壮通饮通过Nrf2-SCL7A11/xCT-Gpx4通路调控铁死亡改善脑缺血再灌注损伤  

Zhuangtongyin Modulates Ferroptosis via the Nrf2-SCL7A11/xCT-Gpx4 Pathway to Im⁃prove Cerebral Ischemia-reperfusion Injury

作　　者：王成毅 蔡粤芳 宁振求 邓敏贞[1] 孙景波[1,3,4] Sookja Kim CHUNG 李岩 程骁 WANG Chengyi;CAI Yuefang;NING Zhenqiu;DENG Minzhen;SUN Jingbo;CHUNG Sookja Kim;LI Yan;CHENG Xiao(The Second Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510006,China;The People's Hospital of Shenzhen,Shenzhen 518020,China;State Key Laboratory of Dampness Syndrome of Traditional Chinese Medicine,Guangzhou 510120,China;Guangdong Provincial Key Laboratory of Research on Emergency in TCM,Guangzhou 510120,China;Faculty of Medicine,Macao University of Science and Technology,Macao 999078,China;Guangxi International Zhuang Medicine Hospital,Nanning 530201,China)

机构地区：[1]广州中医药大学第二临床医学院,广东广州510006 [2]深圳市人民医院、南方科技大学第一附属医院、暨南大学第二临床医学院,广东深圳518020 [3]省部共建中医湿证国家重点实验室,广东广州510120 [4]广东省中医急症研究重点实验室,广东广州510120 [5]澳门科技大学医学院,中国澳门999078 [6]广西国际壮医医院,广西南宁530201

出　　处：《中山大学学报（医学科学版）》2024年第4期539-548,共10页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(82160946,81774042);广西自然科学基金(2024GXNSFAA010231);广西重点研发计划(桂科AB24010222);省部共建中医湿证国家重点实验室专项(SZ2023ZZ04);广东省中医急症重点实验室专项(2023B1212060062);广西国际壮医医院“青苗工程”培育项目(2022001)。

摘　　要：【目的】探究壮通饮通过Nrf2-SCL7A11/xCT-Gpx4通路调控铁死亡对大脑中动脉栓塞模型小鼠脑缺血再灌注损伤的保护作用及其机制。【方法】将C57BL/6J小鼠随机分为假手术组(Sham)、模型组(MCAO)、低剂量壮通饮组(ZTY-L+MCAO),高剂量壮通饮组(ZTY-H+MCAO),每组5只。其中MCAO组采用硅胶线栓法进行造模,小鼠大脑中动脉栓塞1h,拔出线栓再灌注72h后进行心脏灌注取脑,Sham组除了不插入线栓其余操作同MCAO组。采用Zea-Longa法对小鼠神经功能进行评分;采用TTC染色评估小鼠脑梗死体积;采用HE染色、尼氏染色评估脑损伤程度;采用普鲁士蓝染色评价三价铁离子蓄积水平;qPCR检测铁离子转运相关载体受体TfR1和DMT1、脂质过氧化相关基因ACSL4以及铁死亡标志物PTGS2 mRNA表达;ELISA试剂盒检测4-HNE的含量评估小鼠大脑脂质过氧化水平;Western-blot、免疫荧光检测小鼠脑组织中Nrf2、SCL7A11/xCT、Gpx4蛋白水平。【结果】①壮通饮改善了MCAO/R后小鼠神经功能(P<0.05)和大脑梗死体积(P<0.05)及缓解MCAO/R后大脑皮层细胞病理损伤。②壮通饮减弱了MCAO/R后小鼠大脑组织的三价铁离子蓄积状态;减少MCAO/R后小鼠大脑中与铁离子转运入细胞内相关载体TfR1和DMT1 mRNA的表达(P<0.001);下调MCAO/R后小鼠大脑脂质过氧化物4-HNE含量和组织脂质过氧化物酶ACSL4 mRNA表达(P<0.001);降低MCAO/R后小鼠大脑铁死亡标志物PTGS2 mRNA表达(P<0.001)。③壮通饮增加MCAO/R后Nrf2入核表达(P<0.001),以及增加xCT和Gpx4在神经元中的表达(P<0.001)。【结论】壮通饮能够改善MCAO/R小鼠神经功能和脑梗死体积,缓解大脑皮层细胞病理损伤,影响Nrf2-SCL7A11/xCT-Gpx4通路关键信号分子表达,所以壮通饮改善小鼠MCAO/R损伤的作用机制可能是通过Nrf2-SCL7A11/xCT-Gpx4通路调节铁死亡水平达到的。【Objective】To investigate the protective effect of Zhuangtongyin on the Middle Cerebral Artery Occlusion(MCAO)model by modulating ferroptosis through the Nrf2-SCL7A11/xCT-Gpx4 pathway and its underlying mechanism.【Methods】C57BL/6J mice were randomly divided into Sham operation group(Sham),model group(MCAO),low-dose Zhuangtongyin group(ZTY-L),high-dose Zhuangtongyin group(ZTY-H),with 5 mice in each group.The MCAO group was modelled by silica gel embolization,the middle cerebral artery of mice was embolized for 1h,then the silica gel was pulled out and reperfusion was performed after 72 h;and the other operations in the Sham group were the same as those in the MCAO group except that the thread plug was not inserted.The neural function of mice was evaluated by Zea-Longa method.TTC staining was used to evaluate the volume of cerebral infarction.The level of brain injury was evaluated by HE staining and Nissl staining.Prussian blue staining and the expression of iron transport-related carrier receptors TfR1 and DMT1 on mRNA level was detected by qPCR to evaluate the iron ion deposition level in mice brain.The expression of lipid peroxidation-related gene ACSL4 on mRNA level was detected by qPCR,and the content of 4-HNE was detected by ELISA kit to evaluate the lipid peroxidation level of mice brain.The expressions of ferroptosis marker PTGS2 mRNA level was detected by qPCR.The expressions of Nrf2,SCL7A11/xCT,Gpx4 in mice brain tissue were detected by Western-blot and immunofluorescence.【Results】Zhuangtongyin improved the nerve function of mice after MCAO(P<0.05)and the cerebral infarction volume of mice(P<0.05)and alleviate the pathological injury of cerebral cortex cells after MCAO operation.Zhuangtongyin attenuated the accumulation of trivalent iron ions in the brain tissue of mice following MCAO.Additionally,Zhuangtongyin downregulated the expression of TfR1 and DMT1 mRNA(P<0.001),a transporter associated with cellular iron ion uptake,in the brains of post-MCAO mice.Furthermore,Zhuangtongyin reduced lev

关 键 词：缺血性脑卒中 铁死亡 壮通饮 Nrf2-SCL7A11/xCT-Gpx4通路 机制研究 

分 类 号：R285.5[医药卫生—中药学]