Therapeutic effects of Melittin acupoint injection on rheumatoid arthritis through autophagy activation and PI3K/AKT/mTOR pathway inhibition

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that can lead to cartilage and bone damage, ultimately resulting in disability (1). Currently, the pharmacological treatments for RA include non-steroidal anti-inflammatory drugs (NSAIDs), anti-rheumatic drugs, and biologics. However, these medications are often associated with serious side effects (2,3), which drives the search for new and effective treatment methods for RA. Traditional Chinese medicine (TCM) has demonstrated promising results in both the treatment and prevention of this condition (4). Melittin (MEL), which constitutes approximately 40–60% of the dry weight of bee venom (BV), is recognized as an effective anti-arthritic component of BV (5). As a natural product, BV and its primary component, MEL, can regulate the cell cycle, alter cell membrane permeability, inhibit proliferation and migration, and promote both endogenous and exogenous apoptosis, as well as autophagy and other forms of regulated cell death (6). Acupoint injection involves the administration of drugs into specific acupoints, such as Zusanli (ST36), to stimulate these points for therapeutic effects (7). This method serves as an alternative and complementary therapy rooted in the meridian theory of TCM, offering the dual benefits of drug administration and acupuncture, and is sometimes more effective than conventional Western medicine (8). Acupoint injection has been utilized to treat various diseases, including RA (9,10). Both MEL and acupoint injection exhibit therapeutic effects on RA, and melittin acupoint injection (MAI) combines the benefits of both MEL and acupuncture. Preliminary studies have demonstrated the efficacy of MAI in treating RA (11).

In recent years, numerous studies have shown that autophagy plays a key role in the occurrence and development of RA. The regulation of autophagy is strictly controlled to maintain immune homeostasis, and its dysregulation may lead to the occurrence and aggravation of RA (12). Therefore, studying the autophagy level and related pathways in patients with RA may provide a new direction for treatment. Among them, the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway plays a crucial role in the occurrence and development of RA. As an important intracellular regulatory hub, this pathway not only participates in cell proliferation, survival, and metabolic processes but also can inhibit autophagic activity, directly affecting the pathological process of RA (13). Studies have shown that the PI3K/AKT/mTOR pathway is in a state of persistent activation in the synovial tissue of RA patients. This abnormal activation can inhibit the autophagic process, leading to the abnormal proliferation and apoptosis resistance of fibroblast-like synoviocytes (FLSs), thereby promoting synovial inflammation and joint erosion (14,15). Moreover, a variety of drugs exert therapeutic effects on RA through the PI3K/AKT/mTOR pathway. For example, signal protein 5A inhibits ferroptosis by activating PI3K/AKT/mTOR signaling in RA; Wutou Decoction alleviates RA by inhibiting angiogenesis and regulating the PI3K/AKT/mTOR/HIF-1α pathway (16,17).

As a natural component with multiple biological activities, the potential anti-arthritic mechanism of MEL may be closely related to this pathway. However, the interaction between MEL and autophagy in RA remains unclear. Although there have been some studies on the treatment of RA with MEL, systematic research is still lacking, especially regarding the regulation of the PI3K/AKT/mTOR pathway, which has not been fully explored. Elucidating this scientific issue will not only help to deeply understand the pathogenesis of RA, but also provide a theoretical basis for the development of new RA treatment strategies based on autophagy regulation. The uniqueness and novelty of this study lie in exploring the novel mechanism underlying the therapeutic effect of MEL on collagen-induced arthritis (CIA) mice, and further investigating whether MEL can activate autophagy by inhibiting the PI3K/AKT/mTOR pathway, ultimately exerting anti arthritic effects, with the aim of providing a brand-new theoretical basis and treatment strategy for the treatment of RA. We present this article in accordance with the ARRIVE reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-540/rc).

Methods

Drugs and reagents

MEL and methotrexate (MTX; purity ≥99%) were procured from Shanghai Aladdin Biochemical Technology (Shanghai, China). Bovine type II collagen solution, complete Freund’s adjuvant, and incomplete Freund’s adjuvant were sourced from Chondrex (Woodinville, WA, USA). A hematoxylin and eosin (HE) staining kit, as well as a safranin O-fast green (SafO) staining kit, were obtained from Leigen Biotech (Beijing, China). Antibodies against TNF-α, LC3 I/II, LC3B, ULK1, Beclin-1, sequestosome 1 (P62), mTOR, p-mTOR, PI3K, Akt, p-Akt, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and normal rabbit immunoglobulin G (IgG) were acquired from Abmart (Shanghai, China). Additionally, IL-1β, IL-17A, IL-10, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Mlbio (Shanghai, China). Diaminobenzidine (DAB) chromogenic solution was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Other reagents are commercially available.

