首页|Experimental study of TGF-β1/Smads pathway inhibition of macrophage polarization based on miR145-5P negative feedback regulation
Experimental study of TGF-β1/Smads pathway inhibition of macrophage polarization based on miR145-5P negative feedback regulation
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维普
Objective: To investigate the effect of miR145-5P overexpression on the polarization imbalance of synovial macrophages in patients with rheumatoid arthritis (RA). Methods: Human mononuclear cells (THP-1) at logarithmic growth stage were induced into M1-type macrophages, and RA synovial fibroblasts M1-type macrophages were co-cultured into synovial macrophages. Synovial macrophages were divided into four groups :RA group (blank group), TGF-β1 group (model group) and miR145-5P overexpression group (TGF-β1+ miR145-5P mimics group) and miR145-5P overexpression negative control group (TGF-β1+ miR145-5P-mimics -NC group). The blank group did not receive any treatment, and the other three groups were induced by TGF-β1 in the medium for 48 h. Transfection miR145-5p mimic and miR145-5P-mimics-NC were added to co-culture medium, and IL-6, IL-6 and IL-6 of synovial macrophages were detected by ELISA.CD163 expression. Rt-qpcr was used to detect miR145-5p mRNA, TGF-β1mRNA, Smad3mRNA,Smad7mRNA expression level. The expression of TGF-β1/Smads pathway related proteins was detected by Western Blotting. Results: Compared with blank group, IL-6 level was up-regulated (P<0.01) ,and CD163 level was down-regulated in model group(P<0.05) , suggesting that TGF-β1 could induce intensified immune inflammatory response. Compared with the negative miR145-5P overexpression control group and model group, The expression of miR145-5P overexpression group molecule CD163 was significantly increased by ELISA(P<0.01), and the expression of inflammatory factor IL-6 was decreased (P<0.05). PCR showed that miR145-5P mRNA expression level was significantly increased in miR145-5P overexpression group,Smad3 mRNA and TGF-β1 mRNA were significantly decreased, and Smad7 mRNA was significantly increased (P<0.01). WB method showed that the anti-inflammatory protein Smad7 was significantly increased, while TGF-β1 and Smad3 were significantly decreased (P<0.01). Transwell chamber results confirmed that miR145-5P overexpression group significantly reduced macrophage invasion (P<0.01). Correlation analysis showed that miR145-5P was negatively correlated with Smad3 and positively correlated with Smad7 (P<0.01). Conclusion: miR145-5P may inhibit macrophage polarization in RA patients by targeting Smad3 protein, negatively regulating TGF-β1/Smads pathway, and alleviating immune inflammation.
万方数据
维普
