小鼠脑缺血再灌注早期cGAS-STING信号通路与铁自噬的关系

Relationship between cGAS-STING signaling pathway and ferritinophagy in early stage of cerebral ischemia-reperfusion in mice

目的评价小鼠脑缺血再灌注早期环鸟苷酸-腺苷酸合成酶(cGAS)-干扰素基因刺激蛋白(STING)信号通路与铁自噬的关系。方法清洁级健康雄性C57BL/6小鼠24只,6~8周龄,体重21~25 g,采用随机数字表法分为4组(n=6):假手术组(Sham组)、脑缺血再灌注损伤(CIRI)组、CIRI+cGAS抑制剂组(CIRI+RU组)和CIRI+cGAS抑制剂+过表达核受体辅激活因子4(NCOA4)组(CIRI+RU+LV-NCOA4组)。采用大脑中动脉栓塞(MCAO)法制备CIRI模型。CIRI+RU组于再灌注前10 min腹腔注射cGAS抑制剂5 mg/kg;CIRI+RU+LV-NCOA4组于MCAO前7 d脑室注射NCOA4过表达慢病毒(1×10^(9) TU/ml)2μl,其余操作同CIRI+RU组。再灌注6 h后行神经功能缺陷评分,随后处死小鼠并取脑,TTC法检测脑梗死体积,TBA法检测MDA含量,WST-1法检测SOD活性,Western blot法检测cGAS、STING、NCOA4、铁蛋白和微管相关蛋白1轻链3B(LC3B)的表达。结果与Sham组相比,CIRI组神经功能缺陷评分和脑梗死体积增加,脑组织SOD活性降低,MDA含量升高,cGAS、STING、NCOA4和LC3B表达上调,铁蛋白表达下调(P<0.05);与CIRI组相比,CIRI+RU组神经功能缺陷评分和脑梗死体积减小,脑组织SOD活性升高,MDA含量降低,cGAS、STING、NCOA4和LC3B表达下调,铁蛋白表达上调(P<0.05);与CIRI+RU组相比,CIRI+RU+LV-NCOA4组神经功能缺陷评分和脑梗死体积增加,脑组织SOD活性降低,MDA含量升高,NCOA4和LC3B表达上调,铁蛋白表达下调(P<0.05),cGAS和STING表达差异无统计学意义(P>0.05)。结论cGAS-STING信号通路可促进铁自噬的过度激活,增强氧化应激,进而诱发小鼠早期CIRI。

Objective To evaluate the relationship between the second messenger cyclic GMP-AMP(cGAS)-cyclic GMP-AMP receptor stimulator of interferon genes(STING)signaling pathway and ferritinophagy in the early stage of cerebral ischemia-reperfusion(I/R)in mice.Methods Twenty-four clean-grade healthy male C57BL/6 mice,aged 6-8 weeks,weighing 21-25 g,were divided into 4 groups(n=6 each)using a random number table method:sham group,cerebral I/R injury group(CIRI group),cerebral I/R injury+cGAS inhibitor group(CIRI+RU group),and cerebral I/R injury+cGAS inhibitor+overexpressed nuclear receptor coactivator 4(NCOA4)group(MCAO+RU+LV-NCOA4 group).The model of cerebral I/R injury was developed using the middle cerebral artery occlusion(MCAO)in anesthetized animals.In CIRI+RU group,cGAS inhibitor 5 mg/kg was intraperitoneally injected at 10 min before reperfusion.In CIRI+RU+LV-NCOA4 group,NCOA4-overexpressing lentivirus(1×10^(9) TU/ml)2μl was injected into the ventricle at 7 days before MCAO,and the other operations were the same as those previously described in CIRI+RU group.After 6 h of reperfusion,the neurological function deficits were assessed and scored,then the mice were sacrificed,and brains were removed for determination of the cerebral infarct size(by TTC method),MDA content(by TBA method),activity of SOD(by WST-1 method),and expression of cGAS,STING,NCOA4,ferritin,and microtubule-associated protein 1 light chain 3B(LC3B)(by Western blot).Results Compared with Sham group,the neurological function deficit score and cerebral infarct size were significantly increased,SOD activity was decreased,MDA content was increased,the expression of cGAS,STING,NCOA4 and LC3B was up-regulated,and the expression of ferritin was down-regulated in CIRI group(P<0.05).Compared with CIRI group,the neurological function deficit score and cerebral infarct size were significantly decreased,SOD activity was increased,MDA content was decreased,the expression of cGAS,STING,NCOA4 and LC3B was down-regulated,and the expression of ferritin was up-regulated in CIRI+RU group(P<0.05).Compared with CIRI+RU group,the neurological function deficit score and cerebral infarct size were significantly increased,SOD activity was decreased,MDA content was increased,the expression of cGAS,STING,NCOA4 and LC3B was up-regulated,and the expression of ferritin was down-regulated in CIRI group(P<0.05),and no significant change was found in the expression of cGAS and STING in CIRI+RU+LV-NCOA4 group(P>0.05).Conclusions The cGAS-STING signaling pathway can promote the over-activation of ferritinophagy,enhance oxidative stress,and thus induce early CIRI in mice.