Animals

A total of 40 male DBA/1 mice were obtained from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Nanjing, China), classified as specific-pathogen-free (SPF) grade, with each mouse weighing between 18 and 20 grams and aged 6–8 weeks. The mice were housed in an SPF-grade animal room at the Animal Experimental Center of Southern Medical University. The rearing conditions maintained a temperature of 25±1 ℃, humidity of 60%±10%, and a 12-hour light/dark cycle. Mice had unrestricted access to water and food, and bedding was changed biweekly. Experimental studies commenced following a week of adaptive feeding. All animal experiments of this study were conducted under the approval of a project license (No. SMUL202308014) issued by the Ethics Committee of Southern Medical University, and strictly adhered to the guidelines for the care and use of laboratory animals established by Southern Medical University. A protocol was prepared before the study without registration.

Induction of CIA and experimental grouping

Hair was removed from the base of the mouse’s tail using a pet shaver, and the local skin was disinfected with a 0.5% amyl iodine solution prior to subcutaneous injection. On day 0, eight mice were randomly selected and injected subcutaneously with 0.1 mL of normal saline at the base of the tail, serving as the blank group (NC). The CIA model was established in the remaining mice, which received an intradermal injection of 100 µL of an emulsifier containing bovine type II collagen solution mixed with an equal volume of complete Freund’s adjuvant at the base of the tail. On day 21, the NC group was re-injected with 0.1 mL of normal saline, whereas the other mice received a subcutaneous injection of an emulsifier composed of 100 µL of bovine type II collagen and an equal volume of incomplete Freund’s adjuvant at the tail root for booster immunization (18). Following the enhancement of immunization, the morphological changes in the metatarsal joints of the mice were observed. A successful model was indicated by an arthritis index (AI) score of ≥1. By day 28, all mice had stabilized their disease status and were divided into four groups using a completely random method: the MEL acupoint subcutaneous injection group (ST group), the MEL acupoint intramuscular injection group (IT group), the methotrexate group (MTX group), and the model group (MO group), with eight animals in each group.

It should be noted that the safety of the MEL dose (0.5 mg/kg) and the administration regimen (0.02 mL every two days) used in this study has been verified (11). At this dose, no abnormalities in liver and kidney functions were observed in CIA mice [the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (Crea) were all within the normal range], and our treatment plan was strictly implemented in accordance with the safety-verified standards.

In this study, ST36 was selected for MEL injection. ST36 is located on the anterolateral side of the lower leg, 3 cun (body inch) below Dubi (ST35), one finger breadth away from the anterior border of the tibia, and represents the He sea point of the Stomach Meridian of Foot Yangming. Previous studies have shown that manual acupuncture (MA) applied to the ST36 acupoint can significantly increase the gene expression level of tissue repair growth factors in the inflamed joints of adjuvant-induced arthritis (AIA) mice (19) and also promote the expression of the anti-inflammatory cytokine IL-10. To sum up, acupuncture may alleviate the joint symptoms of RA by regulating the tissue repair process. Based on the above findings, we propose to adopt MAI at ST36 as the treatment regimen.

The intervention commenced on the 28th day of the experiment. The ST and IT groups received MAI every other day, specifically at the ST36 point, with an injection of 0.5 mg/kg MEL. The ST36 point is situated near the knee joint of the hind limb, 1.5 mm distal to the anterior tibial tubercle (20). During each treatment session, 0.02 mL of MEL solution was injected into the ST36 acupoint on one side of the mouse; the acupoints on both sides were alternately used for injection to avoid repeated stimulation of the mouse’s injection site, which could otherwise cause pain or swelling. In contrast, the NC and MO groups were administered the same volume of normal saline. For the MTX group, 2 mg/kg of MTX was given intragastrically once a week. All mice underwent treatment for a duration of 28 days, after which they were euthanized under anesthesia, and blood and joint tissues were collected for further analysis.

General condition measurement and arthritis assessment

From the onset of the intervention, the mental state, eating and drinking habits, hair color, urination, and defecation of the mice were monitored daily. Body weight was recorded every four days, and growth changes were observed. Additionally, the thickness of the soles and the diameter of the ankles were measured. The severity of arthritis in the mice was evaluated using the AI index score and joint swelling number scoring standards. SJC scoring criteria: Each paw comprises five finger/toe joints and one ankle/wrist joint (i.e., six joints per paw), resulting in a total of 24 joints per mouse. Each swollen joint is assigned a score of 1 point, allowing each paw to achieve a total score ranging from 0 to 6 points, with a maximum cumulative joint swelling score of 24 points per mouse (21). AI scoring standard: the score for each paw ranges from 0 to 4 points, based on the following criteria: 0 points indicates normal condition with no redness or swelling; 1 point indicates interphalangeal joint swelling and/or redness; 2 points indicate involvement of 3–4 fingers or one larger joint; 3 points indicate severe redness and swelling below the ankle joint; and 4 points indicate severe arthritis affecting the entire paw, including the ankle (22).

