神经调节素1β通过激活Sirt1信号通路抑制自噬改善大鼠脑缺血再灌注损伤研究

Neuregulin 1βImproves Cerebral Ischemia Reperfusion Injury by Inhibiting Autophagy via Sirt1 Signaling Pathway in Rats

目的 探讨神经调节素1β(neuregulin1β,NRG1β)是否通过抑制自噬减轻大鼠大脑中动脉缺血再灌注(middle cerebral artery occlusion reperfusion,MCAO/R)损伤,以及对沉默信息调节因子1(silent information regulator protein 1,Sirt1)信号通路的影响。方法 将210只健康雄性SD大鼠随机分为假手术组(Sham组)、模型组(MCAO/R组)、治疗组(NRG1β组)、激动剂组(SRT501组)、激动剂+治疗组(SRT501+NRG1β组)、抑制剂组(EX527组)和抑制剂+治疗组(EX527+NRG1β组),每组30只。采用改良线栓法建立MCAO/R模型,线栓由颈外动脉插入颈内动脉18~22 mm,堵塞左侧大脑中动脉起始部。缺血2 h后,缓慢拔出线栓,恢复脑血流22 h。EX527(5 mg/kg)、SRT501(100 mg/kg)于术前30 min腹腔注射,NRG1β(2μg/kg)于拔出线栓后由微量注射器注入颈内动脉。脑缺血2 h、再灌注22 h后采用改良神经损伤严重程度评分(modifiedneurologicalseverityscore,mNSS)法评价各组大鼠的神经行为功能,采用2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazoliumchloride,TTC)染色法计算大鼠脑梗死体积比例,苏木精-伊红染色观察神经元形态变化,免疫印迹(westernblot,WB)和免疫荧光(immunofluorescence,IF)法分别检测额叶皮质缺血半暗带(ischemicpenumbra,IP)区Sirt1、LC3、P62蛋白的表达和阳性细胞指数(positive cell index,PCI)。结果 NRG1β组大鼠的mNSS[(10.0±0.8)分vs.(12.8±0.6)分,P<0.001]和脑梗死体积比例[(23.78%±3.52%)vs.(40.24%±1.55%),P<0.001]均优于MCAO/R组,差异具有统计学意义;与MCAO/R组比较,NRG1β组、SRT501组、SRT501+NRG1β组mNSS均有不同程度下降、脑梗死体积比例缩小;各组中SRT501+NRG1β组mNSS最低、脑梗死体积比例最小,EX527组mNSS最高、脑梗死体积比例最大。苏木精-伊红染色显示,NRG1β组大鼠的神经元形态结构损伤较MCAO/R组、EX527组有所改善。WB结果显示,NRG1β组Sirt1表达[(0.81±0.01)vs.(0.67±0.02),P<0.001]和P62表达[(0.92±0.01)vs.(0.78±0.02),P<0.001]均高于MCAO/R组,LC3表达[(0.49±0.02)vs.(0.94±0.03),P<0.001]低于MCAO/R组。IF结果显示,NRG1β组Sirt1PCI[(0.67±0.01)vs.(0.52±0.02),P<0.001]和P62PCI[(0.52±0.02)vs.(0.37±0.01),P<0.001]均高于MCAO/R组,LC3PCI[(0.38±0.01)vs.(0.50±0.01),P<0.001]低于MCAO/R组。WB和IF检测显示,Sirt1与P62表达趋势一致,NRG1β组、SRT501组与SRT501+NRG1β组表达高于MCAO/R组,各组中SRT501+NRG1β组表达最高、EX527组表达最低;LC3蛋白表达趋势与Sirt1、P62相反,在NRG1β组、SRT501组与SRT501+NRG1β组表达较MCAO/R组低;各组中SRT501+NRG1β组表达最低、EX527组表达最高。结论 NRG1β可通过激活MCAO/R损伤大鼠Sirt1信号通路抑制自噬发挥神经保护作用。

Objective To investigate whether neuregulin 1β(NRG1β)can alleviate middle cerebral artery occlusion reperfusion(MCAO/R)injury in rats by inhibiting autophagy,and whether this effect is mediated by the silent information regulator protein 1(Sirt1)signaling pathway.Methods A total of 210 healthy male SD rats were randomly divided into sham group(sham group),model group(MCAO/R group),treatment group(NRG1βgroup),agonist group(SRT501 group)and agonist combined with treatment group(SRT501+NRG1βgroup),inhibitor group(EX527 group)and inhibitor combined with treatment group(EX527+NRG1βgroup),with 30 rats in each group.The MCAO/R model was established by the modified thread occlusion method to occlude the initial part of middle cerebral artery.After 2 hours of ischemia,cerebral blood flow was restored for 22 hours.EX527(5 mg/kg)and SRT501(100 mg/kg)were injected intraperitoneally 30 minutes before surgery,and NRG1β(2μg/kg)was injected into the internal carotid artery with a microsyringe after restoration of reperfusion.Neurological behavioral function was evaluated by modified neurological severity score(mNSS)at 2 hours after cerebral ischemia and 22 hours after reperfusion.The proportion of cerebral infarction volume in rats was calculated by TTC staining.Morphological changes of neurons were observed by hematoxylin-eosin(HE)staining.The western blot(WB)and immunofluorescence(IF)were used to detect the expression of Sirt1,LC3 and P62 proteins in ischemic penumbra of the frontal cortex.Results The mNSS[(10.0±0.8)v s.(12.8±0.6),P<0.001]and TTC staining results[(23.78%±3.52%)vs.(40.24%±1.55%),P<0.001]in NRG1βgroup were better than those in MCAO/R group.Compared with MCAO/R group,mNSS in NRG1βgroup,SRT501 group,SRT501+NRG1βgroup all decreased in different degree,and the proportion of TTC-stained infarct volume reduced.The mNSS and the proportion of infarct volume were the lowest in SRT501+NRG1βgroup,while they were the highest in EX527 group among these groups.HE staining showed that the morphological and structural damage of neurons in NRG1βgroup improved compared with that in MCAO/R group and EX527 group.The WB results showed that the expression of Sirt1[(0.81±0.01)vs.(0.67±0.02),P<0.001]and P62[(0.92±0.01)vs.(0.78±0.02),P<0.001]in NRG1βgroup were higher than those in MCAO/R group,and the LC3 expression[(0.49±0.02)vs.(0.94±0.03),P<0.001]was lower than that in MCAO/R group.The IF results showed that Sirt1 positive cell index(PCI)[(0.67±0.01)vs.(0.52±0.02),P<0.001]and P62 PCI[(0.52±0.02)vs.(0.37±0.01),P<0.001]in NRG1βgroup were higher than those in MCAO/R group,and LC3 PCI[(0.38±0.01)vs.(0.50±0.01),P<0.001]was lower than that in MCAO/R group.The WB and IF results showed that the expression trend of Sirt1 and P62 was consistent as follows,their expression in NRG1β,SRT501 and SRT501+NRG1βgroups were higher than that in MCAO/R group,with the highest expression in SRT501+NRG1βgroup and the lowest expression in EX527 group;the expression trend of LC3 protein was contrary to that of Sirt1 and P62,and the expression of LC3 protein in NRG1β,SRT501 and SRT501+NRG1βgroups were lower than that in MCAO/R group,with the lowest expression in SRT501+NRG1βgroup and the highest expression in EX527 group.Conclusions NRG1βplays a neuroprotective role in MCAO/R rats by activating Sirt1 signaling pathway to inhibit autophagy.