基于调控METTL3介导的m6A甲基化修饰探究针刺治疗类风湿关节炎的机制OA

Objective To observe the mechanism by which acupuncture treats rheumatoid arthritis(RA).Method This study recruited 40 healthy volunteers and 40 RA patients.The symptom score was assessed before and after treatment in the RA patients;the total m6A RNA methylation level in the peripheral blood mononuclear macrophages and the mRNA level of methyltransferase-like 3(METTL3)were detected,and their correlations with the change in the symptom score before and after treatment were analyzed.In vivo,a rat RA model was induced using bovine type Ⅱcollagen and Freund's incomplete adjuvant,followed by acupuncture intervention.The paw swelling degree was measured before and after intervention;hematoxylin-eosin(HE)staining was used to observe the pathological changes in knee joint cartilage tissue,and the symptoms were scored;terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)was adopted to detect apoptosis in rat knee joint cartilage tissue;the expression of METTL3 in rat joint cartilage tissue was observed using immunohistochemistry(IHC),and co-localization of METTL3 and CD68 was visualized by immunofluorescence(IF).In vitro,phorbol ester-induced human monocytic leukemia cells(THP-1)were differentiated into M0 macrophages,followed by polarization into M1 or M2 phenotypes using interferon-γ(IFN-γ)and lipopolysaccharide(LPS),or interleukin-4(IL-4),respectively;M1 and M2 macrophages were co-cultured with human RA fibroblast-like synoviocytes(MH7A),or subjected to METTL3 knockdown or overexpression via plasmid transfection.M1 macrophages were treated with the m6A methylation inhibitor STM2457,while M2 macrophages were treated with the m6A demethylase inhibitor FB23-2.The METTL3 expression level in macrophages was determined using quantitative real-time polymerase chain reaction(qPCR)and Western blot;the total m6A RNA methylation level in rat knee joint cartilage tissue and macrophages was quantified using m6A RNA methylation quantification assay;flow cytometry was used to analyze the ratios of CD68+and CD206+and the apoptosis rate of MH7A in rat knee joint cartilage or in macrophages;the contents of tumor necrosis factor-α(TNF-α)and interleukin-10 in the articular cartilage tissue and macrophages were determined by the enzyme-linked immunosorbent assay;cell counting kit-8(CCK-8)was employed to detect the survival rate of MH7A.Result The clinical trial demonstrated significant reductions in the symptom score after acupuncture intervention in the RA patients,along with reduced METTL3 expression and m6A methylation levels in peripheral blood mononuclear macrophages;there was a positive correlation between the symptom score and the levels of METTL3 expression and m6A methylation before and after intervention.The animal experiments observed that the symptoms improved after acupuncture intervention in RA rats,together with decreased METTL3 expression and m6A methylation levels and improved M1 macrophage polarization in rat knee joint cartilage tissue.In cellular experiments,M1 polarization of macrophages promoted MH7A apoptosis;METTL3 was highly expressed in M1 macrophages,and METTL3 knockdown inhibited m6A methylation in macrophages and M1 polarization while promoting M2 polarization,whereas METTL3 overexpression exerted opposite effects;m6A methylation modification boosted M1 polarization of macrophages.Conclusion Clinical,in vivo,and in vitro evidence primitively suggests that the improving effect of acupuncture on RA may be mediated through the downregulation of METTL3.