Micro-computed tomography (CT) analysis

The right foot of the mouse was isolated from the middle and lower segments of the tibia and fibula, and subsequently fixed in a 4% paraformaldehyde solution at 4 ℃. The paraformaldehyde was replaced every 24 hours. After a total fixation period of 96 hours, the right foot underwent micro-CT detection. The right ankle joint was positioned in the fixator along the long axis. The scanning parameters were set to a voltage of 70 kV and a power of 7 W, with 4 frames superimposed. The angle gain was set to 0.72 degrees, and the exposure time was 100 ms, completing the micro-CT scan after one full rotation.

Immunohistochemical (IHC) staining

The left ankle joint tissues of mice were dissected, fixed, and sectioned. Subsequently, tissue sections were incubated with specific primary antibodies against TNF-α followed by IHC staining to visualize the expression and spatial distribution of TNF-α within the tissue architecture. Finally, the stained sections were systematically examined under a light microscope equipped with image analysis software (e.g., ImageJ; National Institutes of Health, Bethesda, MD, USA) for the quantitative assessment of TNF-α expression levels and precise localization within distinct tissue compartments.

HE staining

The left ankle joints of mice were harvested and fixed in 4% paraformaldehyde solution at 4 ℃ for 24 hours. Following fixation, the specimens were decalcified using ethylenediaminetetraacetic acid (EDTA) bone decalcification agent, embedded in paraffin, and sectioned with a microtome. Tissue sections (5 μm thickness) were prepared and stained with HE for histopathological observation under light microscopy.

Histopathological alterations were evaluated according to established criteria (23). The severity of joint tissue damage was graded as follows: Grade 0, normal architecture; Grade 1, mild synovitis with proliferative membrane formation, without inflammatory infiltration; Grade 2, moderate synovitis without pannus formation, accompanied by localized bone and cartilage destruction; Grade 3, severe synovitis with pannus formation, extensive bone and cartilage erosion, and disruption of joint integrity. Two independent investigators performed blinded histological scoring, and the mean value of both scores was calculated for statistical analysis.

SafO staining

The left ankle joints of mice were processed into paraffin sections and subjected to SafO staining. In this histological analysis, basophilic cartilage components bound to Safranin O appeared red, whereas acidophilic cartilage matrix combined with Fast Green exhibited green or blue coloration, creating distinct chromatic contrast between cartilage and osseous tissues. Cartilage erosion in arthritic joints was graded according to established criteria: Grade 0 indicated normal architecture without pathological changes; Grade 1 featured mild synovitis with hyperplastic synovial membrane; Grade 2 demonstrated joint space narrowing accompanied by loss of non-calcified cartilage layer, with cartilage matrix depletion extending through two-thirds of the middle zone; Grade 3 exhibited complete cartilage denudation exposing the calcified layer, coupled with structural joint destruction and morphological alterations at the calcification front (24). Two independent investigators performed blinded histological evaluations, with final scores representing the mean values from both assessments to ensure objective quantification.

ELISA

Following blood collection from the mice, the samples were allowed to clot at room temperature for 4 hours and subsequently centrifuged at 1,500 rpm for 15 minutes at 4 ℃ using a high-speed centrifuge. The resulting supernatant was carefully aspirated into sterile EP tubes and immediately stored at –80 ℃ until analysis. Serum concentrations of TNF-α, IL-1β, IL-17A, and IL-10 were quantitatively determined using commercially available ELISA kits, with all procedures rigorously adhering to the manufacturer’s standardized protocols to ensure experimental consistency and data reliability.

Transcriptome sequencing and bioinformatics analysis

Cartilage tissues were collected from the NC, MO, and ST groups for research and analysis. The construction of libraries, messenger RNA sequencing (mRNAseq), and bioinformatics analysis were conducted by Shanghai Meiji Biomedical Technology Co., Ltd. The overall workflow was as follows: RNA extraction and detection; messenger RNA (mRNA) enrichment, fragmentation, and reverse transcription into complementary DNA (cDNA); cDNA end repair, A-tailing, and the addition of sequencing adapters; and polymerase chain reaction (PCR) enrichment for library construction. RNA sequencing was performed using the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). Sample quality was first assessed, and a sample was considered qualified only if it met the following quality control requirements: the average error rate (%) of sequenced bases was less than 0.1%, a quality score of 20 (Q20) was higher than 85%, and a quality score of 30 (Q30) was higher than 80%. Cluster analysis was subsequently performed to evaluate gene expression correlations. Using P<0.05 and a fold change of 1.5 as the threshold for significant differential expression, Gene Ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted on the differentially expressed gene (DEG) transcripts. Finally, target genes were selected from the DEGs for protein network interaction analysis.

