Toll样受体4促进子宫腺肌病子宫内膜间质细胞增殖、迁移和侵袭的机制研究

The mechanisms of toll-like receptor 4 in promoting the proliferation,migration and invasion of endometrial stromal cells in adenomyosis

目的探讨Toll样受体4(TLR4)/核因子-κB(NF-κB)通路对子宫腺肌病子宫内膜间质细胞(ESCs)通道蛋白4(aquaporins 4,AQP4)表达的影响,并阐明其可能的分子机制。方法分离并培养原代ESCs,将细胞分为对照组、脂多糖组、TAK-242组,按照分组加入TLR4激活剂脂多糖和抑制剂TAK-242。另外将一批ESCs分为对照组、脂多糖组、脂多糖+si-NC组、脂多糖+si-AQP4组,按照分组,进行si-NC和si-AQP4细胞转染。细胞转染48 h后,向细胞中添加脂多糖。Western blot检测细胞TLR4、p-p65、p65和AQP4蛋白表达;qRT-PCR检测细胞AQP4 mRNA表达;细胞增殖、迁移和侵袭水平通过CCK-8、细胞划痕和Transwell实验测定;细胞划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力。结果成功分离并传代培养ESCs。与对照组相比,TAK-242组ESCs中AQP4 mRNA表达水平和AQP4、TLR4和p-p65/p65蛋白表达水平明显降低(P<0.05),细胞增殖能力、划痕愈合率和侵袭细胞数明显降低(P<0.05);与对照组相比,脂多糖组和脂多糖+si-NC组ESCs中AQP4 mRNA表达水平和AQP4、TLR4、p-p65/p65蛋白表达水平明显升高(P<0.05),细胞增殖能力、划痕愈合率和侵袭细胞数明显升高(P<0.05);与脂多糖组和脂多糖+si-NC组相比,脂多糖+si-AQP4组ESCs中AQP4 mRNA和蛋白表达水平明显降低(P<0.05),而TLR4和p-p65/p65蛋白表达水平差异无统计学意义(P>0.05),细胞增殖能力、划痕愈合率和侵袭细胞数明显降低(P<0.05)。结论通过激活TLR4/NF-κB通路上调AQP4的表达可以促进子宫腺肌病ESCs增殖、迁移和侵袭。

Objective To investigate the effects of toll-like receptor 4(TLR4)/nuclear factor-kB(NF-kB)pathway on the expression of aquaporins 4(AQP4)in adenomyosis endometrial stromal cells(ESCs)and elucidate the possible molecular mechanisms.MethodSs Primary ESCs were isolated and cultured,the cells were divided into control group,lipopolysaccharide(LPS)group and TAK-242 group,and TLR4 activator LPS and inhibitor TAK-242 were added according to the group.In addition,another goups of ESCs were divided into control group,LPS group,LPS+si-NC group,LPS+si-AQP4 group,and transfected si-NC and si-AQP4 according to the grouping.After 48 h the cells were transfected,LPS was added to the cells.TLR4,P-p65,p65 and AQP4 protein expression was detected by Western blot;qRT-PCR was used to detect the expression of AQP4 mRNA expression in cells;cell proliferation,migration,and invasion levels were measured by CCK-8,cell scratch,and Transwell assays.Results ESCs were successfully isolated and subcultured.Compared with control group,AQP4 mRNA expression levels and AQP4,TLR4 and p-p65/p65 protein expression levels of ESCs in TAK-242 group were significantly decreased(P<0.05),with cell proliferation ability,wound closure rate and invasion cell number were significantly decreased(P<0.05).Compared with control group,the AQP4 mRNA expression levels and the AQP4,TLR4 and p-p65/p65 protein expression levels in the ESCs of the LPS group and the LPS+si-NC group were significantly increased(P<0.05),cell proliferation capacity,wound closure rate and number of invasive cells were significantly increased(P<0.05).Compared with the LPS group and the LPS+si-NC group,the expression levels of AQP4 mRNA and protein in the ESCs of the LPS+si-AQP4 group were significantly reduced(P<0.05),while there was no statistical difference in the expression levels of TLR4 and p-p65/p65 protein(P>0.05).Meanwhile the cell proliferation capacity,wound closure rate and number of invasive cells were significantly reduced(P<0.05).Conclusion Activating the TLR4/NF-kB pathway could promote the proliferation,migration and invasion of adenomyosis ESCs by up-regulating the expression of AQP4.