连接蛋白43在吡啡尼酮促进小鼠RAW264.7巨噬细胞M2型极化中的作用

Effects of connexin 43 on pirfenidone-induced M2 polarization of mouse RAW264.7 macrophages

目的探讨连接蛋白43(connexin 43,Cx43)是否能够调节吡啡尼酮(pirfenidone,PFD)介导的小鼠RAW264.7巨噬细胞M2型极化。方法将体外培养的对数生长期小鼠RAW264.7巨噬细胞分为对照组(Control组)、PFD组、PFD+Cx43特异性阻断剂Gap19组(PFD+Gap19组)、吡啡尼酮+阴性慢病毒对照组(PFD+NC组)、吡啡尼酮+Cx43过表达慢病毒组(PFD+OE组)。采用慢病毒转染技术,加入含有过表达慢病毒的培养基,使Cx43在巨噬细胞中过表达。采用Western blot技术检测小鼠RAW264.7巨噬细胞Cx43和M2型极化标记物CD206、Arg-1的蛋白表达水平,采用倒置荧光显微镜观察小鼠RAW264.7巨噬细胞I型精氨酸酶(arginase1,Arg-1)和CD206的蛋白表达和定位,采用实时荧光定量PCR(qRT-PCR)技术检测Cx43,Arg-1和CD206的RNA表达水平。结果PFD组中的Cx43蛋白表达与Control组相比降低;PFD组中的M2型极化标志物CD206和Arg-1与Control组相比大幅升高;与PFD组相比,在PFD+Gap19组中Arg-1和CD206的表达均显著升高;与PFD组相比,在PFD+NC组中Arg-1和CD206的表达无明显改变。与PFD+NC组相比,PFD+OE组中Arg-1和CD206的表达均显著降低。结论PFD可以通过下调Cx43促进小鼠RAW264.7巨噬细胞向M2型极化。通过Gap19和过表达慢病毒调节Cx43的表达,也可调节巨噬细胞M2型极化。

This study was designed to investigate the effect of Cx43 on pirfenidone(PFD)-induced M2 polarization of mouse RAW264.7 macrophages.Macrophages were cultured in vitro and were divided into control group,PFD group,PFD+Cx43 specific blocker Gap19 group(PFD+Gap19 group),PFD+negative lentivirus control group(PFD+NC group),and PFD+Cx43 overexpression lentiviral group(PFD+OE group).Cx43 was overexpressed by lentivirus transfection technique.Western blot was used to detect the protein expression levels of Cx43 and M2-type polarization markers CD206 and Arg-1 in mouse RAW264.7 macrophages,while inverted fluorescence microscopy was used to observe the protein expression and localization of Arg-1 and CD206 in mouse RAW264.7 macrophages.Furthermore,the RNA expression levels of Cx43 and Arg-1 were detected by quantitative real-time PCR(qRT-PCR).Data showed that as compared with the control group,Cx43 expression in PFD group was decreased,and the M2-type polarization markers CD206 and Arg-1 were significantly increased.Compared with PFD group,the expression levels of Arg-1 and CD206 in PFD+Gap19 group were significantly increased,while the expression levels of Arg-1 and CD206 in the PFD+NC group were not significantly changed.Compared with PFD+NC group,the expressions of Arg-1 and CD206 in PFD+OE group were significantly decreased.In summary,PFD can promote M2-type polarization of mouse RAW264.7 macrophages by down-regulating Cx43.Regulation Cx43 expression by Gap19 and overexpressed lentvirus can also regulate M2-type polarization of macrophages.