Immunofluorescence staining

The paraffin-embedded knee joint sections were initially dewaxed by baking in a 65 ℃ oven for 2 hours, followed by sequential immersion in xylene I and xylene II for 15 minutes each to completely remove residual paraffin. Tissue rehydration was achieved through a graded ethanol series (100%, 95%, 80%, 70%) with 5-minute incubations at each concentration. Endogenous peroxidase activity was quenched by incubating with 3% hydrogen peroxide solution at room temperature under light-protected conditions for 10 minutes. Antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0) heated to boiling point in a microwave at medium power, followed by maintenance at sub-boiling temperature for 8 minutes before natural cooling to room temperature. After three 5-minute phosphate-buffered saline (PBS) washes, non-specific binding sites were blocked with 5% normal goat serum in PBS through a 30-minute room temperature incubation. Sections were then sequentially treated with a 1:200 dilution of LC3B primary antibody (overnight incubation at 4 ℃) and fluorescently-labeled secondary antibody (1-hour room temperature incubation protected from light). Nuclear counterstaining was achieved using 4’,6-diamidino-2-phenylindole (DAPI) working solution (1 μg/mL) with 5-minute room temperature incubation. Mounted sections using glycerol-based anti-fade mounting medium were cured under light-protected conditions prior to fluorescence microscopy analysis. Five randomly selected high-power fields (200× magnification) were captured for quantitative assessment of LC3B protein expression levels through fluorescence intensity measurement using ImageJ software, with relative expression levels calculated based on normalized fluorescence values.

Western blot (WB)

The glass plates were thoroughly cleaned with deionized water and absolute ethanol, with particular attention paid to inspecting the integrity of edge sealing strips to prevent gel leakage. A 12% separating gel was prepared by mixing 30% acrylamide stock solution, Tris-HCl buffer, and ammonium persulfate/tetramethylethylenediamine (APS/TEMED) polymerization agents, which was then injected into the interplate space and immediately overlaid with distilled water to exclude oxygen. After complete polymerization (40 min), the water layer was removed and replaced with 5% stacking gel containing lower-concentration acrylamide in Tris-HCl buffer, into which a 1.0-mm comb was inserted to create sample wells. Following gel polymerization, the assembled cassette was mounted in a vertical electrophoresis chamber filled with Tris-glycine running buffer (pH 8.3). Protein samples (10 μg per lane) were loaded and electrophoresed initially at 80 V constant voltage until the bromophenol blue front entered the separating gel, then increased to 120 V for continued separation. For membrane transfer, polyvinylidene fluoride (PVDF) membranes were activated in methanol for 30 seconds and assembled with gel and filter papers into a transfer sandwich within a semi-dry transfer apparatus. Transfer was conducted at 100 mA constant voltage for 120 minutes under ice-cooled conditions. The membranes were subsequently blocked with 5% skimmed milk-Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 hours, followed by target region excision guided by pre-stained protein markers (e.g., PageRuler™, Thermo Scientific, Waltham, MA, USA). Sequential incubations were performed with primary antibody (1:1,000 dilution, 4 ℃ overnight) and horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000 dilution, room temperature, 2 h), with three 10-minute TBST washes after each incubation. Protein signals were visualized using enhanced chemiluminescence (ECL) substrate (e.g., Immobilon, Millipore, Burlington, MA, USA) and captured through a chemiluminescence imaging system. Band intensity quantification was performed using Image Lab software with normalization to GAPDH as internal reference, followed by statistical analysis through one-way analysis of variance (ANOVA) in GraphPad Prism (GraphPad Software, Dan Diego, CA, USA).

Data analysis

Statistical analysis was conducted using SPSS 28.0 software (IBM Corp., Armonk, NY, USA). The results are presented as mean ± standard deviation (SD). For data that met the assumptions of normal distribution and homogeneity of variances, ANOVA was employed to assess statistical significance. Multiple sets of experimental data were evaluated using one-way ANOVA. For data that did not follow a normal distribution, the non-parametric Kruskal-Wallis rank sum test was utilized for analysis. A P value of less than 0.05 was considered indicative of a statistically significant difference. The intervention was administered every other day for a duration of 28 days.

Results

Effect of MAI on paw swelling and AI in CIA mice

The results indicated that the weight of mice in the model group was lower than that of the control group. However, the difference in body weight between the MAI group and the model group was not statistically significant (Figure 1A). Treatment with MAI significantly alleviated arthritis symptoms (Figure 1B-1E). Starting from day 32, both the AI score and sole swelling in the MAI group and the MTX group were significantly lower than those observed in the CIA group. The effects of the MAI via subcutaneous administration were particularly pronounced, demonstrating a statistically significant difference (P<0.05).

Figure 1 Effect of MAI on symptoms in CIA mice. (A) Body weight. (B) AI score. (C) SJC score. (D) Hind paw thickness. (E) Ankle diameter. All data are expressed as mean ± SD (n=8). Compared to the NC group: ^##, P<0.01; compared to the MO group: *, P<0.05; **, P<0.01. NC, normal control group; MO, CIA model group; ST, subcutaneous MAI group; IT, muscle MAI group; MTX, methotrexate group. AI, arthritis index; CIA, collagen-induced arthritis; MAI, melittin acupoint injection; SD, standard deviation; SJC, swollen joint count.

