acetate and ponesimod act on keloids necessitate further detailed exploration.

Despite the lack of prior reports on these drugs as keloid inhibitors, their strong affinity for BAG2 and effectiveness in inhibiting KFs suggest their potential as promising candidates for targeted keloid therapy. Bazedoxifene acetate, in particular, stands out for its lower K_D, lower IC₅₀, and higher binding affinity. As an FDA-approved medication, bazedoxifene acetate may manage antifibrotic activity by affecting both collagen deposition and the viability of KFs. Moreover, due to their relatively high SLogP values, as detailed in Supplementary Table 5, bazedoxifene acetate and ponesimod were more likely to facilitate transdermal absorption, potentially reducing side effects associated with systemic administration. Topical application of these compounds may provide a viable therapeutic option for keloid treatment.

Despite the promising potential of drugs targeting BAG2 in keloid therapy, treatment with these agents may induce unintended off-target effects on other proteins. Therefore, further functional analyses and the development of systems to predict potential side effects are warranted. In this study, we found that inhibiting BAG2 could reduce collagen deposition. Additionally, the system of MMPs and TIMPs was also affected, warranting further investigation. Furthermore, the direct interaction between BAG2 and MEK signaling requires further investigation to elucidate the role of BAG2 in keloid formation. In addition, proliferation is a confounding factor for the scratch assay, the effect of BAG2 in the migration of KFs should be further investigated. Further, in vivo models are warrant for validation of the role of BAG2 in keloids. Compared to the ex-vivo model, the in-vivo model provides more comprehensive information on physiological responses and systemic pathological changes. In addition, as indicated by SPR, the binding affinity of Bazedoxifene acetate and Ponesimod to BAG2 is relatively higher than that of the other tested drugs. To further validate the effect of Bazedoxifene acetate and Ponesimod on keloids via BAG2, silencing experiments should be conducted in future studies. In summary, this study highlighted BAG2’s impact on collagen deposition and the proliferation of KFs, which are critical to the pathogenesis of keloids, benign skin tumors with high recurrence rates. Additionally, we identified compounds with high-affinity ligands to BAG2 that could serve as potential therapeutic targets. Among the compounds tested, Bazedoxifene acetate demonstrated superior affinity for BAG2 and more effective inhibition of keloid fibroblasts and keloid tissue compared to other ligands, suggesting novel approaches for the precise management of keloid disease.

Conclusion

This study revealed the pathogenic role of BAG2 in keloid and identified its high-affinity ligands, Bazedoxifene acetate and Ponesimod. The therapeutic capabilities of these compounds demonstrated their potential to improve targeting therapy for keloids.

Resource Availability

The data underlying this article are available in the article and in its online supplementary material. Single cell sequencing data is available in [GEO database] at [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE181297]. Further data underlying this article will be shared on reasonable request to the corresponding author.

Acknowledgments

We thank all the patients who provided samples for the experiment for their support. This work was supported by National Nature Youth Foundation of China (Grant No. 82402938 & 82102319) Science and Technology Commission of Shanghai Municipality (Grant No. 22MC1940300) and Wuxi Taihu Lake Talent Plan, Supports for Leading Talents in Medical and Health Profession. The funding body did not play a role in the study design, data collection, analyses, interpretation, manuscript preparation, and in the decision to submit the manuscript.

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.

Disclosure

The authors have declared that no conflict of interest exists. The patents associated with this article include patent application number 202411342096.X, credited to Yinmin Wang, Lin Lu, and Jun Yang.

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Introduction

Myofascial Pain Syndrome in Neck or shoulder, as a typical manifestation of myofascial pain syndrome (MPS), has shown significant population heterogeneity and social medical burden in its epidemiological characteristics. A cross-sectional study showed that myofascial trigger points (MTrPs) were present in all 224 patients with chronic nonspecific neck pain. The prevalence of trigger points in the near-horizontal fibers of the trapezius muscle was as high as 82.1% (right) and 79% (left), while the active trigger points in the levator scapularis and sandwicularis cerae reached 82.14% and 62.5%, respectively. This suggests that the neck and shoulder muscle group is the core affected area of the disorder.¹ It is notable that changes in modern lifestyle have exacerbated the prevalence of the disease. A systematic review in 2020 indicated that the prevalence of neck pain among long-term smartphone users could reach 55.8% to 89.9%, with myofascial pain syndrome being the most common pathological type. The risk was significantly dose-response to the length of time spent using electronic devices with the head down every day.² Young people also face severe challenges. An epidemiological study shows that 86.4% of people aged 18–25 have neck and shoulder myofascial trigger points, with women having 164 times higher risk than men, and the number of trigger points is significantly positively correlated with depression and anxiety scale scores.³ The loss of productivity caused by the disease is not to be underestimated in the socio-economic dimension. Data show that patients with chronic neck pain have an average of 23 times more sick leave per year than the general population, with 72% of absenteeism cases attributed to limited neck movement caused by acute attacks of myofascial pain.¹ More notably, cervicoshoulder myofasciitis often affects the outcome of other diseases as comorbidities. Among 2,361 patients with cervicogenic vertigo, 58.5% had Myofascial Pain Syndrome in Neck or shoulder as the underlying cause. Although these patients had less severe vertigo, the proportion of those with anxiety disorders was 18 times that of the standard population.⁴

The current treatment strategies for Myofascial Pain Syndrome in Neck or shoulder are diversified, mainly covering three directions: physical therapy, drug intervention and invasive treatment. In the field of physical therapy, the immediate analgesic effect of transcutaneous electrical nerve stimulation (TENS) as a non-pharmacological intervention has been systematically supported by evidence. For example, a meta-analysis of 381 randomized controlled trials by Johnson et al showed that the intensity of pain during or immediately after TENS treatment was significantly lower than that in the placebo group (SMD = −096, moderate-quality evidence), and there was no significant difference in the incidence of adverse reactions compared with the control group.⁵ However, the study also noted that the small sample sizes of most trials led to insufficient accuracy in effect size estimation, and the lack of long-term follow-up data made it difficult to assess the sustained efficacy of TENS. In the field of manual therapy, dry needle therapy and manual therapy are widely used, but their clinical value remains controversial. Barbero’s clinical observations further revealed that although manual therapy targeting myofascial trigger points can improve local blood supply and muscle tone, the efficacy usually lasts no more than 72 hours and needs to be combined with Exercise rehabilitation to extend the benefit period. In terms of drug treatment, non-steroidal anti-inflammatory drugs (NSAIDs) and muscle relaxants remain the first-line clinical options, but Galasso’s comprehensive study indicates that about 38% of long-term NSAIDs patients have gastrointestinal adverse reactions, and drug dependence is particularly prominent among people with chronic pain.⁶ Although ultrasound-guided local injection therapy has been able to precisely locate the lesion in recent years, Kara’s imaging studies showed transient muscle fibrosis changes after injection in about 25% of cases, suggesting that invasive procedures may exacerbate the mechanical stress imbalance of the fascia structure.⁷

