基于TRPM2/Akt/GSK3β通路探讨24-乙酰泽泻醇A保护脑缺血再灌注损伤脑微血管内皮细胞的机制

Protective Mechanism of Alisol A 24-Acetate on Brain Microvascular Endothelial Cells in Mice with Cerebral Ischemia-Reperfusion Injury by TRPM2/Akt/GSK3βPathway

目的:探讨24-乙酰泽泻醇A(24A)改善小鼠脑缺血再灌注后脑微血管内皮细胞(BMECs)损伤的作用机制。方法:将45只雄性10周龄C57BL/6小鼠按随机数字表法分为假手术组、模型组、24A组,每组15只。模型组、24A组采用20 min双侧颈总动脉结扎后再灌注的方法构建小鼠脑缺血再灌注损伤(CI/RI)模型,假手术组小鼠只分离颈总动脉和迷走神经,不结扎。24 A组采用24 A灌胃,剂量为30 mg/(kg·d);假手术组和模型组则给予等体积的生理盐水进行灌胃,3组均灌胃1次/d,连续干预7 d。分别采用神经功能缺损评分(mNSS)评估小鼠神经功能受损程度;采用新物体识别测试(NORT)和水迷宫(MWM)评价小鼠认知功能;采用免疫荧光法检测内皮细胞CD31表达;采用Western blot法检测脑组织闭锁小带蛋白1(ZO-1)、闭合蛋白(Occludin)、紧密连接蛋白-5(Claudin-5)、磷酸化核因子κB(p-NF-κB)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、瞬时受体电位离子通道-2(TRPM2)、磷酸化苏氨酸蛋白激酶(p-AKT)、磷酸化糖原合成酶激酶-3(p-GSK3β)蛋白表达水平。结果:①神经功能:与假手术组比较,模型组mNSS在第1、3、5、7天均明显升高(P<0.05);与模型组比较,24A组第3、7天mNSS明显降低(P<0.05)。②认知功能:与假手术组比较,模型组NORT 1、24 h识别指数明显降低(P<0.05);与模型组比较,24A组NORT 1、24 h识别指数明显升高(P<0.05)。与假手术组比较,模型组干预后第2~4天逃避潜伏期明显延长,穿越目标平台次数明显减少(P<0.05);与模型组比较,24A组干预后第3、4天逃避潜伏期明显缩短,穿越目标平台次数明显增加(P<0.05)。③内皮细胞CD31、ZO-1、Occludin、Claudin-5蛋白表达:假手术组CD31荧光表达较为密集,呈现红色荧光;与假手术组比较,模型组CD31荧光表达明显降低(P<0.05);与模型组比较,24A组CD31荧光表达明显升高(P<0.05)。与假手术组比较,模型组ZO-1、Occludin、Claudin-5蛋白表达量明显下降(P<0.05);与模型组比较,24A组ZO-1、Occludin、Claudin-5蛋白表达量明显升高(P<0.05)。④p-NF-κB、IL-1β、TNF-α、TRPM2、p-Akt、p-GSK3β蛋白表达:与假手术组比较,模型组p-NF-κB、IL-1β、TNF-α、TRPM2表达明显增加,p-Akt、p-GSK3β表达明显降低(P<0.05);与模型组比较,24A组p-NF-κB、IL-1β、TNF-α、TRPM2蛋白表达水平明显下降,p-Akt、p-GSK3β表达明显增加(P<0.05)。结论:24A可有效改善CI/RI小鼠的神经功能、认知功能,降低炎症反应,改善BMECs损伤,其机制可能与TRPM2/Akt/GSK3β通路的调控有关。

Objective:To explore the mechanism of Alisol A 24-acetate(24A)on improving brain microvascular endothelial cells(BMECs)injury after cerebral ischemia reperfusion in mice.Methods:A total of 4510-week-old male C57BL/6 mice were randomly divided into sham surgery group,model group and 24A group,with 15 cases in each group.The model group and the 24A group were treated with 20 min bilateral common carotid artery ligation followed by reperfusion to establish a cerebral ischemia reperfusion injury(CI/RI)model,but the common carotid artery and vagus nerve were separated without ligation in the sham surgery group.The 24A group was given 24A through intragastric administration with the dosage of 30 mg/(kg·d),and the sham surgery group and the model group were given equal volume of normal saline through intragastric administration.All three groups were given intragastric administration once a day,for seven consecutive days.Modified neurological severity score(mNSS)was used to evaluate neurological deficit;novel object recognition test(NORT)and Morris water maze(MWM)were used to evaluate the cognitive function;immunofluorescence was used to detect the expression of CD31 in cortex;Western blot was used to detect zonula occludens-1(ZO-1),Occludin,Claudin-5,phosphorylated nuclear factor kappa-B(p-NF-κB),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α),transient receptor potential melastatin-2(TRPM2),phosphorylated protein kinase B(p-Akt),phosphorylated glycogen synthetase kinase-3β(p-GSK3β)protein expression levels.Results:(1)Neurological function:compared with the sham surgery group,mNSS in the model group significantly increased at the 1st,3rd,5th and 7th day(P<0.05);compared with the model group,mNSS in the 24A group significantly decreased at the 3rd and 7th day(P<0.05).(2)Cognitive function:compared with the sham surgery group,the NORT recognition index at 1st and 24th hour in the model group was significantly lower(P<0.05);compared with the model group,the 1-hour and 24-hour NORT recognition index in the 24A group significantly increased(P<0.05).Compared with the sham surgery group,the escape latency of the model group significantly prolonged and the number of times of crossing the platform significantly reduced from day 2 to day 4 after intervention(P<0.05);compared with the model group,the escape latency of the 24A group was significantly shortened from day 3 to day 4 after intervention,and the number of platform-site crossovers was significantly increased(P<0.05).(3)The expression of CD31,ZO-1,Occludin,Claudin-5 protein:the fluorescence expression of CD31 in the sham surgery group was dense and showed red fluorescence;compared with the sham surgery group,the fluorescence expression of CD31 in the model group significantly decreased(P<0.05).Compared with the model group,CD31 fluorescence expression in 24A group significantly increased(P<0.05).Compared with the sham group,ZO-1,Occludin and Claudin-5 protein expression levels in the model group significantly decreased(P<0.05).Compared with the model group,protein expression levels of ZO-1,Occludin and Claudin-5 in the 24A group significantly increased(P<0.05).(4)Protein expression of p-NF-κB,IL-1β,TNF-α,TRPM2,p-Akt,p-GSK3β:compared with the sham surgery group,the expression levels of p-NF-κB,IL-1β,TNF-αand TRPM2 in the model group significantly increased,while the expression levels of p-Akt and p-GSK3βsignificantly decreased(P<0.05).Compared with the model group,the expression levels of p-NF-κB,IL-1β,TNF-αand TRPM2 of the 24A group significantly decreased,while the expression levels of p-Akt and p-GSK3βsignificantly increased(P<0.05).Conclusion:24A can effectively improve the neurological function and cognitive function,reduce inflammatory response and ameliorate BMECs damage of CI/RI mice,which may be related to the regulation of TRPM2/Akt/GSK3βpathway.