ObjectiveTo observe the effects of moxibustion at “Shenshu” （BL23） and “Zusanli” （ST36） on the T-cell immunoglobulin and mucin domain-containing molecule 3/galectin-9 （Tim-3/Gal-9） signaling pathway and macrophage polarization in rats with rheumatoid arthritis （RA）， so as to explore the mechanism by which moxibustion ameliorates synovial inflammation in RA.MethodsMale Sprague-Dawley （SD） rats were randomly divided into the blank control， model， moxibustion， and Tim-3 knockdown （Tim-3 KD） group， with 8 rats in each group. The RA model was established by subcutaneous injection of Freund’s complete adjuvant （FCA） into the plantar region. The Tim-3 KD group received a single multi-point subcutaneous injection of Tim-3 lentivirus into the plantar region. Both the moxibustion group and Tim-3 KD group were treated with grain-sized moxibustion at BL23 and ST36， 5 cones per acupoint， once daily， with 6 treatments as one course and 1 day of rest after each course， totaling 3 courses. The thickness of bilateral plantar regions was measured on days 1， 7， 14， 21， 28， and 35 of the experiment. HE staining was used to observe joint pathological changes and pathological scores were evaluated. ELISA was employed to detect the serum contents of interleukin （IL）-4 and vascular endothelial growth factor （VEGF）. Immunofluorescence staining was used to determine the expression of Tim-3， Gal-9， and macrophage polarization-related molecules （CD86， CD206） in synovial tissue.ResultsCompared with the blank control group， the model group showed increased thickness of bilateral plantar regions （P<0.01）， narrowed joint space， proliferation of synovial and fibrous tissues， elevated scores of synovial hyperplasia， fibrous tissue proliferation， and inflammatory cell infiltration （P<0.01）， decreased serum IL-4 content （P<0.05）， increased serum VEGF content （P<0.01）， up-regulated expression of Tim-3， Gal-9， and CD86 （P<0.01， P<0.05） and down-regulated expression of CD206 （P<0.01） in synovial tissue. Compared with the model group， the moxibustion group exhibited reduced thickness of bilateral plantar regions （P<0.01）， alleviated joint space narrowing， decreased proliferation of synovial and fibrous tissues， lowered scores of synovial hyperplasia， fibrous tissue proliferation， and inflammatory cell infiltration （P<0.01）， increased serum IL-4 content （P<0.05）， decreased serum VEGF content （P<0.01）， up-regulated expression of Tim-3， Gal-9， and CD206 （P<0.05， P<0.01） and down-regulated expression of CD86 （P<0.05） in synovial tissue. Compared with the moxibustion group， the Tim-3 KD group showed narrowed joint space， increased score of synovial hyperplasia and inflammatory cell infiltration （P<0.05）， decreased serum IL-4 content， and down-regulated expression of Tim-3， Gal-9， and CD206 （P<0.05， P<0.01）， along with up-regulated CD86 expression （P<0.05） in synovial tissue.ConclusionMoxibustion can ameliorate synovial inflammation in RA rats， and its mechanism may be related to regulating the expression of the Tim-3/Gal-9 signaling pathway， inhibiting M1 macrophage polarization， and promoting M2 macrophage polarization.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovitis. Moxibustion is a natural therapy that exhibits anti-inflammatory bioactivity. However, the mechanism of moxibustion in the treatment of RA still needs further study. Objective: This study aimed to observe the effects of moxibustion on macrophage polarization and the STAT1/miR-155/SOCS1 signaling pathway in rats with RA. Methods: The rats' right hind paws were injected with freundʼs complete adjuvant (FCA) to establish the model of RA. Seven days post-FCA injection, moxibustion therapy was performed on the acupoints of Shenshu (BL23) and Zusanli (ST36) once a day for three weeks. The thickness of the foot pad, histopathological changes and inflammatory cytokines were then analyzed. The Immunofluorescence and Elisa were employed to evaluate the impact of moxibustion on the expression of macrophage's markers and the expression of molecules related to the STAT1/miR-155/SOCS1 signaling pathway. Results: Following the injection of FCA, it was observed that the thickness of the foot pad increased, the joint space narrowed, and a significant number of inflammatory cells infiltrated the area. However, after the moxibustion treatment, these symptoms were improved. Additionally, the levels of IL-23 were down-regulated, while the levels of IL-4, IL-10, and Arginase (Arg)-1 were up-regulated. The polarization balance of macrophages in the RA rats was disrupted, with a stronger polarization towards M1 and a weaker polarization towards M2. Post-moxibustion treatment, the polarization of macrophages was corrected, characterized by an increase in M2-polarized macrophages and a decrease in M1-polarized macrophages. Furthermore, moxibustion inhibited the expression of STAT1 and miR-155 within the STAT1/miR-155/SOCS1 pathway and promoted the expression of SOCS1, which was conducive to regulating the polarization phenotype of macrophages. Conclusion: The results demonstrated that moxibustion regulated the polarization phenotype of macrophages and exhibited anti-inflammatory effects, which may be related to the regulation of macrophage polarization through the STAT1/miR-155/SOCS1 pathway.