In the clinical practice of Myofascial Pain Syndrome in Neck or shoulder, acupuncture therapy has shown significant advantages due to its unique mechanism of action. Compared with traditional treatment, acupuncture can achieve both short-term analgesia and long-term functional improvement through multiple pathways of regulating local microcirculation, inhibiting the release of inflammatory factors, and releasing myofascial trigger points. The research by Guo et al⁸ indicates that MTrPs are somewhat close to the locations of acupuncture points and some overlap. However, the anatomical positioning must be on the muscles (as there is a muscle twitch reaction during acupuncture). Guo et al used the concept of referred pain to locate MTrPs and found that their distribution paths are similar to the course of meridians. They also discovered that if the trigger points are accurately touched or needled, local muscle twitch reactions will occur, that is, local muscle twitching, which will have a very good analgesic effect.For example, a reticular meta-analysis confirmed that gua sha combined with warm acupuncture had a significant advantage in improving pain intensity, while small Needle knife therapy was highly effective in raising the tenderness threshold, and this targeted therapeutic effect made personalized programming possible.⁹ However, the limitations of this therapy are also worth noting. The differences in manipulation techniques among different practitioners may affect the stability of therapeutic effects, especially when it comes to deep myofascial treatment.Meanwhile, the mechanism of acupuncture in treating musculoskeletal pain also includes non-specific mechanisms, such as placebo/situational effects. A meta-analysis has shown that in terms of pain treatment, the effect of acupuncture is superior to that of the placebo.¹⁰

Currently, there are significant differences in the evidence levels of different acupuncture therapies. There is a lack of direct comparison data among different therapies, and traditional meta-analyses are difficult to establish a complete evidence chain. Additionally, the combined therapies commonly used in clinical practice have not yet established an efficacy grading evaluation system, and there is a lack of evidence for comparing the effects of different acupuncture therapies. There is no consensus on the standardization of needle selection and insertion depth, which leads to a significant increase in heterogeneity among clinical studies. Therefore, it is of urgent necessity to conduct a network meta-analysis on acupuncture therapy for cervical and shoulder myofascial inflammation. This study uses a network meta-analysis to investigate the differences in efficacy among various acupuncture therapies, aiming to identify the most effective acupuncture therapy and provide evidence-based evidence for clinical practice.

Methods

Register

This study was written in accordance with the PRISMA guidelines.¹¹ This study has been prospectively registered with PROSPEO under the number CRD420251040965.

Inclusion Criteria

(1) Study subjects: Patients with myofascial pain syndrome (the study description includes neck or shoulder). Age, gender, duration of the disease, etc. are not restricted. (2) Intervention measures: acupuncture-related therapies (including Internal heat acupuncture, Electroacupuncture, Moxibustion, Acupoint injection, etc).; (3) Control group: Conventional acupuncture. (4) Outcome measure: Efficacy (effective number/total number *100%); Visual Analog Scales(VAS) score: A commonly used subjective tool for assessing pain intensity. The score ranges from 0 to 10, with a lower score indicating less pain. Reporting one outcome measure can be included. (5) Study type: Randomized controlled trial. The language is limited to Chinese and English.

Exclusion Criteria

(1) Duplicate references. (2) Non-randomized controlled trials (reviews, animal trials, abstracts, etc). (3) The intervention measures do not match. (4) Incomplete or incorrect data. (5) Unavailable documentation. (6) Network meta-analysis cannot be formed.

Search Strategy

Randomized controlled trials of acupuncture for Myofascial Pain Syndrome in Neck or shoulder published in PubMed, Web of Science, Embase, Cochrane Library, VIP, SinoMed, CNKI and Wanfang were searched. Using a combination of subject words and free words. The time period is from establishment to April 30, 2025. The search strategy is in the supplementary materials 1.

Literature Screening and Data Extraction

Two researchers independently used Endnote to screen the literature respectively. The study was initially screened by checking for duplicates, reading titles and abstracts, and then re-screened by reading the full text. Data were entered using Excel. Extract ① Basic information: author, journal, region, publication year. ② Baseline data: patient information, intervention measures, course of treatment, outcome indicators. ③ Risk of bias information: randomization method, allocation hiding, blinding, etc. If the data is incomplete, contact the original study authors for detailed data. After completion, check each other, and if there are any issues, discuss and decide within the group.

Quality Evaluation

The quality was evaluated independently by two evaluators using the Cochrane Risk Bias Evaluation Tool, and cross-checked after completion. It included six aspects: random sequence, allocation concealment, blinding, completeness of outcomes, selective reporting of results, and other sources of bias. Each was evaluated as Low risk, High risk, and Unclear. The criteria for judging the quality of the literature were as follows: high grade if all were low risk, medium grade if no high risk but with uncertain risk, and low grade if high risk.

Evaluation of Evidence Quality

We used CINeMA¹² to assess the quality of evidence. CINeMA graded the quality of evidence in six areas, namely intra-study bias, inter-study bias, indirectness, imprecision, heterogeneity, and inconsistency, and the final quality of evidence was classified as high, medium, and Low and very low quality grades.

Statistical Analysis

Literature quality was evaluated using RevMan 5 4 and a literature risk bias map was created. Meta-analysis was performed using Stata 160. Binary variables used relative risk (RR), continuous variables used the mean difference (MD), and 95% confidence interval (CI) was calculated. The I2 statistic test was used to determine the size of heterogeneity, and when I2 < 50%, it indicated no statistical heterogeneity; When I2>50%, statistical heterogeneity was indicated, and sensitivity analysis was conducted to explore the source of heterogeneity. Plot the network of evidence for each outcome measure. When closed loops appear, perform the inconsistency test. If P > 005, use the consistency model for analysis; If P < 005, the inconsistencies will be reported and the node splitting method will be used for the inconsistencies test. Sort each outcome measure to obtain the surface under the cumulative ranking (SUCRA). SUCRA is expressed as a percentage, with a higher percentage indicating a better intervention.¹³ When the number of included literature for outcome measures was ≥10, a “comparation-correction” funnel plot was plotted and Egger’s test was performed to determine the possibility of publication bias.

Results

Study Selection

A preliminary search yielded 702 relevant articles, 196 duplicate articles were excluded, 466 articles were excluded by reading the title and abstract, and 11 articles were excluded by reading the full text (2 studies had incomplete data and 9 studies did not meet the intervention measures), and ultimately 29 studies were included. Figure 1 shows the screening process diagram.

Figure 1 Literature screening process.

