橘皮素调控PI3K/Akt信号通路介导的自噬对脑缺血/再灌注损伤大鼠神经细胞焦亡的影响  

Effects of Tangeretin on Neuronal Pyroptosis in Rats with Cerebral Ischemia/Reperfusion Injury Through PI3K/Akt Signaling Pathway-Mediated Autophagy

作　　者：孙军[1] 温昌明[1] 张丽阳 高军 张在行 陈笛[1] 时彩丽[3] 兰端云 张保朝[1] SUN Jun;WEN Changminga;ZHANG Liyang;GAO Jun;ZHANG Zaixing;CHEN Di;SHI Caili;LAN Duanyun;ZHANG Baochao(Neurology Departmentt,Henan Nanyang Central Hospital,Nanyang 473009,China;Outpatient Department of Superior Consulting Roomt,Henan Nanyang Central Hospital,Nanyang 473009,China;Nursing Departmentt,Henan Nanyang Central Hospital,Nanyang 473009,China;Endocrine Department,Henan Nanyang Central Hospital,Nanyang 473009,China)

机构地区：[1]河南省南阳市中心医院神经内科,河南南阳473009 [2]河南省南阳市中心医院优诊室门诊,河南南阳473009 [3]河南省南阳市中心医院护理部,河南南阳473009 [4]河南省南阳市中心医院内分泌科,河南南阳473009

出　　处：《中国药学杂志》2024年第6期511-521,共11页Chinese Pharmaceutical Journal

基　　金：河南省重点研发与推广专项资助(192102310349);南阳市科技攻关计划项目资助(KJGG2018082)。

摘　　要：目的探究橘皮素(tangeretin,TAN)对脑缺血/再灌注损伤(cerebral ischemia-reperfusion injury,CIRI)大鼠神经元细胞焦亡的影响及作用机制。方法采用线栓法阻塞大鼠大脑中动脉2 h后再灌注复制CIRI模型。实验1:将SD大鼠随机分为假手术组(sham)、CIRI组、TAN-5 mg·kg^(-1)组、TAN-10 mg·kg^(-1)组、TAN-20 mg·kg^(-1)组、阳性对照药依达拉奉组(EDA-10 mg·kg^(-1)),每组各10只,各组大鼠于再灌注24 h后进行指标检测及病理取材。Zea-Longa法进行神经功能缺失评分;2,3,5-三苯基氯化四氮唑(TTC)法检测脑梗死比率;苏木素-伊红(HE)染色观察脑组织形态结构,筛选出TAN最佳给药剂量;碘化丙啶染色(PI)检测神经元焦亡比率;免疫荧光双染检测神经元微管相关蛋白轻链3-Ⅱ(microtubule-associated proteins light chain 3-Ⅱ,LC3-Ⅱ)阳性表达率;Western blot检测脑组织焦亡相关蛋白、磷脂酰肌醇3激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K/Akt)通路相关蛋白和自噬相关蛋白(LC3-Ⅱ/LC3-Ⅰ、p62)表达。实验2:将SD大鼠随机分为sham组、CIRI组、TAN组、TAN+PI3K抑制剂组(LY294002),每组各10只,各组大鼠于再灌注24 h后进行指标检测及病理取材。Zea-Longa法进行神经功能缺失评分;TTC染色法检测脑梗死比率;Western blot检测脑组织焦亡相关蛋白和自噬相关蛋白表达。结果与CIRI组相比,各给药剂量组大鼠神经功能缺失评分降低、脑梗死体积比率降低、脑组织病理性损伤减轻,筛选得到TAN-20 mg·kg^(-1)为最佳给药剂量组。与CIRI组相比,TAN-20 mg·kg^(-1)组大鼠神经元细胞焦亡率降低,且焦亡相关蛋白表达下调,神经元LC3-Ⅱ阳性细胞率和LC3-Ⅱ/LC3-Ⅰ表达均降低,p-PI3K/PI3K、p-Akt/Akt、p62表达升高。与TAN-20 mg·kg^(-1)组相比,PI3K抑制剂LY294002能够抑制上述指标变化,逆转TAN对CIRI大鼠的神经功能保护作用。结论TAN能够通过调控PI3K/Akt介导的细胞自噬,抑制脑缺血再OBJECTIVE To investigate the effect of tangeretin(TAN)on neuronal pyroptosis in rats with cerebral ischemia/reperfusion injury(CIRI)and its mechanism.METHODS The CIRI model of rats was established by reperfusion after occlusion of the middle cerebral artery for 2 h using thread occlusion method.Experiment 1:SD rats were randomly divided into sham group,CIRI group,TAN-5 mg·kg^(-1),TAN-10 mg·kg^(-1),TAN-20 mg·kg^(-1),and positive control edaravone group(EDA-10 mg·kg^(-1)),with 10 rats in each group.The rats in each group were subjected to index detection and pathological sampling 24 hours after reperfusion.Zea Longa method was used to score neurological deficits;the rate of cerebral infarction was detected using the 2,3,5-triphenyl tetrazolium chloride(TTC)method;hematoxylin eosin(HE)staining was used to observe the morphology and structure of brain tissue,and the optimal dosage of TAN was selected;propidium iodide staining(PI)was used to detect the ratio of neuronal focal death;immunofluorescence double staining was used to detect the positive expression rate of LC3-II in neurons;Western blot was performed for detection of the expressions of pyroptosis-related proteins,PI3K/Akt pathway-related proteins,and autophagy-related proteins(LC3-II/LC3-I,p62)in brain tissue.Experiment 2:SD rats were randomly divided into sham group,CIRI group,TAN group,and TAN+PI3K inhibitor group(LY294002),with 10 rats in each group.After 24 hours of reperfusion,indicators were tested and pathological samples were taken from each group of rats.Zea Longa method was used to score neurological deficits;TTC staining was used to detect the rate of cerebral infarction;Western blot was used to detect the expressions of pyroptosis-related proteins and autophagy-related proteins in brain tissue.RESULTS Compared with the CIRI group,the neurological deficit score,cerebral infarction volume ratio,and pathological damage to brain tissue of rats in each dose group were reduced.The TAN-20 mg·kg^(-1) was selected as the optimal dose group.Compared w

关 键 词：脑缺血/再灌注损伤 橘皮素 细胞焦亡 自噬 磷脂酰肌醇3激酶/蛋白激酶B通路 

分 类 号：R966[医药卫生—药理学]