OBJECTIVE: To explore whether moxibustion exerts therapeutic effects on rheumatoid arthritis(RA) by regulating the expression of T-cell immunoglobulin and mucin-containing protein-3(TIM-3) and subsequently modulating the macrophage M1 polarization toll-like receptor 4(TLR4)-myeloid differentiation factor 88(My D88)-nuclear factor kappa B(NF-κB) signaling pathway. METHODS: We utilized moxibustion treatment in RA rat models using the Zusanli(ST36) and Shenshu(BL23) acupoints. Hematoxylin and eosin(HE) staining was used to observe the pathological changes of the synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease. Enzyme-linked immunosorbent assay(ELISA) was applied to verify the efficacy of moxibustion in reducing inflammation. Quantitative real-time polymerase chain reaction(q RTPCR) was used to detect the expression of the TIM-3/TLR4-My D88-NF-κB signaling pathway-related molecules, and Western blot was used to detect the contents of synovial NF-κB. RESULTS: We established the Freund's complete adjuvant(FCA)-induced RA model in rats. The expression level of M1 polarization signaling pathway TLR4-My D88-NF-κB and the inflammatory factors interleukin-12(IL-12), tumor necrosis factor alpha(TNF-α), and tumor necrosis factor beta(TNF-β) were significantly increased in the RA model. After moxibustion treatment, the expression level of TLR4-My D88-NF-κB was significantly decreased, and the inflammatory factors IL-12, TNF-α, and TNF-β were decreased, but the expression level was significantly increased in the RA model. When TIM-3 expression was inhibited, the expression level of TLR4-My D88-NF-κB, and the inflammatory factors IL-12, TNF-α, and TNF-β were not suppressed, even after moxibustion treatment. CONCLUSIONS: Moxibustion regulates the key target TIM-3 by acting on the Zusanli(ST36) and Shenshu(BL23) points, thereby inhibiting the M1 polarization of macrophages; that is, it inhibits the TLR4-My D88-NF-κB signaling pathway, and finally achieves alleviation of pathological changes and anti-inflammatory effects.