Study Characteristics

This study included 29 studies^14–42 with a total of 2424 patients (1215 in the experimental group and 1209 in the control group). There were a total of 13 interventions in the experimental group: Fu’s subcutaneous needling, Internal heat acupuncture, Electroacupuncture, Round-point needle, Needle knife, Moxibustion Youdaoplaceholder0 cord, Acupoint injection, Conventional acupuncture+Moxibustion, Conventional acupuncture+Bloodletting Conventional acupuncture+Traditional Chinese Medicine,Conventional acupuncture+Tuina,Conventional acupuncture+Exercise. The control group was Conventional acupuncture. Table 1 shows the basic characteristics of the included studies.

Table 1 Basic Characteristics of the Included Studies

Risk of Bias in Studies

In terms of randomization methods, 25 studies used the low-risk randomization method, and 1 study used the wrong randomization method. In terms of allocation hiding, 13 studies employed a low-risk approach. In terms of blinding (performance bias and detection bias), five studies used blinding. Due to the particularity of acupuncture, blinding was difficult to implement but had little effect on the results. Data from all studies were complete, and no sources of selective reporting and other publication biases were found. Risk of bias results indicated moderate overall quality of the studies. Eight studies were “high risk” (27%), and 21 studies were “medium risk” (73%). Figures 2a and b show the risk of bias results of the included studies.

Figure 2 (a) Risk of bias graph (b) Risk of bias summary.

Meta Analysis

Vas

12 studies reported VAS, including 11 acupuncture treatments: Internal heat acupuncture, Acupoint injection, Electroacupuncture, Moxibustion Conventional acupuncture+Traditional Chinese Medicine, Conventional acupuncture+Moxibustion Conventional acupuncture+Exercise, Conventional acupuncture+Bloodletting, Fire acupuncture, Needle knife Round-point needle. There is no closed loop in the figure The inconsistency test results show P > 005, and the surface network is consistent, analyzed using the consistency model (Figure 3). Heterogeneity test results showed I2=5%, indicating low heterogeneity. Compared with Conventional acupuncture, Fire acupuncture (MD=−1.42, 95% CI (−2.45, −0.40)), Internal heat acupuncture (MD=−2.18, 95% CI (−3.37, – 0.99)), Moxibustion (MD = – 2.21,95% CI (−3.46, −0.97)), but these acupunctureandMoxibustion (MD = – 2.02,95% CI (−3.12, −0.92)), the effect is good (Figure 4). The therapeutic effects of different acupuncture treatments vary (Table 2). SUCRA sorting the top three in turn is: Moxibustion (0.84), Internal heat acupuncture (0.84), but these acupunctureandMoxibustion (0.79) (Table 3 and Figure 5).

Table 2 League Table (VAS) Comparing the Effectiveness of Different Acupuncture Treatments for Myofascial Pain Syndrome in Neck or Shoulder

Table 3 SUCRA Sort Results

Figure 3 Network Diagram of acupuncture for MPS in Neck or shoulder(VAS).

Figure 4 Forest map of acupuncture for MPS in Neck or shoulder(VAS).

Figure 5 The comprehensive ranking result of SUCRA.

Efficacy

Twenty-three studies reported Efficacy, including 11 acupuncture treatment methods: Internal heat acupuncture, Acupoint injection, Moxibustion, Conventional acupuncture+Traditional Chinese Medicine Conventional acupuncture+Tuina, Conventional acupuncture+Moxibustion, Sunken cord, Fu’s subcutaneous needling Conventional acupuncture+Bloodletting, Fire acupuncture, Needle knife. There is no closed loop in the picture. The inconsistency test results show P > 005, and the surface network is consistent, analyzed using the consistency model (Figure 6). Heterogeneity test results showed I2=5%, indicating low heterogeneity. Compared with Conventional acupuncture, Acupoint injection (RR=1.10,95% CI (1.00,1.20) Conventional acupuncture+Traditional Chinese Medicine (RR=1.15, 95% CI (1.03,1.29)), acupuncture and Tuina (RR = 1.31,95% CI (1.07,1.61)), acupuncture and Moxibustion (RR = 1.10,95% CI (1.01,1.19)), Fire acupuncture (RR=1.20, 95% CI (1.09,1.33)), Fu’s subcutaneous needling (RR=1.20, 95% CI (1.02,1.41)) Internal heat acupuncture (RR=1.27, 95% CI (1.12,1.45)) and Needle knife (RR=1.12, 95% CI (1.04,1.19)) worked better (Figure 7). There were differences in the efficacy of different acupuncture (Table 4). The top three SUCRAs in order are Conventional acupuncture+Tuina (0.98), Internal heat acupuncture (0.85), and Fire acupuncture (0.72) (Table 3). Combining the SUCRA results of the two outcome measures, it was found that Internal heat acupuncture had the best combined effect (Table 3 and Figure 5).

Table 4 League Table (Efficacy) Comparing the Effectiveness of Different Acupuncture Treatments for Myofascial Pain Syndrome in Neck or Shoulder

Figure 6 Network diagram of acupuncture for MPS in Neck or shoulder(Efficacy).

Figure 7 Forest map of acupuncture for MPS in Neck or shoulder(Efficacy).

Publication Bias Analysis

The egger test for VAS and Efficacy results showed a low likelihood of publication bias (Egger’s P > 005) (Figure 8a and b).

Figure 8 (a) Funnel plot of acupuncture for MPS in Neck or shoulder(VAS) (b) Funnel plot of acupuncture for MPS in Neck or shoulder(Efficacy).

Evaluation of Evidence Quality

The results of each acupuncture method compared with conventional acupuncture showed a lower quality of evidence. The downgrading was mainly due to selective reporting and imprecision (Supplementary Material 2).

Discussion

This study employed a meta-analysis approach to evaluate the effectiveness of various acupuncture methods in treating Myofascial Pain Syndrome in Neck or shoulder. A total of 29 studies were included, involving 2,424 patients (1,215 in the experimental group and 1,209 in the control group). The experimental group received 13 different interventions: Fu’s subcutaneous needling, Internal heat acupuncture, Electroacupuncture, Round-point needle, Needle knife, Moxibustion, Acupoint injection, Conventional acupuncture + Moxibustion, Conventional acupuncture + Bloodletting, Conventional acupuncture + Traditional Chinese Medicine, Conventional acupuncture + Tuina, and Conventional acupuncture + Exercise. The control group received Conventional acupuncture. VASand efficacy rates were used as primary outcome measures for assessing pain-related symptom improvement in patients with Myofascial Pain Syndrome in Neck or shoulder. In terms of VAS scores, the meta-analysis results indicated that Fire acupuncture, Internal heat acupuncture, Moxibustion, and Conventional acupuncture + Moxibustion demonstrated superior effects compared to Conventional acupuncture alone. The SUCRA ranking revealed that Moxibustion (SUCRA = 0.84) was the most effective intervention. Regarding efficacy rates, the meta-analysis showed that Acupoint injection, Conventional acupuncture + Traditional Chinese Medicine, Conventional acupuncture + Tuina, Conventional acupuncture + Moxibustion, Fire acupuncture, Fu’s subcutaneous needling, Internal heat acupuncture, and Needle knife all exhibited better outcomes than Conventional acupuncture. The SUCRA ranking identified Conventional acupuncture + Tuina as the best intervention in this category. The results were stable, with no significant publication bias observed. A comprehensive analysis of the SUCRA rankings indicated that Internal heat acupuncture had the overall best therapeutic effect.

