Fractalkine通过激活p38MAPK信号通路调控狼疮性肾炎小鼠Treg细胞凋亡

Fractalkine regulates Treg cell apoptosis in lupus nephritis mice by activating p38MAPK signaling pathway

目的:探讨趋化因子Fractalkine(FKN)对狼疮性肾炎(LN)小鼠Treg细胞凋亡的影响及机制。方法:构建LN小鼠模型,使用FKN中和抗体(Anti-FKN)、p38MAPK信号通路的抑制剂(SB203580)、p38MAPK信号通路的激活剂(U-46619)作用于正常小鼠及模型小鼠,采用免疫组化检测小鼠肾组织中FKN、磷酸化p38(p-p38)及Foxp3的蛋白表达;采用免疫磁珠法分选模型小鼠脾脏CD4^(+)CD25^(+)Treg细胞,使用腺病毒转染细胞的方式敲低FKN的表达水平,同时使用p38MAPK信号通路抑制剂(SB203580)及激活剂(U-46619)干预细胞。采用Western blot验证FKN敲低的成功转染、检测p38、p-p38、Foxp3、凋亡相关因子(Bax、Bcl-2及Cyt-c)的蛋白表达。结果:LN小鼠肾组织中的FKN及磷酸化p38MAPK的表达明显上调,低表达FKN可降低LN小鼠肾组织及Treg细胞中FKN和p-p38的表达,增加Foxp3的表达,同时增加细胞内抗凋亡因子Bcl-2蛋白的表达,而减少促凋亡因子Bax及Cyt-c蛋白的表达。p38MAPK信号通路抑制剂对上述因子的作用与低表达FKN相似,其激活剂可以逆转这些因子在LN小鼠Treg细胞中的表达。结论:FKN可能通过激活p38MAPK信号通路参与调控LN小鼠Treg细胞凋亡,为阐明LN的发病机制提供了实验依据。

Objective:To investigate the effect of Fractalkine(FKN)on apoptosis of Treg cells in mice with lupus nephritis(LN).Methods:The LN mice model was established.The Anti-FKN,p38MAPK signal pathway inhibitors(SB203580)and activator of p38MAPK signaling pathway(U-46619)were used to act on the normal mice and the model mice.The protein expressions of FKN,phosphorylated p38(p-p38)and Foxp3 were detected by immunohistochemistry;CD4^(+)CD25^(+)Treg cells in spleen were sorted by immunomagnetic bead method,and the expression level of FKN was knocked down by adenovirus transfection at the same time,the p38MAPK signal pathway inhibitor(SB203580)and activator(U-46619)were used to intervene the cells.Western blot was used to verify the successful transfection of FKN knockdown,and the protein expression of p38,p-p38,Foxp3 and apoptosis related factors(Bax,Bcl-2 and Cyt-c)were detected by Western blot.Results:Downregulation of FKN decreased the expression of FKN and p-p38,increased the expression of Foxp3,increased the expression of anti-apoptotic factor Bcl-2,but decreased the expression of pro-apoptotic factors Bax and Cyt-c.The effect of p38MAPK signaling pathway inhibitor on the above-mentioned factors was similar to that of FKN,and its activator could reverse the expression of these factors in Treg cells of LN mice.Conclusion:FKN can regulate the apoptosis of Treg cells in LN mice,which may be involved in the activation of p38MAPK signaling pathway,and provides experimental basis for elucidating the pathogenesis of LN.