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

doi:10.3969/j.issn.1673-5765.2023.03.010

Abstract

Abstract: 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 (NRG1β group), agonist group (SRT501 group) and agonist combined with treatment group (SRT501+NRG1β group), inhibitor group (EX527 group) and inhibitor combined with treatment group (EX527+NRG1β group), 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 (2 μg/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) vs. (12.8±0.6), P<0.001] and TTC staining results [(23.78%±3.52%) vs. (40.24%±1.55%), P<0.001] in NRG1β group were better than those in MCAO/R group. Compared with MCAO/R group, mNSS in NRG1β group, SRT501 group, SRT501+NRG1β group 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+NRG1β group, while they were the highest in EX527 group among these groups. HE staining showed that the morphological and structural damage of neurons in NRG1β group 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 NRG1β group 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 NRG1β group 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+NRG1β groups were higher than that in MCAO/R group, with the highest expression in SRT501+NRG1β group 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+ NRG1β groups were lower than that in MCAO/R group, with the lowest expression in SRT501+NRG1β group and the highest expression in EX527 group.
Conclusions NRG1β plays a neuroprotective role in MCAO/R rats by activating Sirt1 signaling pathway to inhibit autophagy.

Key words: Cerebral ischemia reperfusion injury; Neuromodulin 1β; Rat; Autophagy; Sirt1 signaling pathway

摘要： 目的探讨神经调节素1β（neuregulin 1β，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（2 μg/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（modified neurological severity score，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-triphenyltetrazolium chloride，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（western blot，WB）和免疫荧光（immunofluorescence，IF）法分别检测额叶皮质缺血半暗带（ischemic penumbra，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Sirt1 PCI[（0.67±0.01）vs.（0.52±0.02），P<0.001]和P62 PCI[（0.52±0.02）vs.（0.37±0.01），P<0.001]均高于MCAO/R组，LC3 PCI[（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。

关键词: 脑缺血再灌注损伤; 神经调节素1β; 大鼠; 自噬; Sirt1信号通路

LIANG Xiaoyan, HOU Feng, LIU Xiaoqing, NI Qinshuai, GUO Yunliang, ZHANG Rui.

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

[J]. Chinese Journal of Stroke, 2023, 18(03): 301-314.

梁晓燕, 侯峰, 刘小青, 倪钦帅, 郭云良, 张睿. 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[J]. 中国卒中杂志, 2023, 18(03): 301-314.

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