Internal heat acupuncture is a novel therapeutic method that combines traditional warm acupuncture techniques, such as silver needles, with modern technology. The procedure involves connecting the resistance wire at the tail of the Internal heat acupuncture needle to an Internal heat acupuncture treatment device. The needle is then inserted into the tender points of the affected soft tissue, and the device is activated to heat the needle, delivering continuous and steady heat deep into the tissue. This process enhances local microcirculation, alleviates inflammatory responses, and accelerates lesion repair.⁴³ Patients with long-standing Myofascial Pain Syndrome in Neck or shoulder, characterized by palpable hard nodules or cords in the affected area, typically exhibit slower responses to treatment, are prone to recurrence, and are more challenging to manage.⁴⁴ In contrast, Internal heat acupuncture passively releases soft tissue lesions caused by Myofascial Pain Syndrome in Neck or shoulder, particularly those with prominent tenderness, palpable hard nodules, or cords. It reduces local fascial pressure, improves blood circulation, increases interstitial oxygen partial pressure, and eliminates local inflammatory edema, thereby promoting tissue repair and achieving remarkable therapeutic effects with a low recurrence rate.

Multiple basic and clinical studies have demonstrated that Internal heat acupuncture is significantly more effective than conventional acupuncture. The mechanisms underlying Internal heat acupuncture include the following: (1) Muscle spasm relief and tension reduction. Internal heat acupuncture treats soft tissue injuries through puncturing, where the micro-trauma induced by the puncture shortens muscle contraction after chronic injury and alleviates it, improving high tissue tension in the acupuncture area. Animal experiments⁴⁵ have shown that using an Internal heat acupuncture needle with a diameter of 1.1 mm to treat soft tissue injuries at 42°C with a needle spacing of 1.0 cm resulted in significantly less inflammatory edema compared to untreated controls. This may be attributed to the heat energy delivered deep into the muscle via Internal heat acupuncture, reducing γ-neuron excitability and weakening α-motor neuron efferent impulses through negative feedback, thereby decreasing muscle and fibrous tissue tension.⁴⁶ (2) Reduction of inflammatory responses and alleviation of local pain in soft tissue and muscle tissue damage. Yang Zhili et al⁴⁷ found through animal experiments that Internal heat acupuncture treatment reduced malondialdehyde (MDA) levels in the muscles of rats with chronic skeletal muscle injuries while increasing superoxide dismutase (SOD) activity. This suggests that Internal heat acupuncture can mitigate inflammatory responses after chronic soft tissue muscle injuries, enhance antioxidant capacity in injured muscles, and relieve pain. (3) Promotion of angiogenesis and improvement of blood circulation. Internal heat acupuncture significantly increases vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (BFGF) activity in chronically injured rat skeletal muscle and nerve tissues, promoting angiogenesis and enhancing local blood circulation.⁴⁸ (4) Facilitation of muscle repair and restoration of bodily functions. Internal heat acupuncture markedly affects vimentin and albumin expression in the muscle tissue of animals with soft tissue injuries, playing a critical role in muscle repair.⁴⁹ (5) Nourishment of peripheral nerves and promotion of wound healing. Internal heat acupuncture achieves the shortest recovery time for peroneal nerve function in cases of tissue injury, indicating its effectiveness not only in treating soft tissue injuries but also in promoting peripheral nerve injury healing.⁵⁰

Internal heat acupuncture demonstrates efficacy in treating chronic soft tissue injury-induced pain. For instance, Wang et al⁵¹ randomly divided 60 patients into an experimental group and a control group, evaluating therapeutic effects using the visual analog scale (VAS) and clinical efficacy. The total Efficacy of the Internal heat acupuncture group was significantly higher than that of the control group. In terms of short-term and long-term efficacy tracking, Zhang Zhaowen et al⁵² assessed efficacy at the end of treatment, 1 month, and 3 months post-treatment. The total Efficacy of the Internal heat acupuncture group was 92.9%, higher than the 75.9% of the Electroacupuncture group. The Internal heat acupuncture group maintained higher efficacy rates than the Electroacupuncture group during 1-month and 3-month follow-ups. Zhang Ding⁵³ divided 26 patients with periarthritis of the shoulder into a treatment group and a control group, administering Internal heat acupuncture and conventional acupuncture, respectively. Results showed that the total Efficacy of the treatment group was significantly higher than that of the control group, with significantly lower VAS scores, demonstrating a marked effect in reducing patient pain. In terms of long-term efficacy, Ding et al⁵⁴ treated 60 cases of periarthritis of the shoulder in groups, revealing that the total clinical Efficacy of the observation group was better than that of the control group in the second and third weeks post-treatment. Wang Rong⁵⁵ selected 48 patients with lumbar intervertebral disc protrusion, randomly dividing them into Internal heat acupuncture and Electroacupuncture groups. Retrospective analysis of changes in lumbar JOA score, VAS score, and LFR after treatment showed that the total Efficacy of Internal heat acupuncture at the lumbar intervertebral point was significantly higher than that of Electroacupuncture. Internal heat acupuncture offers advantages such as minimal trauma, rapid recovery, green safety, and low cost, with significant and lasting therapeutic effects, making it worthy of promotion.

Luo et al’s⁵⁶ meta-analysis compared the effects of different acupuncture therapies on MPS, and the results showed that electroacupuncture was the most effective in improving pain. However, it did not include Internal heat acupuncture. Our research proved that Internal heat acupuncture was more effective than electroacupuncture. Ge’s study⁵⁷ was a network meta-analysis of different acupuncture treatments for myofascial pain in the lumbar and dorsal muscles, and the probability of acupuncture thread embedding being the best therapy was the highest. Our research included myofascial pain syndrome in the neck and shoulders, and no relevant studies on acupuncture thread embedding were found. We will closely follow related research in the future and hope to obtain more evidence.

