基于PERK/TXNIP/NLRP3通路探讨左归降糖通脉方对糖尿病合并脑梗死大鼠神经元细胞焦亡的影响

Exploring the Effect of Zuogui Jiangtang Tongmai Prescription on the Pyroptosis of Neuron in Rats with Diabetes Mellitus Complicated with Cerebral Infarction Based on PERK/TXNIP/NLRP3 Pathway

目的基于蛋白激酶R样内质网激酶(PERK)/硫氧还蛋白互作蛋白(TXNIP)/NOD样受体蛋白3(NLRP3)通路,探讨左归降糖通脉方对糖尿病合并脑梗死(diabetes mellitus complicated with cerebral infarction,DM-CI)大鼠神经元细胞焦亡的影响。方法将95只大鼠随机分为假手术组,模型组,左归降糖通脉方低(12 g·kg^(-1))、高剂量(24 g·kg^(-1))组,阳性对照(吡格列酮二甲双胍片+阿司匹林肠溶片)组。除假手术组外,其余各组以高糖高脂饲料喂养4周后联合35 mg·kg^(-1)1%链脲佐菌素(STZ)腹腔注射合并大脑中动脉闭塞(MCAO)术建立DM-CI大鼠模型。用蒸馏水,左归降糖通脉方低、高剂量,吡格列酮二甲双胍片和阿司匹林肠溶片进行相应干预,Zea-Longa评分法对大鼠进行神经功能缺损(NIHSS)评分,TTC染色法测量大鼠脑梗死体积,检测各组大鼠随机血糖和果糖胺水平,HE染色观察大鼠脑组织病理学变化,Western Blot法检测大鼠缺血侧脑皮质、海马区PERK、p-PERK、真核起始因子2α(eIF2α)、p-eIF2α、TXNIP、NLRP3、半胱天冬酶-1(Caspase-1)、消皮素D(GSDMD)、白细胞介素(IL)-1β和IL-18蛋白的表达,免疫荧光双染法检测缺血侧脑皮质、海马区组织中神经元特异性烯醇化酶(NSE)与NLRP3蛋白的共定位表达。结果与假手术组比较,模型组大鼠随机血糖、果糖胺水平、NIHSS评分、脑梗死体积、缺血侧脑皮质和海马区p-PERK/PERK、p-eIF2α/eIF2α、TXNIP、NLRP3、Caspase-1、GSDMD、IL-1β、IL-18蛋白水平和NSE与NLRP3双阳性信号细胞数量均明显升高(P<0.05,P<0.01)。与模型组比较,左归降糖通脉方高剂量组的随机血糖、果糖胺水平、NIHSS评分、脑梗死体积、缺血侧脑皮质和海马区p-PERK/PERK、p-eIF2α/eIF2α、TXNIP、NLRP3、Caspase-1、GSDMD、IL-1β、IL-18蛋白水平和NSE与NLRP3双阳性信号细胞数量均明显降低(P<0.05,P<0.01)。结论左归降糖通脉方可改善DM-CI大鼠糖代谢紊乱,减轻神经功能损伤,其机制可能与左归降糖通脉方抑制PERK/TXNIP/NLRP3通路,减轻神经元细胞焦亡有关。

Objective To invesitigate the effect of Zuogui Jiangtang Tongmai Prescription(ZJTP)on the pyroptosis of neuron in rats with diabetes mellitus complicated with cerebral infarction(DM-CI)based on protein kinase R-like endoplasmic reticulum kinase(PERK)/thioredoxin-interacting protein(TXNIP)/NOD-like receptor protein 3(NLRP3)pathway.Methods Ninety-five rats were randomly divided into sham operation group,model group,low-(12 g·kg^(-1))and high-(24 g·kg^(-1))doses of ZJTP groups,and positive control group(Pioglitazone/Metformin Tablets+Aspirin Enteric-Coated Tablets).Except for the sham operation group,the other groups were fed with high sugar and high fat fodder for 4 weeks.DM-CI rat models were established by intraperitoneally injection of 35 mg·kg^(-1)1%streptozotocin(STZ)combined with middle cerebral artery occlusion(MCAO)and then were treated with distilled water,low-and high-doses of ZJTP,Pioglitazone/Metformin Tablets,and Aspirin Enteric-Coated Tablets,respectively.The Zea-Longa score was used to evaluate the neurological function defect(NIHSS).TCC staining was used to measure cerebral infarction volume.The levels of random blood glucose and fructosamine in each group were also determined.Morphological changes of brain tissue in rats with DM-CI were detected by HE staining.The protein expression levels of PERK,p-PERK,eukaryotic initiation factor 2α(eIF2α),p-eIF2α,TXNIP,NLRP3,Caspase-1,GSDMD,IL-1βand IL-18 in the ischemic cerebral cortex and hippocampus were detected by Western Blot.The co-localization expressions of NSE and NLRP3 proteins in the ischemic cerebral cortex and hippocampus was also detected by immunofluorescence double staining.Results Compared with the sham operation group,the NIHSS score,cerebral infarction volume,random blood glucose concentration,fructosamine content,the expressions of p-PERK/PERK,p-eIF2α/eIF2α,TXNIP,NLRP3,Caspase-1,GSDMD,IL-1βand IL-18 proteins in the ischemic cerebral cortex and hippocampus,and the number of NSE and NLRP3 double positive cells in the model group were significantly increased(P<0.05,P<0.01).Compared with the model group,the NIHSS score,cerebral infarction volume,random blood glucose concentration,fructosamine content,the expressions of p-PERK/PERK,p-eIF2α/eIF2α,TXNIP,NLRP3,Caspase-1,GSDMD,IL-1βand IL-18 proteins in the ischemic cerebral cortex and hippocampus,and the number of NSE and NLRP3 double positive cells in the highdose group of ZJTP were significant decreased(P<0.05,P<0.01).Conclusion ZJTP can improve glucose metabolism disorder and alleviate neurological damage in rats with DM-CI.Its mechanism may be related to the inhibition of the PERK/TXNIP/NLRP3 pathway and the reduction of the pyroptosis of neuron.