二甲双胍通过AMPK/STAT3信号通路抑制星形胶质细胞的反应性

Metformin inhibits the reactivity of astrocytes through AMPK/STAT3 signaling pathway

目的探讨二甲双胍对体外培养的大鼠脊髓星形胶质细胞反应性的作用及机制。方法使用三因子(TNF-α、IL-1α、C1q)诱导产生反应性星形胶质细胞,RT-qPCR及Westernblot检测补体3(C3)的表达;使用5、10、20 mM浓度的二甲双胍处理,Westernblot检测星形胶质细胞中腺苷酸激活蛋白激酶(AMPK)与信号转导和转录激活因子3(STAT3)磷酸化水平;采用AMPK抑制剂Compound C处理,检测AMPK和STAT3磷酸化水平。结果形态学及RT-qPCR结果显示:与对照组相比,三因子处理改变了星形胶质细胞形态并显著提高了C3的表达(P<0.05),表明成功建立反应性星形胶质细胞模型。RT-qPCR结果及Westernblot结果显示:与对照组相比,二甲双胍对C3表达有明显的抑制作用,呈现浓度依赖性(P<0.05)。在诱导星形胶质细胞反应性过程中,与对照组相比,AMPK的磷酸化水平显著降低(P<0.05),使用不同浓度二甲双胍处理后,AMPK的磷酸化水平显著增加且呈浓度依赖性(P<0.05),同时信号传导及STAT3的磷酸化水平显著降低且呈浓度依赖性(P<0.05)。AMPK抑制剂Compound C处理后显著抑制了二甲双胍导致的AMPK活性升高及STAT3活性降低,同时抑制了C3表达下调(P<0.05)。结论二甲双胍通过AMPK/STAT3信号通路抑制星形胶质细胞的反应性。

Objective The purpose of this study was to investigate the effect and mechanism of metformin on the reactivity of rat spinal cord astrocytes in vitro.Methods The reactive astrocytes were induced by three factors(TNF-α,IL-1α,C1q),and the expression of complement component 3(C3)was detected by RT-qPCR and Western blot.The phosphorylation levels of AMPK and STAT3(signal transduction and transcription activator 3)in astrocytes were detected by Western blot after treatment with different concentrations(5mM,10 mM and 20 mM)of metformin.The Adenosine 5'-monophosphate-activated protein kinase(AMPK)and signal transduction and transcription activator 3(STAT3)phosphorylation(p-AMPK and p-STAT3)levels were detected by an AMPK inhibitor Compound C.Results Morphology and RT-qPCR results showed that compared with the control group,the triple-factor treatment altered the morphology of astrocytes and significantly increased the expression of C3(P<0.05),indicating the successful establishment of a reactive astrocyte model.RT-qPCR and Western blot results showed that compared with the control group,metformin had a significant inhibitory effect on C3 expression,showing concentration dependence(P<0.05).During the induction of reactive astrocytes,compared with the control group,the phosphorylation level of AMPK was significantly decreased(P<0.05).After treatment with different concentrations of metformin,the phosphorylation level of AMPK was significantly increased and showed concentration dependence(P<0.05),while the phosphorylation level of STAT3 was significantly decreased and showed concentration dependence(P<0.05).Treatment with the AMPK inhibitor Compound C significantly suppressed the metformin-induced increase in AMPK activity and decrease in STAT3 activity,and also suppressed the downregulation of C3 expression(P<0.05).Conclusion This study revealed metformin could inhibit three factors(TNF-α,IL-1α,C1q)induced astrocytes activation through AMPK/STAT3 signaling pathway.