OBJECTIVE: To test the hypothesis that moxibustion may inhibit rheumatoid arthritis(RA) synovial inflammation by regulating the expression of macrophage migration inhibitory factor(MIF)/glucocorticoids(GCs). METHODS: Fifty male Sprague-Dawley rats were randomly divided into five groups(n = 10 each): blank Control(CON) group, RA Model(RA) group, Moxibustion(MOX) group, MIF inhibitor(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester(ISO-1) group, and Moxibustion+MIF inhibitor ISO-1(MOX+ISO-1) group. Rats in the ISO-1 group and ISO-1+MOX group were intraperitoneally injected with the inhibitor ISO-1. The rats in the RA group, ISO-1 group, MOX group, and ISO-1+MOX group were injected with Freund’s complete adjuvant(FCA) in the right hind footpad to establish an experimental RA rat model. In the MOX group and MOX+ISO-1 group, rats were treated with Moxa. The thickness of the footpads of the rats in each group was measured at three-time points before, after modeling and after moxibustion treatment. The contents of serum MIF, corticosterone(CORT), tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by enzyme-linked immunosorbent assay; and the contents of synovial MIF were detected by Western blot. Hematoxylin-eosin(HE) staining method was used to observe the pathological changes of synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease. RESULTS: Moxibustion was found to reduce the level of MIF and alleviate inflammation in RA rats in this study. In addition, after inhibiting the expression of MIF, the level of CORT increased, and the level of TNF-α decreased. Treating RA rats with inhibited MIF by moxibustion, the level of CORT was almost unchanged, but the level of TNF-α further decreased. The correlation analysis data suggested that MIF was positively related to the expression of TNF-α and negatively correlated with the expression of CORT. CONCLUSION: Reducing MIF to increase CORT and decrease TNF-α by moxibustion treatment in RA. MIF may be a factor for moxibustion to regulate the expression of CORT, but the expression of TNF-α is due to the incomplete regulation of the MIF. This study added to the body of evidence pointing to moxibustion’s antiinflammatory mechanism in the treatment of RA.

To observe the effects of moxibustion on T cells and T cell immunoglobulin and mucin-domain-containing molecule-3/galectin-9 (Tim-3/Gal-9) pathway in rats with rheumatoid arthritis (RA). To further explore the possible anti-inflammatory mechanism of moxibustion in the treatment of RA. Thirty Sprague Dawley rats were randomly divided into three groups, including a control group, an RA model group, and a moxibustion group. An RA model was created through the injection of Freund's complete adjuvant. In the moxibustion group, rats were treated with moxibustion at acupoints of "Shenshu" and "Zusanli." A total of three courses of treatment were conducted. Then the thickness of foot pad was measured, joint pathological changes were observed by hematoxylin-eosin (HE) staining, the proportion of CD4+T and CD8+T in peripheral blood was detected by flow cytometry, the expression levels of Tim-3 and Gal-9 in synovium were detected by polymerase chain reaction (PCR), and the expressions of CD4+T and CD8+T in synovium were detected by immunofluorescence. HE staining showed that the synovial tissue of the control group was smooth and neatly arranged without inflammatory cell infiltration. In the model group, the joint space was narrowed, the synovial tissue had congestion and edema, and a large number of inflammatory cells infiltrated. Compared with the model group, in the moxibustion group, the joint space narrowed with synovium hyperemia and edema, and the level of inflammatory cell infiltration decreased. Flow cytometry showed that compared with the model group, CD4+T expression in the moxibustion group was downregulated, while CD8+T expression was upregulated. PCR results showed that compared with the model group, the expressions of Tim-3 and Gal-9 in the moxibustion group were upregulated. Immunofluorescence results showed that compared with the model group, CD4+T expression in the moxibustion group was decreased, while CD8+T expression was increased. The results demonstrate that moxibustion not only suppressed the expression of CD4+T but also promoted the expression of CD8+T. The anti-inflammatory effect of moxibustion may be related to the regulation of T cell expression through the Tim-3/Gal-9 signaling pathway.