The range of motion of the neck and shoulders is an important indicator. However, many studies have not reported this indicator separately, making it impossible to conduct a meta-analysis. Many studies have included the range of motion in the category of “efficacy”. Currently, there is evidence that acupuncture can improve the range of motion of the neck and shoulders. For instance, Zhan’s study⁵⁸ randomly assigned 9090 patients with myofascial pain syndrome of the neck and shoulders to receive acupuncture treatment. The study observed the range of functional limitation, muscle spasm, and cervical range of motion before and after treatment. The results showed that acupuncture could significantly improve the clinical symptoms and signs of patients with myofascial pain syndrome of the neck and shoulders and increase the range of motion of the shoulder and neck joints. Future research should pay more attention to the range of motion of the neck and shoulders and provide more evidence. In addition, patient satisfaction should also be regarded as an important indicator. However, many studies have not reported this indicator separately, making it impossible to conduct a meta-analysis. Currently, there is evidence of patient satisfaction. For example, Wang’s study⁵⁹ found that the acupuncture group combined with nursing had a higher satisfaction rate. Currently, acupuncture is accepted and adopted to varying degrees as a method of pain management in different cultures and medical systems around the world. Over the past 20 years, with the continuous accumulation of evidence supporting its efficacy, acupuncture has gradually become a popular choice for treating pain in rehabilitation patients. For instance, an article published by the Canadian Physiotherapy Association emphasized the important role of acupuncture in relieving pain and advocated for the inclusion of pain-related knowledge and treatment methods in physiotherapy professional education to improve treatment efficacy and patient satisfaction.⁶⁰ Future research should place more emphasis on studies related to humanistic care and provide more evidence.

We conducted a systematic analysis of the differences in the effects of different acupuncture therapies for MPS in the neck and shoulders and found that Internal heat acupuncture was effective, providing additional evidence for clinical research. However,There are certain limitations in this study: (1) The quality of the evidence from the included literature is relatively low, potentially reducing result credibility; (2) The included literature has average quality, with most lacking large sample sizes, high-quality, multicenter randomized controlled trials; (3) Efficacy criteria across studies were inconsistent, with varying grades of efficacy, determination standards, operation methods, and treatment durations, which may impact results and increase heterogeneity among studies; (4) A considerable number of studies did not specify randomization methods, allocation concealment, or blinding procedures, resulting in lower quality evaluations; (5) Outcome indicators in the included studies were predominantly subjective, lacking authoritative and scientific metrics to validate result reliability and reduce heterogeneity. These factors may influence results and lead to greater heterogeneity among studies. (4) The quality of evidence results show that the confidence interval of the estimated effect is wide, indicating imprecision. This may be due to the significant differences among the included studies. The included studies have significant differences in terms of population, intervention measures, and outcome measurements. For example, different acupuncture operation techniques (needle depth, retention time); inconsistent pain assessment tools; and differences in the design of the control group. Some studies did not mention or only mentioned the randomization method without specifying the specific operation, did not explain the allocation concealment method and blinding, and had a low quality evaluation, all of which may affect the estimated effect. (5) The outcome indicators of the included studies are mostly subjective indicators, lacking authoritative and scientific indicators to verify the reliability of the results, and there is a possibility of selective reporting. These factors may affect the results and even lead to significant heterogeneity among studies. Although we reduced the risk of publication bias by searching seven major databases, the funnel plot and Egger’s test confirmed no significant publication bias. In addition, we extracted the pre-specified primary outcomes of all included studies and found no evidence of selective reporting. However, the conclusions of this study still need to be further verified by high-quality, multi-center, and scientifically based RCT studies.

Conclusions

Based on the existing evidence mainly from Chinese studies, various acupuncture methods have significant advantages compared to conventional acupuncture. Comprehensive analysis shows that Internal Heat Acupuncture has the best effect. The current conclusion is mainly applicable to the Chinese (or similar background) population. Its promotion to other races or regions requires caution and needs further research to confirm. Future research should prioritize the design and implementation of high-quality, large-sample, multi-center, multi-ethnic/population randomized controlled trials to verify the efficacy and safety of acupuncture therapy in different populations, and assess the possible impacts of culture and race factors, so as to obtain more universal and persuasive evidence-based evidence. We will also actively follow and incorporate future published international studies that meet the standards.

Abbreviations

(MPS), Myofascial Pain Syndrome; (RCTs), Randomized controlled trials; (TCM), Traditional Chinese Medicine; (SUCRA), Cumulative Ranking Curve; (MTrPs), myofascial trigger points; (TENS), transcutaneous electrical nerve stimulation; (NSAIDs), non-steroidal anti-inflammatory drugs; (VEGF), vascular endothelial growth factor; (BFGF), basic fibroblast growth factor.

Acknowledgments

We would like to express our gratitude to all the participants in this research.

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 Hunan Provincial Clinical Medical Technology demonstration base of chronic musculoskeletal pain rehabilitation (grant number:2023SK4074),the Chronic musculoskeletal pain rehabilitation technology research and development center of Chenzhou City (grant number:2021-02-31).

Disclosure

The authors declare that they have no conflicts of interest.

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Story Continues

There are growing fears of an imminent stock market crash – one that will transform from a dip to a dive when euphoric headlines about the wonders of artificial intelligence begin to wane.

Shares in US tech stocks have fallen in recent weeks and the prospect is that a flood of negative numbers will become the norm before the month is out.

It could be 2000 all over again, and just like the bursting of the dotcom bubble it may be ugly, with investors junking businesses that once looked good on paper but now resemble a huge liability.

Jerome Powell, the Federal Reserve chair, is one of the policymakers tasked with keeping the wolf from the door. Speaking on Friday at the annual Jackson Hole gathering of central bank governors in Wyoming, he tried to calm nerves.

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Stock markets are on Powell’s radar even more than usual now that so many personal pensions in the US are directly invested in listed companies. And, more particularly, tech stocks making huge AI investments that have yet to make a dollar of profit.

A recent Massachusetts Institute of Technology report revealed that 95% of companies investing in generative AI have yet to see any financial returns.

This revelation came after Sam Altman, the boss of the ChatGPT owner OpenAI, warned that some company valuations were “insane”.

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Microsoft’s Copilot and the many other “helpful” AI tools on offer are becoming embedded in office life and beginning to perform large numbers of low-level tasks.

If this trend takes off, and in many parts of the economy it already has, there is a soft landing waiting for the tech industry even as some of the flakier, more speculative businesses are weeded out and fail.

If anything, a downturn helps the big firms pick up new technological breakthroughs from the wreckage and on the cheap.

Palantir’s price-to-earnings ratio is north of 500 when most investors would find themselves getting panicky at anything above 50. Nvidia has a price-to-earnings ratio of 56.

The Palantir/Nvidia ratios could decline as their share price falls more into line with a reasonable prospect of likely earnings, but the businesses are not going to go bust, even in the most severe equity market storms.

