GRSF1在小鼠脑缺血再灌注损伤中的作用:与铁死亡的关系

Role of GRSF1 in cerebral ischemia-reperfusion injury in mice:relationship with ferroptosis

目的评价富含鸟嘌呤序列的结合因子1(GRSF1)在小鼠脑缺血再灌注损伤中的作用及其与铁死亡的关系。方法清洁级雄性C57BL/6小鼠24只,8~10周龄,体质量20~25 g,采用随机数字表法分为4组(n=6):假手术组(Sham组)、脑缺血再灌注组(IR组)、脑缺血再灌注+GRSF1过表达组(IR+LV-GRSF1组)、脑缺血再灌注+GRSF1过表达+谷胱甘肽过氧化物酶4(GPX4)抑制剂组(IR+LV-GRSF1+RSL3组)。采用大脑中动脉栓塞(MCAO)法制备小鼠脑缺血再灌注损伤模型。IR+LV-GRSF1组于造模前7 d时侧脑室注射GRSF1过表达慢病毒2μl;IR+LV-GRSF1+RSL3组造模前连续2 d腹腔注射GPX4抑制剂RSL35 mg/kg。再灌注24 h后TTC法确定脑梗死体积百分比,Nissl染色计数缺血区存活神经元,取缺血区脑组织,RT-qPCR法检测衰老标志物p16、p21及衰老相关分泌表型TNF-α的mRNA表达,ELISA法检测MDA、SOD和谷胱甘肽(GSH)含量,Western blot法检测GRSF1、GPX4、长链脂酰辅酶A合成酶4(ACSL4)和铁蛋白的表达水平。结果与Sham组相比,IR组脑梗死体积百分比升高,存活神经元计数降低,缺血区脑组织p16 mRNA、p21 mRNA及TNF-αmRNA表达上调,SOD和GSH含量降低,MDA含量升高,GRSF1和GPX4表达下调,ACSL4和铁蛋白表达上调(P<0.05);与IR组相比,IR+LV-GRSF1组脑梗死体积百分比降低,存活神经元计数升高,缺血区脑组织p16 mRNA、p21 mRNA及TNF-αmRNA表达下调,SOD和GSH含量升高,MDA含量降低,GRSF1和GPX4表达上调,ACSL4和铁蛋白表达下调(P<0.05);与IR+LV-GRSF1组相比,IR+LV-GRSF1+RSL3组脑梗死体积百分比升高,存活神经元计数降低,缺血区脑组织p16 mRNA、p21 mRNA及TNF-αmRNA表达上调,SOD和GSH含量降低,MDA含量升高,GPX4表达下调,ACSL4和铁蛋白表达上调(P<0.05)。结论GRSF1可通过上调GPX4表达,减轻氧化应激,进而抑制铁死亡,参与小鼠脑缺血再灌注损伤的内源性保护机制。

Objective To evaluate the role of G-rich RNA sequence binding factor 1(GRSF1)in cerebral ischemia-reperfusion(I/R)injury in mice and the relationship with ferroptosis.Methods Twenty-four clean-grade male C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups(n=6 each)using a random number table method:sham operation group(Sham group),cerebral I/R group(IR group),cerebral I/R+GRSF1 overexpression group(IR+LV-GRSF1 group),and cerebral I/R+GRSF1 overexpression+glutathione peroxidase 4(GPX4)inhibitor group(IR+LV-GRSF1+RSL3 group).The model of middle cerebral artery occlusion was developed by thread-occlusion method in anesthetized animals.In IR+LV-GRSF1 group,GRSF1-overexpressed lentivirus 2μl was injected into the lateral ventricle at 7 days before the development of the model.GPX4 inhibitor RSL35 mg/kg was intraperitoneally injected for 2 consecutive days before the development of the model in IR+LV-GRSF1+RSL3 group.After 24 h of reperfusion,the percentage of cerebral infarction volume was determined by TTC assay,the survival neurons in ischemic area were detected by Nissl staining,and brain tissues in ischemic area were obtained for determination of the expression of p16,p21(markers of senescence)and tumor necrosis factor-alpha(TNF-α,senescence-associated secretory phenotype)mRNA(by quantitative real-time polymerase chain reaction),contents of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)(by enzyme-linked immunosorbent assay)and expression of GRSF1,GPX4,Acyl-CoA synthetase long-chain family member 4(ACSL4)and ferritin(by Western blot).Results Compared with Sham group,the percentage of cerebral infarction volume was significantly increased,the count of viable neurons was decreased,the expression of p16,p21 and TNF-αmRNA in ischemic brain tissues was up-regulated,SOD and GSH contents were decreased,the MDA content was increased,the expression of GRSF1 and GPX4 was down-regulated,and the expression of ACSL4 and ferritin was up-regulated in IR group(P<0.05).Compared with IR group,the percentage of cerebral infarction volume was significantly decreased,the count of viable neurons was increased,the expression of p16,p21 and TNF-αmRNA in ischemic brain tissues was down-regulated,SOD and GSH contents were increased,the MDA content was decreased,the expression of GRSF1 and GPX4 was up-regulated,and the expression of ACSL4 and ferritin was down-regulated in IR+LV-GRSF1 group(P<0.05).Compared with IR+LV-GRSF1 group,the percentage of cerebral infarction volume was significantly increased,the count of viable neurons was decreased,the expression of p16,p21 and TNF-αmRNA in ischemic brain tissues was up-regulated,SOD and GSH contents were decreased,the MDA content was increased,the expression of GRSF1 and GPX4 was down-regulated,and the expression of ACSL4 and ferritin was up-regulated in IR+LV-GRSF1+RSL3 group(P<0.05).Conclusions GRSF1 is involved in the endogenous protective mechanism against cerebral I/R injury by up-regulating GPX4 expression,attenuating oxidative stress,and thus inhibiting ferroptosis in mice.