MAI can improve arthritis symptoms in CIA mice

Micro-CT and histological scores were employed to evaluate the impact of MAI on bone destruction in CIA mice. Figure 2A illustrates the efficacy of MAI on bone and joint destruction. Compared to the NC group, the MO group exhibited a rough bone surface, reduced bone density, severe bone destruction, and narrowed joint space. IHC staining of TNF-α protein in the right ankle joint of the mice revealed that the MO group exhibited a substantial increase in TNF-α protein expression compared to the NC group (Figure 2B, P<0.01).

Figure 2 Effect of MAI on ankle joint and cartilage pathology in CIA mice. (A,E) Representative micro-CT three-dimensional images of the ankle joint (n=3) along with Micro-CT analysis results. (B,F) IHC staining of the ankle joint (magnification ×100) and the percentage of TNF-α positive cells. (C,G) HE staining of the ankle joint (magnification ×100) and the histopathological inflammation score. (D,H) SafO staining of the ankle joint (magnification ×100) and the corresponding histopathological inflammation score. Results in parts (E-H) are expressed as mean ± SD (n=8). ^##, significance compared with the NC group (P<0.01); significance compared with the MO group: *, P<0.05; **, P<0.01. NC, normal control group; MO, CIA model group; ST, subcutaneous MAI group; IT, intramuscular MAI group; MTX, methotrexate group. CIA, collagen-induced arthritis; CT, computed tomography; HE, hematoxylin and eosin; IHC, immunohistochemical; MAI, melittin acupoint injection; SafO, saffron O-fast green; SD, standard deviation.

The pathological changes in the joint tissue were observed and scored under a microscope. HE staining revealed that the joint cavity of the MO group contained numerous tissue fragments (Figure 2C). The cartilage surface appeared uneven, with instances of connective tissue eroding down to the subchondral bone. The original structure of the underlying bone was lost, replaced by proliferating connective tissue, which exhibited significant inflammatory cell infiltration, pronounced synovial proliferation and thickening, as well as severe connective tissue hyperplasia. The results from SafO stained ankle joint tissue sections (Figure 2D) corroborated these findings. In the MO group, cartilage tissue was stained red, osteogenic tissue green, and an increased number of tissue fragments were observed in the joint cavity, alongside substantial cartilage tissue loss and erosion, indicating severe degradation and destruction of articular cartilage. Conversely, cartilage damage in the ST, IT, and MTX groups was less pronounced, suggesting that both MAI and MTX can mitigate articular cartilage erosion.

Meanwhile, the pathological scoring chart also showed the same results. In the BS/BV chart, both MAI and MTX treatments were effective in reducing joint destruction, particularly in the ST group (Figure 2E). Furthermore, following MAI and MTX intervention, TNF-α protein expression levels decreased in comparison to the MO group (both P<0.01), with the ST group demonstrating the most pronounced effects (Figure 2F). The results shown in the pathological scoring charts for HE staining and SafO staining were similar—both MAI and MTX effectively reduced these histological severity scores compared to the MO group (Figure 2G,2H).

The results indicate that MAI can mitigate joint pathological damage in mice with CIA.

MAI can inhibit serum levels of pro-inflammatory cytokines while increasing levels of anti-inflammatory cytokines in CIA mice

The imbalance between pro-inflammatory and anti-inflammatory cytokine activities is widely regarded as a contributing factor in the induction of RA autoimmunity (25). Various pro-inflammatory factors, such as IL-1β, TNF-α, and IL-17A, can induce and exacerbate inflammation in RA, leading to cartilage damage and bone destruction; conversely, IL-10 has a protective role, reducing inflammation and the degree of bone erosion in RA. Thus, we investigated whether MAI has a regulatory effect on the function of pro- and anti-inflammatory cytokines. As illustrated in Figure 3A-3C, serum levels of IL-1β, TNF-α, and IL-17A in the MO group increased, whereas MAI and MTX significantly reduced the levels of these cytokines. Compared to the NC group, serum levels of IL-4 and IL-10 in the MO group were significantly lower (P<0.01). Furthermore, when compared to the MO group, serum IL-10 levels in the MAI group and the MTX group increased significantly (P<0.01), with statistical significance also noted (P<0.05). Notably, three different doses of MAI and positive control drugs elevated IL-10 levels (Figure 3D). These results indicate that MAI can modulate the secretion levels of pro- and anti-inflammatory cytokines in CIA models.

Figure 3 Effect of MAI on the expression of inflammatory cytokines. (A) TNF-α, (B) IL-1β, (C) IL-17A, and (D) IL-10 levels in the serum of mice following treatment. Data are presented as mean ± SD (n=8). Comparison with the NC group: ^##, P<0.01; Comparisons with the MO group: *, P<0.05; **, P<0.01. NC, normal control group; MO, CIA model group; ST, subcutaneous MAI group; IT, intramuscular MAI group; MTX, methotrexate group. CIA, collagen-induced arthritis; IL, interleukin; MAI, melittin acupoint injection; SD, standard deviation; TNF, tumor necrosis factor.

