Molecular mechanism of Meihua Shengjin decoction in inhibiting M1 macrophage polarization via METTL3-regulated m6A methylation of STAT1
Objective:To explore the effect of Meihua Shengjin decoction on METTL3-mediated m6A methylation of STAT1,and to elucidate its molecular mechanism in treating primary sjögren's syndrome by modulating macrophage polarization.Methods:An in vitro inflammation model was established using LPS-induced RAW264.7,and Meihua Shengjin decoction-containing serum was administered for intervention.Flow cytometry was used to assess the proportion of M1 macro-phages,ELISA to measure the levels of inflammatory factors(IL-6,IL-23,TNF-α)in cell supernatants,Western blot and RT-qPCR to detect the expression of STAT1,METTL3,and glycolysis-related proteins and genes,and dot blotting to evaluate the level of m6A methylation level of STAT1.Results:Compared with the control group,LPS induction significantly increased the proportion of M1 macrophages and the levels of IL-6,IL-23,and TNF-α in the cell culture supernatant,and upregulated the mRNA and protein expression of METTL3,STAT1,GP1,and ALDOA,with statistically significant differences(P<0.01).Compared with the model group,Meihua Shengjin decoction-containing serum significantly reduced the proportion of M1 macro-phages and the levels of IL-6,IL-23,and TNF-α in the cell culture supernatant,and downregulated the mRNA and protein ex-pression of METTL3,STAT1,GP1,and ALDOA,with statistically significant differences(P<0.05 and P<0.01).Additional-ly,LPS induction significantly increased the m6A methylation modification level of STAT1 compared to the control group(P<0.01),while Meihua Shengjin decoction-containing serum significantly reduced the m6A methylation level in macrophages,with statistically significant differences compared to the model group(P<0.01).Conclusion:Meihua Shengjin decoction inhibits MET-TL3 expression and reduces STAT1 m6A methylation,thereby suppressing M1 macrophage polarization and inflammation by low-ering glycolysis levels in macrophages.
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