OBJECTIVE: To determine whether moxibustion had an anti-inflammatory effect on rheumatoid arthritis(RA) by regulating Annexin 1 expression and interfering with the phospholipaseA2 signaling pathway. METHODS: Thirty male Sprague-Dawley rats were randomly categorized into five groups(six rats per group): blank control(CON) group, RA model(RA) group, moxibustion(MOX) group, Annexin 1 lentiviral intervention(RNAi-Anxa1) group, and Annexin 1 lentiviral intervention + moxibustion(RNAi-Anxa1 + MOX) group. The rats in the RNAi-Anxa1 and the RNAi-Anxa1 + MOX groups were injected with the lentiviral vector-mediated RNAi-Anxa1 into the rat foot pad. An experimental RA rat model was established by injecting Freund's complete adjuvant(FCA) into the RA, MOX, RNAi-Anxa1, and RNAi-Anxa1 + MOX groups. Rats in the MOX and RNAiAnxa1 + MOX groups received moxibustion treatment. After modeling, using moxibustion “Shenshu(BL23)” and “Zusanli(ST36)”, each point is 5 times, bilateral alternating, once a day, 6 times for a course of treatment, between the courses of rest for a one day. A total of three treatment courses were conducted. Both bilateral pad thicknesses were measured using Vernier calipers on experimental days 1, 7, 14, 21, and 28. The expression of cPLA2α signaling in the synovium of diseased joints was observed using Western blot. The pathology of the rat ankle synovium was observed using hematoxylineosin(HE) staining. Interleukin(IL)-1β, IL-10, prostaglandin E2(PGE2), and leukotriene B4(LTB4) were detected using enzyme-linked immunosorbent assay. RESULTS: Moxibustion increased the levels of Annexin 1 and decreased the inflammatory response in rats with RA. After increasing the expression of Annexin 1, the phosphorylated expression of cPLA2α was inhibited, the serum levels of IL-1β, PGE2, and LTB4 decreased, and the level of IL-10 increased. In moxibustion treated RA rats after the Annexin 1 lentiviral intervention, the serum levels of IL-1β, PGE2, LTB4, and IL-10 were almost unchanged. CONCLUSION: Moxibustion enhanced the negative regulation of the cPLA2α signaling pathway, increased the synovial Annexin 1 expression, inhibited the cPLA2α signaling pathway, indirectly inhibited the expression of downstream inflammatory factors, and played a role in reducing inflammation.

There is a potential link between rheumatoid arthritis (RA) and idiopathic pulmonary fibrosis (IPF). The aim of this study is to investigate the molecular processes that underlie the development of these two conditions by bioinformatics methods. The gene expression samples for RA (GSE77298) and IPF (GSE24206) were retrieved from the Gene Expression Omnibus (GEO) database. After identifying the overlapping differentially expressed genes (DEGs) for RA and IPF, we conducted functional annotation, protein–protein interaction (PPI) network analysis, and hub gene identification. Finally, we used the hub genes to predict potential medications for the treatment of both disorders. We identified 74 common DEGs for further analysis. Functional analysis demonstrated that cellular components, biological processes, and molecular functions all played a role in the emergence and progression of RA and IPF. Using the cytoHubba plugin, we identified 7 important hub genes, namely COL3A1, SDC1, CCL5, CXCL13, MMP1, THY1, and BDNF. As diagnostic indicators for RA, SDC1, CCL5, CXCL13, MMP1, and THY1 showed favorable values. For IPF, COL3A1, SDC1, CCL5, CXCL13, THY1, and BDNF were favorable diagnostic markers. Furthermore, we predicted 61 Chinese and 69 Western medications using the hub genes. Our research findings demonstrate a shared pathophysiology between RA and IPF, which may provide new insights for more mechanistic research and more effective treatments. These common pathways and hub genes identified in our study offer potential opportunities for developing more targeted therapies that can address both disorders.

OBJECTIVE: To explore whether moxibustion exerts therapeutic effects on rheumatoid arthritis (RA) by regulating the expression of T-cell immunoglobulin and mucin-containing protein-3 (TIM-3) and subsequently modulating the macrophage M1 polarization toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-nuclear factor kappa B (NF-kappa B) signaling pathway. METHODS: We utilized moxibustion treatment in RA rat models using the Zusanli (ST36) and Shenshu (BL23) acupoints. Hematoxylin and eosin (HE) staining was used to observe the pathological changes of the synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease. Enzyme-linked immunosorbent assay (ELISA) was applied to verify the efficacy of moxibustion in reducing inflammation. Quantitative real-time polymerase chain reaction (qRTPCR) was used to detect the expression of the TIM3/TLR4-MyD88-NF-kappa B signaling pathway-related molecules, and Western blot was used to detect the contents of synovial NF-kappa B. RESULTS: We established the Freund's complete adjuvant (FCA)-induced RA model in rats. The expression level of M1 polarization signaling pathway TLR4-MyD88-NF-kappa B and the inflammatory factors interleukin-12(IL-12), tumor necrosis factor alpha (TNF-alpha), and tumor necrosis factor beta (TNF-beta) were significantly increased in the RA model. After moxibustion treatment, the expression level of TLR4-MyD88-NF-kappa B was significantly decreased, and the inflammatory factors IL- 12, TNF-alpha, and TNF-beta were decreased, but the expression level was significantly increased in the RA model. When TIM-3 expression was inhibited, the expression level of TLR4-MyD88-NF-kappa B, and the inflammatory factors IL-12, TNF-alpha, and TNF-beta were not suppressed, even after moxibustion treatment. CONCLUSIONS: Moxibustion regulates the key target TIM-3 by acting on the Zusanli (ST36) and Shenshu (BL23) points, thereby inhibiting the M1 polarization of macrophages; that is, it inhibits the TLR4-MyD88-NF-kappa B signaling pathway, and finally achieves alleviation of pathological changes and anti-inflammatory effects. (c) 2024 JTCM. All rights reserved.