Trump is another significant supporter, clearing the path for AI to make even deeper inroads into corporate life. His support for cryptocurrencies, including his own, and deregulated social media platforms, including his own, are indications of where his sympathies lie.

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Welcome to The Brink. This is Giulia Morpurgo and Constantine Courcoulas in London, where we looked at turmoil in the wind industry. We also have news on Rent the Runway and Greek GDP warrants. Follow this link to subscribe. Send us feedback and tips at debtnews@bloomberg.net.

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How Drivio Play Saves Drivers Money in 2025

One of the strongest reasons consumers are turning to Drivio Play in 2025 is financial practicality. Vehicle prices have climbed to record highs, leaving many households unwilling to commit to costly dealership upgrades or long-term financing. Instead, people are searching for affordable ways to make their existing cars smarter, safer, and more enjoyable to drive.

Drivio Play enters this conversation as a cost-effective alternative. Instead of replacing a car to gain advanced infotainment, navigation, or wireless streaming, drivers can install this device at a fraction of the cost. For families, this means keeping reliable vehicles on the road longer without sacrificing access to modern features.

Fuel savings also connect to the product’s value. With real-time navigation and traffic data available through connected apps, drivers can plan more efficient routes and reduce wasted fuel. Over time, these savings compound, making Drivio Play not just a tech upgrade but also a financial tool for smarter driving.

Another cost advantage is its portability. Unlike permanent installations, Drivio Play can move from one vehicle to another. This flexibility eliminates the need to buy multiple systems or pay for expensive dealer-specific hardware. Households with more than one car especially value this feature, as it stretches the utility of a single purchase.

By combining affordability, portability, and efficiency, Drivio Play allows drivers to access the technology they want without stretching their budgets. It represents a smarter way to upgrade, proving that financial caution and modern convenience can go hand in hand.

Safety and Convenience Benefits Drivers Value Most

Modern drivers want more than entertainment—they want tools that make every trip safer and easier. This is one of the reasons Drivio Play is gaining attention in 2025. It blends convenience with real-world safety features that respond to the demands of today’s road conditions.

Hands-free calling is one of the most practical benefits. With Drivio Play, drivers can take calls or send voice commands without handling a phone. This reduces distraction, aligning with the growing focus on safer driving practices.

Navigation support is another advantage. Clear display visuals combined with real-time map access mean fewer wrong turns and faster route adjustments when traffic shifts. Families and commuters alike value how this feature saves time and lowers stress behind the wheel.

Entertainment integration adds to convenience. Music streaming, podcasts, and audiobook playback all become accessible in seconds, helping transform long commutes into smoother experiences. Drivers appreciate that these features work seamlessly with both iOS and Android devices.

For many households, the ability to consolidate these functions into a single device eliminates clutter and improves overall driving confidence. Instead of juggling multiple gadgets, Drivio Play centralizes everything into one clear interface, giving drivers what they need without unnecessary complication.

By focusing on both safety and convenience, Drivio Play positions itself as more than an upgrade—it becomes an essential travel companion for everyday life.

See the safety and convenience benefits of Drivio Play here

Who Should Consider Drivio Play in 2025

The appeal of Drivio Play stretches across a wide range of drivers. While originally viewed as a simple aftermarket upgrade, it has quickly become relevant for households, commuters, and even tech adopters who want smarter travel without overpaying for a new car.

Families are among the strongest adopters. Parents often look for safer, distraction-free tools that keep them focused while transporting children. Features like voice command, navigation, and easy entertainment access make Drivio Play a reliable solution for busy households balancing safety with convenience.

Commuters also gravitate toward the device. Spending hours on the road each week means drivers want efficiency and comfort. With Drivio Play, they gain tools that simplify daily routes, improve connectivity, and make long drives less stressful.

Road trip enthusiasts and travelers find unique value too. Portability ensures that the system can be used across multiple vehicles, meaning families can bring the same connected experience whether driving their own car, a rental, or even sharing vehicles within a household.

Tech adopters form another segment of interest. This group enjoys trying modern gadgets that make daily life easier. For them, Drivio Play represents an opportunity to modernize an older car quickly, showing how technology can extend value from an existing vehicle.

Finally, cost-conscious households consider Drivio Play a practical investment. Rather than financing expensive new vehicles, they choose an affordable option that introduces current features without long-term debt.

In short, Drivio Play serves multiple audiences—from families to commuters to tech-focused drivers—by providing affordable connectivity that matches modern expectations.

How Drivio Play Compares to Dealership Installs

For many drivers, the decision to upgrade technology comes down to a choice between expensive dealership installations or practical aftermarket solutions. Drivio Play offers a clear alternative that avoids the high costs and limitations often tied to dealer systems.

Dealership upgrades can cost thousands of dollars and are usually restricted to the specific vehicle where they are installed. Once in place, they cannot be transferred, meaning drivers must pay again if they switch cars. By contrast, Drivio Play provides a portable option that can move between vehicles, making it a far more flexible investment.

Another difference lies in installation. Dealership systems often require professional setup, which means additional labor charges and long waiting times. Drivio Play is designed for quick, consumer-friendly setup, allowing most users to begin using the system without paying extra fees.

Functionality is also broader with Drivio Play. While dealership systems are tied to manufacturer updates, this device integrates seamlessly with both Android and iOS, giving users consistent access to navigation, music, and voice commands. This flexibility ensures that drivers do not lose features as technology evolves.

Maintenance is another factor. If a dealership-installed unit fails, drivers must return to the dealer for costly repairs or replacements. With Drivio Play, the process is simpler, affordable, and does not tie drivers to dealership contracts.

When comparing affordability, adaptability, and ease of use, Drivio Play emerges as the smarter option for households that want modern driving features without unnecessary financial strain.

The Growing Market for Vehicle Tech Upgrades in 2025

The automotive market in 2025 is defined by one clear trend: more drivers are upgrading the cars they already own rather than purchasing new models. Rising prices for new vehicles and limited availability of dealer inventory have pushed consumers to explore aftermarket solutions that deliver the same conveniences at a fraction of the cost.

This shift has created explosive growth in the car technology upgrade sector. Drivers are searching for ways to bring streaming, navigation, and voice control into older cars that are still reliable on the road. Products like Drivio Play have become central to this expansion by offering an accessible bridge between traditional vehicles and modern digital features.

Industry data reflects this momentum. Online searches for terms like “car infotainment upgrade,” “wireless driving tech,” and “retrofit vehicle display” have climbed steadily throughout 2025. Forums, social media groups, and review platforms consistently feature discussions about devices that make older cars feel new again. Drivio Play is often mentioned in these conversations as a leading example of affordable innovation.

