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 m...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.展开更多
Objective:To explore the relationship between the changes of miR145‑5p/Smads pathway and macrophage polarization in adjuvant arthritis rats.Methods:Twelve rats were divided into normal group and model group induced by...Objective:To explore the relationship between the changes of miR145‑5p/Smads pathway and macrophage polarization in adjuvant arthritis rats.Methods:Twelve rats were divided into normal group and model group induced by freund's complete adjuvant(0.1 mL/mouse)by random number table method,with 6 rats in each group.The expression of inflammatory polarization markers IL‑8 and CD206 in synovial tissue was detected by enzyme‑linked immunosorbent assay on the 12th day after the formation of arthritis in rats.Western blotting was used to detect the expression of TGF‑β1/Smads pathway factors in synovial tissues.The expression of miR145‑5P,Smads3 and Smads7 in synovial tissue was detected by RT‑qPCR.Results:Compared with normal group,the expression levels of IL‑8,TGF‑β1 and Smad3 in model group were significantly increased(P<0.05);The expression levels of CD206,Smad7 and miR145‑5P were significantly decreased(P<0.01).The correlation results showed that IL‑8 was positively correlated with Smad3(P<0.01),IL‑8 was negatively correlated with Smad7(P<0.05),CD206 was negatively correlated with Smad3(P<0.01)and positively correlated with Smad7(P<0.05).miR145‑5p was negatively correlated with Smad3(P<0.01)and positively correlated with Smad7(P<0.01).Conclusion:miR145‑5p may inhibit the overactivation of TGF‑β1/Smads pathway,regulate macrophage polarization,and inhibit the development of adjuvant arthritis by inhibiting Smad3 expression.展开更多
基金Natural Science Research Project of Colleges and Universities in Anhui Province(No.kj2020a0396)。
文摘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.
基金Natural Science Research Project of Universities in Anhui Province(NO.KJ2020A0396)。
文摘Objective:To explore the relationship between the changes of miR145‑5p/Smads pathway and macrophage polarization in adjuvant arthritis rats.Methods:Twelve rats were divided into normal group and model group induced by freund's complete adjuvant(0.1 mL/mouse)by random number table method,with 6 rats in each group.The expression of inflammatory polarization markers IL‑8 and CD206 in synovial tissue was detected by enzyme‑linked immunosorbent assay on the 12th day after the formation of arthritis in rats.Western blotting was used to detect the expression of TGF‑β1/Smads pathway factors in synovial tissues.The expression of miR145‑5P,Smads3 and Smads7 in synovial tissue was detected by RT‑qPCR.Results:Compared with normal group,the expression levels of IL‑8,TGF‑β1 and Smad3 in model group were significantly increased(P<0.05);The expression levels of CD206,Smad7 and miR145‑5P were significantly decreased(P<0.01).The correlation results showed that IL‑8 was positively correlated with Smad3(P<0.01),IL‑8 was negatively correlated with Smad7(P<0.05),CD206 was negatively correlated with Smad3(P<0.01)and positively correlated with Smad7(P<0.05).miR145‑5p was negatively correlated with Smad3(P<0.01)and positively correlated with Smad7(P<0.01).Conclusion:miR145‑5p may inhibit the overactivation of TGF‑β1/Smads pathway,regulate macrophage polarization,and inhibit the development of adjuvant arthritis by inhibiting Smad3 expression.
