基于GPX4通路的益气解毒方抗脑缺血铁死亡保护机制研究  

Protective mechanism of Yiqi Jiedu formula against ferroptosis in cerebral ischemia via GPX4 pathway

作　　者：蔡冰洁 李淑婷 高艳华 张琪曼 杨家霖 张滢 李韶菁[2] CAI Bing-jie;LI Shu-ting;GAO Yan-hua;ZHANG Qi-man;YANG Jia-lin;ZHANG Ying;LI Shao-jing(Anhui University of Traditional Chinese Medicine,Hefei 230012;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700;Beijing City University,Beijing 100191)

机构地区：[1]安徽中医药大学,合肥230012 [2]中国中医科学院中药研究所,北京100700 [3]北京城市学院,北京100191

出　　处：《中南药学》2024年第10期2551-2559,共9页Central South Pharmacy

基　　金：国家科技重大专项(No.2019ZX09201-004);中国中医科学院科技创新工程项目(No.CI2021A02001)。

摘　　要：目的研究益气解毒方抗急性缺血性脑卒中铁死亡相关保护机制。方法采用大脑中动脉栓塞(MCAO)法复制大鼠急性脑缺血模型,同时构建PC12细胞糖氧剥夺(OGD)模型。缺血24 h后进行神经行为学评分和脑梗死体积测定。ELISA法检测MCAO大鼠结肠内容物中短链脂肪酸(SCFA)含量,普鲁士蓝染色观察缺血侧脑组织Fe^(2+),JC-1和刃天青分别检测缺血侧脑组织线粒体膜电位和活力,并对缺血侧脑组织中亚铁离子、谷胱甘肽(GSH)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平,及血清中三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量进行测定。采用分子对接预测益气解毒方与GPX4蛋白的结合度。Western blot法检测大鼠缺血侧脑组织G蛋白偶联受体41(GPR41)、谷胱甘肽过氧化物酶4(GPX4)、p53蛋白表达水平,流式细胞术和Western blot法检测细胞GPX4蛋白表达水平。结果在动物实验中,与模型组相比,益气解毒方中、高剂量组大鼠神经行为评分与脑梗死体积显著降低(P<0.01),结肠内容物中SCFA含量增加(P<0.01),缺血侧脑组织铁离子颗粒物沉积减少,线粒体膜电位升高(P<0.05),线粒体活力显著增加(P<0.01),缺血侧脑组织中GSH、SOD、GPR41、GPX4水平显著升高(P<0.05或P<0.01),Fe^(2+)、MDA、p53水平显著降低(P<0.01),TG、LDL-C显著降低(P<0.05,P<0.01),HDL-C显著增加(P<0.05);在细胞实验中,与OGD组相比,益气解毒方组和Fer-1组能使细胞GPX4蛋白表达增加(P<0.01)。结论益气解毒方抑制铁死亡可能是其发挥急性脑缺血保护作用的又一关键作用机制,其作用可能与GPX4相关通路密切相关。Objective To determine the protective mechanism of Yiqi Jiedu formula(YQ)against ferroptosis in acute ischemic stroke.Methods The rat model of acute cerebral ischemia was established by middle cerebral artery occlusion(MCAO).The PC12 cell glucose and oxygen deprivation(OGD)model was also established.The neurobehavioral scores and infarct volume were measured 24 h after the ischemia.The content of short-chain fatty acids(SCFA)in the colonic content of MCAO rats was detected by ELISA.The Fe^(2+)in the ischemic brain tissue was observed by Prussian blue staining.The mitochondrial membrane potential and activity in the ischemic brain tissue were detected by JC-1 and resazurin,respectively.The content of ferrous ion,glutathione(GSH),malondialdehyde(MDA),superoxide dismutase(SOD)in the ischemic brain tissue and triglyceride(TG),high density lipoprotein cholesterol(HDL-C),and low density lipoprotein cholesterol(LDL-C)in the serum were measured.Molecular docking was used to predict YQ binding to the GPX4 protein.Western blot was used to detect the protein expression levels of G protein-coupled receptor 41(GPR41),glutathione peroxidase 4(GPX4)and p53 in the ischemic brain tissue of rats.Flow cytometry and Western blot were used to detect the protein expression level of GPX4 in the cells.Results Compared with the model group,the neurobehavioral scores and cerebral infarction volume of the middle and high dose YQ groups were decreased(P<0.01),SCFA content in colon contents was increased(P<0.01),iron particle deposition in the ischemic brain tissue was decreased,and the mitochondrial membrane potential increased(P<0.05),while the mitochondrial viability was increased(P<0.01).The levels of GSH,SOD,GPR41 and GPX4 were increased(P<0.05 or P<0.01),and those of Fe^(2+),MDA and p53 in the ischemic brain tissue were decreased(P<0.01).The serum TG and LDL-C were significantly decreased(P<0.05,P<0.01),while HDL-C increased(P<0.05).Compared with the OGD group,the expression of GPX4 protein in both the YQ group and Fer-1 group was incr

关 键 词：益气解毒方 急性缺血性脑卒中 铁死亡 短链脂肪酸 GPX4通路 

分 类 号：R286[医药卫生—中药学]