MAI treatment influences the expression of genes associated with the PI3K/AKT/mTOR differentiation pathway

Based on the aforementioned research findings, we selected the MEL (ST) group as representative for comparative ankle joint mRNA sequencing studies with the NC and MO groups. mRNA sequencing was performed on triplicate samples from each group. All samples met stringent quality control criteria (Table 1), ensuring the reliability of subsequent experimental results and data analyses.

Cluster analysis revealed distinct expression patterns among the three groups. Heatmap visualization (Figure 4A) demonstrated close similarity in transcriptional profiles between the NC and ME groups, whereas the MO group exhibited inverse expression trends compared to both the NC and ME groups. This divergence supports the therapeutic efficacy of MAI intervention, as post-treatment gene expression patterns in the ST subgroup progressively normalized toward NC group levels, a phenomenon absent in the MO group.

Figure 4 Activation of autophagy by MAI in CIA mice (n=3). (A) Heat map. (B) GO annotation analysis. (C) Top 20 KEGG pathway enrichment analysis. MO, CIA model group; NC, normal control group; ME (ST), subcutaneous MAI group. CIA, collagen-induced arthritis; DEGs, differentially expressed genes; GO, Gene Ontology; IL, interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAI, honey bee toxin acupoint injection; SD, standard deviation; TNF, tumor necrosis factor.

Screening for DEGs using thresholds of P<0.05 and fold change ≥1.5 identified 1,021 significant DEGs between the MO and NC groups (491 upregulated, 530 downregulated). MAI intervention induced substantial transcriptomic remodeling, with 139 upregulated and 508 downregulated genes observed in the ME group compared to the MO group (Figure 4B). KEGG enrichment analysis of the top 20 pathways (Figure 4C) revealed critical involvement of osteoclast differentiation, extracellular matrix (ECM)-receptor interaction, PI3K/AKT signaling, TNF signaling, IL-17 signaling, and calcium signaling pathways—all mechanistically linked to autoimmune and inflammatory processes. Focused analysis on autophagy-related pathways identified 27 target DEGs (21 upregulated, 6 downregulated) associated with autophagy activation (Table 2).

GO annotation categorized these DEGs into three functional domains: biological processes (BP) primarily involved cellular regulation and metabolic activities; cellular components (CC) highlighted organelle and membrane structures; molecular functions (MF) emphasized catalytic activity and protein binding (Figure 5). Protein-protein interaction (PPI) network analysis via the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database revealed extensive functional connectivity among 26 autophagy-related genes, including IL-10, mTOR, STAT3, Atg7, Atg4a, ULK1, Akt1, GSK3β, and Mapk8 (Figure 6). Notably, IL-10ra displayed minimal interaction with other network components.

Figure 5 Volcano plot of differential genes. P<0.05, fold change =1.5. (A) NC vs. MO volcano diagram; (B) NC vs. ME volcano diagram; (C) MO vs. ME volcano diagram. MO, CIA model group; NC, normal control group; ME, subcutaneous MAI group. CIA, collagen-induced arthritis; FC, fold change; MAI, honey bee toxin acupoint injection.

Figure 6 Protein-protein interaction network analysis of differentially expressed genes within the autophagy pathway in CIA mice. CIA, collagen-induced arthritis.

Our findings demonstrate that MAI-mediated activation of autophagy pathways predominantly involves PI3K/AKT/mTOR signaling modulation. The data collectively support the hypothesis that MAI ameliorates CIA in mice by suppressing PI3K/AKT/mTOR signaling to activate autophagy, providing a mechanistic foundation for its therapeutic effects in autoimmune arthritis.

MAI can inhibit the PI3K/AKT/mTOR signaling pathway and promote autophagy in the ankle joints of CIA mice

High-resolution microscopy revealed that MAI intervention markedly upregulated the fluorescence intensity of LC3B (Figure 7A,7B). The WB bands showed that in the MAI intervention group, the activity of autophagy-related factors was increased, whereas the activity of the PI3K/AKT/mTOR pathway was decreased (Figure 7C,7D). WB analysis further demonstrated that MAI intervention significantly suppressed the expression levels of p-mTOR, PI3K, and p-Akt pathway (Figure 7E-7G; P<0.05). Figure 7H shows that in the MAI intervention group, there was significant downregulation of P62 protein levels in the treatment group compared to the normal model group, a degradation pattern strongly associated with effective activation of the autophagolysosomal pathway. Notably, MAI intervention not only enhanced the expression levels of autophagy-related proteins Beclin-1 and ULK1 (Figure 7I,7J) but also induced a substantial increase in the LC3-II/I ratio (Figure 7K; P<0.05), collectively establishing a comprehensive molecular signature of autophagic activation. These results collectively suggest that MAI exerts its biological effects through inhibition of the PI3K/AKT/mTOR signaling axis in FLSs, while concomitantly promoting autophagic flux via coordinated regulation of multiple regulatory nodes in the autophagy cascade.

