二甲双胍通过AMPK/FoxO3a信号通路对创伤性脑损伤后炎症因子的调节作用

Metformin Regulates Inflammatory Factors after Traumatic Brain Injury through AMPK/FoxO3a Signaling

为探究二甲双胍是否通过腺苷单磷酸活化蛋白激酶(AMPK)/FoxO3a途径发挥对创伤性脑损伤(traumatic brain injury,TBI)后神经细胞分泌炎症因子的影响。通过将实验小鼠(雄性,C57BL/6)随机分为二甲双胍组、生理盐水组、复合物C组、野生型组和基因敲除组(FoxO3a敲除小鼠),每组20只。并建立伪手术模型,6、12、24 h的TBI模型。应用蛋白质印迹法(Western Blot)、免疫荧光双染检测的方法,研究了AMPK、FoxO3a、自噬相关蛋白(LC3,P62)和炎症因子(NLRP3,IL-1β)的表达情况,以及LC3和IL-1β的细胞核、细胞质的分布情况。结果表明:与生理盐水组相比较,二甲双胍组p-AMPK/AMPK比值增加,p-FoxO3a/FoxO3a比值降低,自噬水平升高,炎症因子表达降低,而复合物C组则相反;与野生型组相比较,结果提示FoxO3a基因敲除组自噬水平降低,而炎症因子表达水平显著增高。LC3主要表达在细胞质,IL-1β在整个细胞均有分布。可见AMPK的激活和FoxO3a蛋白有利于自噬的发生,二甲双胍可能通过AMPK/FoxO3a信号通路发挥对TBI后炎症因子的抑制作用。

The research aimed to investigate whether the metformin affects the secretion of inflammatory cytokines in neurocytes after traumatic brain injury(TBI).Mice(male C57BL/6)were randomly divided into metformin group,saline group,Compound C group,wild-type group,and knockout group(FoxO3a knockout).Twenty mice were assigned for each group.Then,Pseudo-surgical model,6,12 and 24 hour TBI model were established.Western Blot and immunofluorescence double-staining were used to investigate the expression of AMPK,FoxO3a,autophagy-related proteins(LC3,P62)and inflammatory factors(NLRP3,IL-1β),as well as the distribution of LC3 and IL-1βin the cells.Compared with the saline group,the results show that the ratio of p-AMPK/AMPK and the autophagy level are increased.However,the ratio of p-FoxO3a/FoxO3a and the inflammatory cytokines expression are decreased in the metformin group.Interestingly,the Compound C can reverse the effects of the metformin after TBI.Compared with the wild-type group,the Immunofluorescence staining shows that autophagy level is up-regulated,and the inflammatory factor expression is down-regulated in the knockout group after TBI.It is also found the LC3 are mainly expressed in the cytoplasm,however,the IL-1βare distributed in the whole cell after TBI.It is concluded that both AMPK and the FoxO3a contribute to autophagy after TBI,and the metformin may play an inhibitory role in inflammation after TBI through AMPK/FoxO3a signaling pathway.