二甲双胍调节细胞自噬和焦亡发挥脑缺血再灌注损伤保护作用的机制研究

Mechanism Research of Metformin for Protecting Brain Tissue from Ischemia-Reperfusion Injury by Regulating Autophagy and Pyroptosis

目的研究二甲双胍(metformin,Met)调节细胞自噬和焦亡保护脑缺血再灌注损伤(CIRI)的机制。方法采用线栓法阻塞SD大鼠大脑中动脉2 h后再灌注制备CIRI模型。分两组实验,第1组:采用3个剂量的Met(50、100、200 mg·kg^(-1)·d^(-1),ig)进行Met药效学和血糖评价。第2组:Met对损伤脑组织的细胞自噬和焦亡影响,采用腺苷酸活化蛋白激酶(AMPK)抑制剂化合物C(compound C,Cc,20 mg·kg^(-1)·d^(-1),ip)、自噬抑制剂三甲基腺嘌呤(3-MA,200 mmol·L^(-1)2μL·只-1,icv),caspase-1抑制剂Z-YVAD-FMK(Z-YVAD,8μg·kg-12μL·只-1,icv)分别阻断AMPK通路、自噬和细胞焦亡,考察Met对这些通路和脑损伤的影响。采用Zea-Longa评分评价神经功能,水迷宫实验考查大鼠记忆情况,TTC染色检测脑梗死体积,HE染色观察受损脑组织病理变化,Western blot测定受损脑组织AMPK、p-AMPK、微管相关蛋白1轻链3(LC3-Ⅰ、LC3-Ⅱ)、选择性自噬接头蛋白(p62)、炎症小体NLRP3、pro-caspase-1、半胱氨酸蛋白酶-1(caspase-1)的表达。结果各剂量的Met均使CIRI大鼠的神经功能评分降低,记忆力改善,脑梗死体积减小,脑神经元坏死数降低,对血糖没有影响。Met预处理能显著激活脑中AMPK通路(AMPK表达升高)和自噬活性(LC3-Ⅱ/LC3-Ⅰ升高,p62的表达降低),该效应被Cc抑制,提示Met所致自噬活性升高由AMPK所介导。用3-MA可阻断Met引起的自噬活性和脑损伤缓解作用,对AMPK表达没有影响,说明Met通过自噬发挥保护作用。CIRI大鼠高表达的caspase-1被Z-YVAD阻断,Met与其联用后进一步加强这个作用。说明Met通过抑制炎症小体表达来抑制细胞焦亡。结论Met对缺血再灌注脑损伤有保护作用,其作用机制包括激活AMPK通路,促进细胞自噬,通过自噬去除损伤的线粒体,减少了损伤的线粒体引起的NLRP3炎症小体活化导致的细胞焦亡,从而对缺血再灌注脑损伤起到保护作用。

OBJECTIVE To study the mechanism of metformin for protecting brain tissue from cerebral ischemia-reperfusion injury(CIRI)by regulating autophagy and pyroptosis.METHODS The CIRI model was prepared by perfusion of blood in the middle cerebral artery after 2 h of occlusion in SD rats.Two groups of experiments were performed.In the first group,the therapeutic effect and blood glucose of metformin were evaluated using three doses of metformin(50,100,200 mg·kg^(-1)·d^(-1),ig).In the second group,the effect of metformin on autophagy,pyroptosis and their pathways in brain tissue of rats with ischemia-reperfusion injury were investigated using adenosine-activated protein kinase(AMPK)inhibitor compound C(Cc,20 mg·kg^(-1)·d^(-1),ip),autophagy inhibitor trimethyladenine(3-MA,200 mmol·L^(-1)2μL,icv),and caspase-1 inhibitor Z-YVAD-FMK(Z-YVAD,8μg·kg-12μL,icv)to block AMPK pathway,autophagy and pyroptosis,respectively.The Zea-Longa score was used to evaluate the neurological function.The water maze test was used to examine the memory of the rats.The infarct volume was detected by TTC staining.The pathological changes of the damaged brain tissue were observed by HE staining.Western blot was used to detect the expression of AMPK,p-AMPK,LC3-I,LC3-II,p62,NLRP3 P,pro-caspase-1,and caspase-1 of damaged brain tissue.RESULTS Each dose of metformin reduced the neurological function score in CIRI rats,improved memory,decreased cerebral infarction volume,decreased neuronal necrosis,without effect on blood glucose.Pretreatment by metformin significantly activated AMPK pathway(increased AMPK expression)and autophagy activity(increased LC3-Ⅰ/LC3-Ⅱ,decreased expression of p62)in the rat brain,and the effect was inhibited by Cc.It was shown that metformin elevated autophagy activity by AMPK.3-MA blocked the autophagic activity and brain damage alleviation induced by metformin,but showed no effect on the expression of AMPK,which indicated that metformin exerted protective effects through autophagy.The expression of caspase-1,which are highly expressed in CIRI rats,was blocked by Z-YVAD,and this effect was further enhanced by metformin,which indicated that metformin inhibits cell death by inhibiting the expression of inflammasome.CONCLUSION Metformin has protective effect on cerebral ischemia-reperfusion injury.Its mechanism includes activation of the AMPK pathway,promotion of autophagy,removal of damaged mitochondria by autophagy,and reduction of pyroptosis caused by NLRP3 inflammasome which is activated by damaged mitochondria.