Figure 7 The effect of MAI on autophagy and the PI3K/AKT/mTOR signaling pathway in RA FLSs. Panels A and B present results from immunofluorescence staining (magnification ×200), which was employed to assess the impact of MAI on LC3B expression within the synovial tissue of CIA mice. Additionally, WB (C-K) was utilized to evaluate the expression levels of autophagy-related and PI3K/AKT/mTOR signaling pathway proteins in the joint tissues of CIA mice, with GAPDH serving as the normalization control. Data are expressed as mean ± SD from three independent experiments. Compared to the control group: ^#, P<0.05; ^##, P<0.01; compared to the model group: *, P<0.05; **, P<0.01. ST, subcutaneous MAI group; IT, intramuscular MAI group; MTX, methotrexate group. CIA, collagen-induced arthritis; DAPI, 4’,6-diamidino-2-phenylindole; FLSs, fibroblast-like synoviocytes; WB, western blot; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAI, mellitin acupoint injection; RA, rheumatoid arthritis; SD, standard deviation.

Discussion

Main findings and their implications

RA is a chronic autoimmune disease driven by abnormal proliferation of FLSs and autophagic dysfunction, characterized by joint destruction closely associated with synovial inflammation and chondrocyte death (26). Abnormal excessive proliferation of FLSs and autophagic dysfunction further induce synovial hyperplasia and decreased autophagic activity of chondrocytes, ultimately leading to cartilage destruction and bone damage, forming a vicious cycle that accelerates RA progression (12,27). BV acupuncture improves RA joint symptoms through the synergistic anti-inflammatory and analgesic effects of acupuncture and BV, but its BV components may cause adverse reactions such as local hyperalgesia and swelling (28), and there is a need to optimize the dosage and administration strategy to balance efficacy and safety. The main components of BV peptides include MEL, bee venom phospholipase A2 (bvPLA2), and apamin, among which MEL accounts for 40–60% of the total (29), making it an effective anti-arthritic drug.

ST36 acupoint belongs to the Stomach Meridian of Foot Yangming in TCM, with the effects of dispelling wind dampness and dredging meridians and collaterals, and is a commonly used acupoint for the treatment of RA. Studies have shown that MA applied to the ST36 acupoint can enhance the gene expression of tissue repair growth factors in the inflamed joints of AIA mice, with a significant therapeutic effect on arthritis symptoms (19). Injection through the ST36 acupoint can synergize the “wind-dispelling and collateral-dredging” effect of acupuncture with drug activity; therefore, we selected MAI at ST36 as the treatment regimen.

Although there are currently dedicated drugs for the treatment of RA, most of them need to be taken for a long time, have significant side effects, and can only alleviate the progression of the disease. In recent years, numerous studies have found that TCM has a significant therapeutic effect on RA (30). For example, paeoniflorin can downregulate the nuclear factor-kappa B (NF-κB) signaling pathway and inhibit osteoclast differentiation, thereby preventing bone destruction and alleviating arthritis symptoms (31). Vanillic acid can improve arthritis in CIA mice by inhibiting the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways to suppress the inflammatory response (32). In our experiment, we selected MTX as the positive control drug and found that MAI could achieve better effects. This study demonstrated that MAI can reduce the degree of joint swelling, improve cartilage erosion and bone destruction, inhibit synovial hyperplasia and inflammatory cell infiltration, decrease the levels of inflammatory factors, and increase the levels of anti-inflammatory factors, indicating that MAI is indeed an effective therapy for RA.

Recent studies have found that the combined treatment with modified acupuncture needles and BV protein hydrogel restores T helper 17 cell/regulatory T cell (Th17/Treg)-mediated immune balance, reduces the release of inflammatory factors (TNF-α, IL-6, IL-1β), and alleviates RA symptoms (33). This therapy also combines the dual effects of drugs and acupuncture, similar to our treatment method. In our experiment, ELISA results showed that after intervention with MAI, the levels of inflammatory factors in mice in the treatment group decreased significantly, and the levels of anti-inflammatory factors increased, indicating that MAI can effectively control the release of inflammatory factors, thereby alleviating RA symptoms.