Consumer behavior also shows that drivers prefer flexible, portable solutions over permanent installations. With families sharing cars, commuters leasing vehicles, and travelers using rentals, adaptability has become as important as functionality. Drivio Play aligns with this trend by offering portability without compromising performance.

The rapid growth of this market signals a long-term change in how consumers think about car ownership. Instead of relying on dealerships for upgrades, they are turning to independent solutions that combine affordability with control. Drivio Play sits at the center of this new movement, showing how everyday drivers are shaping the future of automotive technology.

See why the vehicle tech upgrade market is growing with Drivio Play

Real-World Experiences and Consumer Feedback

The most persuasive insights about any product often come from everyday users. In 2025, Drivio Play has attracted attention across forums, social platforms, and review sites where drivers share how the system has impacted their daily commutes and long-distance travel.

Many reviews highlight ease of installation as a standout feature. Drivers note that they were able to set up the device quickly without paying dealership labor costs. This user-friendly design has made it especially popular with households that want results without technical headaches.

Clarity of the display is another recurring theme. Commuters appreciate the crisp visuals when navigating busy streets or long highway stretches, while families point out that the sharp screen makes trip planning smoother and safer. Positive feedback often emphasizes that the clear interface reduces stress behind the wheel.

Hands-free functionality is also praised. Users report that calls and voice commands work smoothly, allowing them to stay connected without fumbling with a phone. For many, this convenience doubles as a safety upgrade, aligning with modern expectations for distraction-free driving.

Of course, not all reviews are the same. Some drivers note that no aftermarket system can replace the full integration of high-end dealership upgrades. These comments typically suggest setting realistic expectations—understanding that Drivio Play is built for affordability and flexibility, not luxury exclusivity. Yet even these balanced perspectives reinforce the value of the device as a practical, budget-friendly alternative.

What unites most user feedback is satisfaction with the balance of price and performance. Drivio Play is frequently described as a solution that makes older vehicles feel modern again without the cost of a trade-in. This perception has fueled ongoing conversations and positioned it as one of the standout aftermarket solutions of 2025.

Read real-world feedback and experiences with Drivio Play here

Why 2025 Is the Right Time to Upgrade With Drivio Play

The decision to modernize an older vehicle often comes down to timing. In 2025, that timing could not be better. Rising car prices, longer loan terms, and the ongoing cost of ownership have forced many drivers to rethink whether buying new is the smartest choice. Instead, upgrading with affordable tech has become the more logical path forward.

Drivio Play is positioned perfectly within this shift. It allows households to enjoy premium features like real-time navigation, streaming, and voice-command functionality without trading in a vehicle that still performs well. For families balancing financial caution with modern expectations, this device is a straightforward solution.

Another factor is lifestyle change. More commuters are back on the road, and road trips have surged as families choose driving over costly flights. With people spending more time behind the wheel, the demand for convenient, connected, and safe driving tools is higher than ever. Drivio Play answers this demand with a system that installs quickly and enhances any journey.

Technology adoption is also a reason 2025 is the right time. Portable, wireless devices are now a standard part of everyday life, and drivers expect the same ease of use in their vehicles. Drivio Play leverages these trends, making advanced driving tech accessible through a familiar, intuitive design.

With affordability, accessibility, and adaptability aligned, the market momentum suggests that this year is the ideal time for drivers to upgrade their vehicles without financial strain. Drivio Play delivers modern features when they are needed most—right now.

Market Outlook and the Future of Retrofit Car Technology

The retrofit car technology market is entering a new phase in 2025. As drivers hold onto their vehicles longer, demand for affordable upgrades has accelerated. Instead of replacing a car every few years, households are investing in devices that extend functionality and align with modern expectations. This shift is creating strong growth for aftermarket solutions like Drivio Play.

Industry forecasts indicate that retrofit technology will continue expanding over the next five years. Rising car prices, combined with consumer reluctance to take on large loans, point to sustained interest in cost-effective upgrades. Products that add value without requiring professional installation will lead this movement.

Drivio Play reflects the qualities that analysts highlight as drivers of growth: affordability, portability, and compatibility with both iOS and Android systems. By giving households a way to keep older vehicles relevant, it positions itself within a consumer market that is expected to expand steadily as adoption becomes mainstream.

Cultural momentum also plays a role in shaping the outlook. Online forums, auto review sites, and consumer tech discussions increasingly feature conversations about extending vehicle life rather than replacing it. This cultural shift toward sustainability and financial caution reinforces why retrofit solutions resonate with today’s drivers.

The broader future of this niche suggests that products combining convenience, safety, and entertainment will dominate. Drivio Play aligns with this direction, providing a model for how car technology can evolve outside dealership channels.

For drivers, this means greater choice, more control, and access to features that make cars smarter without breaking household budgets. For the industry, it means sustained growth as retrofit solutions become standard in everyday driving.

Final Thoughts and Consumer Takeaway

Drivers are redefining what it means to upgrade their vehicles in 2025. Rather than trading in reliable cars for expensive new models, more households are turning to retrofit solutions that add modern features without overwhelming costs. Drivio Play has emerged as one of the most talked-about devices in this category, offering a blend of affordability, convenience, and compatibility that resonates with everyday drivers.

From families looking for safer commutes, to commuters seeking efficiency, to tech adopters wanting smarter features in older vehicles, Drivio Play delivers practical solutions. Its portability, hands-free functionality, navigation support, and easy installation make it a device that fits multiple lifestyles and budgets.

The broader market confirms this trend. Retrofit technology is no longer an afterthought—it is becoming the mainstream choice for consumers who want to extend vehicle value. By focusing on affordability and usability, Drivio Play reflects the direction of the entire automotive upgrade sector.

For consumers evaluating their options in 2025, the takeaway is simple: upgrading your current car does not have to be costly or complicated. Drivio Play provides an accessible path to smarter, safer, and more enjoyable driving without dealership prices.

Get started with Drivio Play today

Contact

• Company: DrivoPlay– Supporting smarter consumer solutions in 2025

• Email: support@drivioplay.com

• Phone (US): +1 (888) 458-0473

Final Disclaimer

This press release is for informational purposes only. The content herein does not constitute financial, legal, medical, or professional advice. Drivio Play is not intended to diagnose, treat, predict, or guarantee any result or outcome. Individual experiences may vary, and outcomes are not assured.

Some links in this release may be promotional in nature and may lead to third-party websites. The publisher or author may receive compensation through affiliate commissions if a purchase is made through these links. This compensation does not affect the price you pay and helps support continued research and content publication.

All statements made about product features, consumer strategies, or technology outlook reflect publicly available information, user discussions, or historical trends, and are not endorsed or validated by regulatory bodies. Readers are encouraged to perform their own research before making financial, technological, or purchasing decisions.