The goal of this work was to use bibliometric analysis to help guide future research on macrophage polarization in RA. We looked for studies on macrophage polarization in RA published between January 1, 2000, and December 31, 2022, in the WoSCC database. Research trends and hotspots were shown and assessed using VOSviewer and CiteSpace. A total of 181 articles were gathered. Belgium was among the early adopters of the field. Chinese institutes have produced the most research. Researchers such as Angel Luis Corb, Amaya Puig-Kröger, and Lizbeth Estrada-Capetillo have made major contributions to the field. Frontiers in Immunology has published the most study findings. According to VOSviewer, the most investigated immune cells, biomarkers, and signaling pathways in the previous three years have been "T cells", "gm-csf", and "nf-κb" in that order. We discovered that the most often used terms in the previous three years were "pathway", "oxidative stress", "extracellular capsule" and "nlrp3 inflammasome" using Citespace. We emphasize these concepts in our findings, presenting the exact mechanisms of pathophysiology related to macrophage polarization in RA, as well as current breakthroughs in therapy strategies.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by systemic inflammation, especially synovitis, leading to joint damage. It is important to explore potential biomarkers and therapeutic targets to improve the clinical treatment of RA. However, the potential underlying mechanisms of action of available treatments for RA have not yet been fully elucidated. The present study investigated the potential biomarkers of RA and identified specific targets for therapeutic intervention. A comprehensive analysis was performed using mRNA files downloaded from the Gene Expression Omnibus. Differences in gene expression were analyzed and compared between the normal and RA groups. In addition, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed genes (DEGs). A protein-protein interaction network, Molecular Complex Detection and cytoHubba network were evaluated to identify hub genes. Finally, using an experimental RA rat model induced by Freund's complete adjuvant (FCA), the expression of potential biomarkers or target genes in RA were verified through reverse transcription-quantitative PCR. The results of the mRNA dataset processing revealed 195 DEGs in patients with RA when compared with the healthy controls. Moreover, 10 hub genes were identified in patients with RA and four candidate mRNAs were identified, as follows: Discs large homolog-associated protein 5 (DLGAP5), kinesin family member 20A (KIF20A), maternal embryonic leucine zipper kinase (MELK) and nuclear division cycle 80 (NDC80). Finally, the bioinformatics analysis results were validated by quantifying the expression of the DLGAP5, KIF20A, MELK and NDC80 genes in the FCA-induced experimental RA rat model. The findings of the present study suggested that the treatment of RA may be successful through the inhibition of DLGAP5, KIF20A, MELK and NDC80 expression. Therefore, the targeting of these genes may result in more effective treatments for patients with RA.