Autophagy plays a complex and multifaceted role in the pathogenesis of RA, involving multiple links such as the initiation of synovial inflammation, abnormal activation of immune cells, progression of joint destruction, and regulation of treatment response. In the pathological environment of RA, the functional state of this process changes significantly; it may play a protective role by removing harmful substances, or it may exacerbate the disease process due to overactivation or dysfunction (34). Regulating autophagy levels is an important pathway for the treatment of RA. Multiple studies have shown that under physiological conditions, the PI3K/AKT/mTOR signaling pathway plays a key role in multiple aspects of cell growth and survival (35). Autophagy is closely related to this pathway (36). This pathway can be activated by growth factors, nutrients, and energy, and inhibits autophagy through negative regulation, playing an important role in cell proliferation (37). The activation of mTOR reduces the level of the downstream molecular complex ULK1, leading to negative regulation of autophagy. As a core mechanism of autophagy regulation, inhibition of mTOR activity leads to dephosphorylation of ULK1, thereby activating the autophagic process. In addition, the PI3K/AKT/mTOR signaling pathway hinders the fusion of autophagosomes and lysosomes by inhibiting the production of LC3-II, thereby reducing autophagy levels (38-40).

Our mRNA transcriptomics experiment performed whole-genome sequencing on joint tissues from the NC group, MO group, and ME group. It was found that the gene expression profile of the ME group was highly similar to that of the NC group, whereas the MO group showed a significant differential gene expression pattern. This trend suggests that MEL can effectively reverse the gene expression disorder in CIA mice and restore a molecular regulatory network close to that of the physiological state. Through GO functional annotation and KEGG pathway enrichment analysis, we screened the most critical signaling pathway, PI3K/AKT/mTOR, from autophagy-related differential genes as the core regulatory axis. This pathway showed a significant activation state in the MO group, whereas its activity was significantly inhibited in the ME group. This finding forms an important echo with a study on osteoarthritis (OA): the OA model also showed that excessive activation of the PI3K/AKT/mTOR pathway leads to decreased autophagic activity, thereby accelerating cartilage degeneration and the release of inflammatory factors (40). This indicates that the PI3K/AKT/mTOR signaling pathway plays an important role in the treatment of CIA mice with MAI.

Many drugs exert therapeutic effects on RA through this pathway. For example, Astragalus polysaccharides treat RA by inhibiting PI3K/AKT/mTOR to enhance autophagy, and inhibiting the growth and pro-inflammatory response of FLSs cells stimulated by IL-1β (33). Celastrol can inhibit the PI3K/AKT/mTOR signaling pathway and activate autophagy to treat RA (41). These conclusions are similar to the results of our experiment. The results of immunofluorescence and WB experiments on the knee joint synovial tissue of mice showed that MAI can treat RA by inhibiting the activity of the PI3K/AKT/mTOR pathway in FLSs, thereby activating and increasing the levels of autophagy-related factors.

Study strengths and limitations

The core advantage of MAI lies in the innovative integration of acupoint synergism and precise attenuation of components: by selecting MEL, the core active component of BV (accounting for 40–60% of the dry weight of BV), to replace whole BV, the risk of sensitizing components such as bvPLA2 is avoided, and a safe administration dose has been established based on previous hepatotoxicity and nephrotoxicity experiments (11); Meanwhile, MAI combines the meridian-dredging effect of the ST36 acupoint with the pharmaceutical action of MEL, inhibits the PI3K/AKT/mTOR signaling pathway, activates autophagy, and achieves multi-level treatment of suppressing synovial inflammation and delaying bone erosion. This therapy breaks through the limitation of single targets of traditional drugs and combines meridian theory with autophagy regulation to form a new paradigm of “component optimization-acupoint synergy”.

The limitations include that the risk of adverse reactions to local injection still needs clinical verification, the number of animal samples is small, which may affect the statistical significance and universality of the results, the difference between the animal model and the human disease course, and the insufficient depth of autophagy mechanism research, requiring further exploration and optimization of the scheme.

Future research perspectives

The above results indicate that MAI has potential roles in the prevention and treatment of RA. Future studies should focus on improving the standardized treatment protocols of MAI and its clinical translation. Moreover, it is unclear whether MAI treats RA through other pathways, and its mechanism of action remains to be further studied.

Conclusions

The current research results provide substantial evidence that MAI induces autophagy by inhibiting the activity of the PI3K/AKT/mTOR pathway, thereby playing a protective role in RA. Furthermore, MAI appears to exert a more pronounced therapeutic effect when administered via subcutaneous injection. Consequently, our study offers preclinical evidence supporting MAI as a novel strategy for the treatment of RA.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-540/rc

Data Sharing Statement: Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-540/dss

Funding: This study was supported by the National Natural Science Foundation of China (No. 81873382), the Natural Science Foundation of Guangdong Province (No. 2024A1515011109), and the Scientific Research Project of Guangdong Provincial Administration of Traditional Chinese Medicine (No. 20241196) to L.Y.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-540/coif). L.Y. reports the funding from the National Natural Science Foundation of China (No. 81873382), the Natural Science Foundation of Guangdong Province (No. 2024A1515011109), and the Scientific Research Project of Guangdong Provincial Administration of Traditional Chinese Medicine (No. 20241196). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were conducted under the approval of a project license (No. SMUL202308014) issued by the Ethics Committee of Southern Medical University, and strictly adhered to the guidelines for the care and use of laboratory animals established by Southern Medical University.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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