CONTACT: Email: support@drivioplay.com Phone (US): +1 (888) 458-0473

DELRAY BEACH, Fla.  , Aug. 23, 2025 /PRNewswire/ — The global Targeted Protein Degradation Market, valued at US$0.01 billion in 2024, stood at US$0.48 billion in 2025 and is projected to advance at a resilient CAGR of 35.4% from 2025 to 2035, culminating in a forecasted valuation of US$9.85 billion by the end of the period. This is mainly due to various factors such as Priority/Fast Track designations and expected first-in-class approvals & and multi-indication expansion. However, the CMC & scale-up complexity for heterobifunctional & IP disputes are predicted to cause market growth barriers.

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Browse in-depth TOC on “Targeted Protein Degradation Market“

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By formulation, the global targeted protein degradation market is segmented by formulation into two main categories: oral and injections. In 2024, the oral formulations segment holds the highest share in the formulation landscape of the novel TPD market, driven by its favorable pharmacokinetics, patient compliance, and manufacturing scalability. Oral delivery remains the preferred route due to its non-invasive nature and ease of administration, which significantly enhances patient adherence, especially in chronic or long-term treatments. The advancements in medicinal chemistry have enabled the development of orally bioavailable degraders, overcoming early limitations of poor solubility and permeability. Many first-generation PROTACs faced challenges with oral bioavailability due to their large molecular weight and polarity; however, recent innovation in structural optimization has yielded degrader candidates suitable for oral dosing without compromising efficacy. From a commercial perspective, oral formulations offer cost-efficiency in distribution and storage, adding to their attractiveness. Oral TPD candidates in oncology, immunology, and neurology have shown promising clinical progress, reinforcing confidence in this route. As the TPD field matures, oral formulations are expected to dominate clinical pipelines and product launches, setting the standard for patient-friendly, scalable therapeutic solutions. The continued investment in optimizing oral bioavailability across heterobifunctional and molecular glue degraders is anticipated to fuel segment growth.

By Type, the PROTACs (PROteolysis TArgeting Chimeras) segment was the fastest-growing within the targeted protein degradation (TPD) market due to their ability to selectively eliminate, rather than inhibit, disease-causing proteins. By leveraging the natural ubiquitin-proteasome system of cells, PROTACs redirect E3 ligases to tag specific proteins for degradation, offering a novel approach to address previously undruggable targets. Unlike conventional inhibitors, PROTACs can achieve greater potency and sustained therapeutic effects, often requiring lower doses and showing potential to overcome resistance mechanisms. This has sparked intense interest from the pharmaceutical industry, leading to a rapidly expanding pipeline with numerous candidates advancing through preclinical and clinical development. Clinical-stage PROTACs like ARV-471, ARV-110, and NX-2127 are being evaluated in oncology, particularly in hormone-driven cancers. Ongoing innovation in linker chemistry, E3 ligase selection, and optimization for oral delivery is driving improvements in drug-like properties. With increasing investments, strategic collaborations, and a broadening scope across therapeutic areas, PROTACs are poised to become a cornerstone of next-generation drug discovery. Their versatility and ability to precisely degrade specific proteins continue to position them as a leading modality within the growing TPD market.

By geography, the market for targeted protein degradation is divided into six segments: North America, Europe, Asia Pacific, Latin America, the Middle East, and Africa. North America holds the largest share in the targeted protein degradation (TPD) market, driven by strong R&D infrastructure, robust funding ecosystems, and a favorable regulatory environment. The region is home to several pioneering biotechnology and pharmaceutical companies that are leading the development of TPD platforms, particularly PROTACs and molecular glues. Major players such as Arvinas, C4 Therapeutics, and Nurix Therapeutics are headquartered in the US, benefiting from access to top-tier research institutions and a highly skilled scientific workforce. In addition, consistent capital inflow from venture capital firms, public markets, and strategic pharmaceutical partnerships has accelerated innovation and clinical progression of TPD candidates. The US Food and Drug Administration (FDA) has also played a critical role by granting fast track and orphan drug designations to promising TPD candidates, thereby expediting their development and regulatory pathways. Increasing investment in oncology, neurodegenerative diseases, and autoimmune indications, which are the key therapeutic areas for TPDs, will continue to drive demand in the region. Academic-industry collaborations, the presence of large-scale clinical trial networks, and a strong intellectual property framework further enhance the leadership position of North America. These combined factors position the region to achieve the highest growth trajectory in the global TPD market.

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Key players in the targeted protein degradation market include Bristol Myers Squibb (US), Arvinas (US), BeiGene (US), Nurix (US), Kymera (US), C4 Therapeutics (US), Stemline Therapeutics (US), AstraZeneca (UK), F. Hoffmann-La Roche Ltd (Switzerland), Bayer (Vividion) (Germany), Captor Therapeutics (Poland), Ranok Therapeutics (US), Pfizer (US), Novartis (Switzerland), and Foghorn Therapeutics (US).

Bristol Myers Squibb:

Bristol Myers Squibb (BMS) is a key player in the targeted protein degradation (TPD) market, leveraging its extensive drug development capabilities to advance next-generation therapeutics. BMS was among the earliest large pharmaceutical companies to recognize the potential of TPD, particularly through molecular glues that reprogram E3 ligases to degrade disease-causing proteins. The company inherited significant TPD assets and expertise by acquiring Celgene, including its partnership with Evotec and initial collaborations with startups such as C4 Therapeutics. BMS continues to build a robust pipeline of TPD candidates, especially in oncology and immunology, where traditional small molecules or biologics have limited efficacy. Its approach combines in-house discovery with strategic partnerships to broaden target scope and accelerate clinical progression. With ongoing investments in E3 ligase biology and protein degradation mechanisms, BMS is positioning itself as a long-term leader in the TPD space. Its focus on molecular glues and degrader programs highlights a commitment to expanding and offering new therapeutic avenues for complex diseases.

Arvinas:

Arvinas is a pioneer in the targeted protein degradation (TPD) field, recognized for being the first company to bring a PROTAC (PROteolysis TArgeting Chimera) degrader into clinical trials. Founded on technology from Yale University, Arvinas has developed a proprietary platform that harnesses the ubiquitin-proteasome system to selectively degrade disease-causing proteins. Its clinical pipeline includes ARV-110 and ARV-471, which target androgen and estrogen receptors in prostate and breast cancers, respectively. Its strong scientific foundation and first-mover advantage have positioned it as a leader in the TPD space. In addition to internal programs, Arvinas has formed high-profile collaborations with Pfizer and Bayer, leveraging its platform to explore new targets and therapeutic areas beyond oncology. These partnerships validate its technology and provide significant funding and development support. As the TPD field matures, Arvinas stands out for its clinical progress, strategic alliances, and expanding expertise in rational degrader design, which could lead to transformative therapies for patients with limited treatment options.

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