OBJECTIVES:To observe the similarities and differences of effects of moxibustion at "Zusanli" (ST36) on target tissues and macrophages polarization in knee osteoarthritis (KOA) and rheumatoid arthritis (RA) rats, and to summarize its efficacy and characteristics.METHODS:Thirty rats were equally and randomly divided into control, KOA, RA, KOA+Moxi and RA+Moxi groups. The KOA model and RA model were induced by injection of sodium monoiodoacetate or Freund's complete adjuvant into the rats' knee joints, respectively. Rats of the KOA+Moxi and RA+Moxi groups received moxibustion stimulation at bilateral ST36 for 30 min, once a day for 21 days, beginning from the 7th day on after modeling. The contents of serum interleukin(IL)-1β and IL-10 were detected by ELISA. Histopathological changes (Markin score of the knee cartilage and synovial pathology score) of the knee joints were observed after HE staining. The polarization state of M1 and M2 macrophages in the synovial tissue of the knee joints was assessed by detecting the expression of CD86 and CD206 after immunofluorescence staining.RESULTS:Compared with the control group, the content of serum IL-1β, synovial pathology score, and synovial CD86 expression were significantly increased (P<0.01, P<0.05), while the content of serum IL-10 and synovial CD206 expression markedly decreased (P<0.01) in both KOA and RA groups；the Markin score was increased (P<0.01) in the KOA group. In comparison with the KOA group, the Markin score was obviously decreased (P<0.01), while the content of serum IL-10 and CD206 expression were apparently increased (P<0.01) in the KOA+Moxi group. No significant changes were found in the content of serum IL-1β, synovial pathology score and CD86 expression in the KOA+Moxi group relevant to the KOA group. In comparison with the RA group, the content of serum IL-1β, synovial pathology score, and CD86 expression were considerably decreased (P<0.01) in the RA+Moxi group. No marked differences were found in the serum IL-10 level, Markin score, and CD206 expression between RA+Moxi and RA model groups. The increased Markin score was significantly higher in the KOA group than in the RA group (P<0.01), but the increased synovial pathology score was significantly lower in the KOA group than in the RA group (P<0.01). Correspondingly, the effect of moxibustion at ST36 was significantly better in RA model than in KOA model in reducing serum IL-1β (P<0.05).CONCLUSIONS:Moxibustion at ST36 can effectively reduce cartilage injury of knee joint in rats with KOA and reduce synovial injury in rats with RA, which may be related with its effects in lowering IL-1β level in RA model by inhibiting the polarization of M1 macrophages, and up-regulating level of IL-10 in KOA model by promoting the polarization of M2 macrophages. However, the relevant mechanism needs to be further studied.

Abstract Background Rheumatoid arthritis (RA) is a refractory autoimmune disease characterized by chronic synovitis. Moxibustion is a natural therapy that possesses anti-inflammatory bioactivity. However, the anti-inflammatory mechanism of moxibustion for the treatment of RA has not yet been clarified. Macrophage infiltration is a prominent characteristic in the pathological process of RA synovitis. Objective The aim of the present research is to investigate the effect of moxibustion on the polarization of macrophage. Methods The rats' right hind paws were injected with freundʼs complete adjuvant (FCA) to establish the model of RA. Seven days after the injection of FCA, moxibustion therapy was performed on the acupoints of Shenshu (BL23) and Zusanli (ST36) once a day for three weeks. The Immunofluorescence, Western bolt and Elisa were used to analyze the influence of moxibustion on the expression of STAT1/miR-155/SOCS1 signaling pathway and macrophage's marker. Results We observed that moxibustion decreased the thickness of foot pad and arthritis scores. Moxibustion up-regulated the exression of Tim-3, promoted the polarization of macrophages to M2 type and inhibited the polarization of macrophages to M1 type. Treatment with moxibustion decreased the level of interleukin (IL)-23 and increased the level of IL-4, IL-10 and Argininase (Arg)-1. Moreover, moxibustion blocked the activation of STAT1/miR-155/SOCS1 signaling pathway and promoted the polarization of macrophages to M2 type. Furthermore, when Tim-3 was knocked down, the anti-inflammatory effect of moxibustion was reduced and the effect of regulating macrophage polarization was inhibited. Conclusion moxibustion may regulate the expression of related molecules in the STAT1/miR-155/SOCS1 signaling pathway through Tim-3, promote the polarization of macrophages M2, and exhibit anti-inflammatory effects.