西红花酸调控Nrf2/HO-1通路抑制高糖诱导人肾小球系膜细胞铁死亡  

Crocetin Inhibits High Glucose-Induced Ferroptosis in Human Glomerular Mesangial Cells Through Regulating Nrf2/HO-1 Pathway

作　　者：陆江华 刘爱军[1] LU Jianghua;LIU Aijun(School of Basic Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510006 Guangdong,China)

机构地区：[1]广州中医药大学基础医学院,广东广州510006

出　　处：《中药新药与临床药理》2023年第1期8-15,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(82174368)。

摘　　要：目的 探讨西红花酸抑制高糖诱导人肾小球系膜细胞(HGMCs)铁死亡的作用机制。方法 (1)体外培养HGMCs,将细胞分为空白对照组、阴性对照组(60 mmol·L^(-1)甘露醇)和葡萄糖实验组(30、60、90 mmol·L^(-1)),干预培养48 h后,采用CCK-8法检测细胞存活率,采用RT-qPCR法检测谷胱甘肽过氧化物酶4(GPX4)mRNA表达,以确定高糖诱导HGMCs的造模条件。(2)体外培养HGMCs,分为空白对照组和西红花酸给药组(5、25、125μmol·L^(-1)),干预培养24 h后,采用CCK-8法检测细胞存活率。(3)体外培养HGMCs,将细胞分为空白对照组、模型组(60 mmol·L^(-1)葡萄糖)、西红花酸组(60 mmol·L^(-1)葡萄糖+5、25、125μmol·L^(-1)西红花酸)及阳性对照组[60 mmol·L^(-1)葡萄糖+1μmol·L^(-1)铁死亡抑制剂(Ferrostatin-1,Fer-1)]。干预培养48 h后,采用CCK-8法检测细胞存活率;流式细胞术检测细胞内活性氧(ROS)水平;Western Blot法检测细胞GPX4、血红素加氧酶-1(HO-1)、转铁蛋白受体(TFRC)蛋白表达;采用免疫荧光法与Western Blot法检测HGMCs细胞核中核因子E2相关因子2(Nrf2)蛋白表达。结果 (1)与空白对照组比较,阴性对照组的细胞存活率及GPX4mRNA表达水平无明显差异(P>0.05);30、60、90 mmol·L^(-1)葡萄糖实验组的细胞存活率及GPX4 mRNA表达水平均显著降低(P<0.01),且以60 mmol·L^(-1)浓度组作用最为明显。(2)与空白对照组比较,5μmol·L^(-1)西红花酸组的HGMCs细胞存活率无明显影响(P>0.05),25、125μmol·L^(-1)西红花酸组的HGMCs细胞存活率显著升高(P<0.01),该浓度西红花酸对HGMCs无明显细胞毒性。(3)与空白对照组比较,模型组的HGMCs细胞存活率显著降低(P<0.01);细胞内ROS水平显著上升(P<0.01);GPX4蛋白表达水平明显下降(P<0.05);细胞核内Nrf2蛋白表达显著下调(P<0.001)。与模型组比较,25、125μmol·L^(-1)西红花酸组的HGMCs细胞存活率明显升高(P<0.05);细胞内ROS水平均显著下降(P<0.01),并呈浓度依赖性;�Objective To investigate the mechanism of action of crocetin in inhibiting high glucose-induced ferroptosis in human glomerular mesangial cells(HGMCs).Methods (1)HGMCs were cultured in vitro and the cells were divided into blank control,negative control(60 mmol·L^(-1)mannitol)and glucose experimental groups(30,60,90 mmol·L^(-1)).After 48-hour cell culture,cell survival rate was detected by CCK-8 method and mRNA expression of glutathione peroxidase 4(GPX4) was detected by RT-qPCR to determine the modeling conditions of HGMCs induced by high glucose.(2)HGMCs were cultured in vitro and divided into blank control group and croceti administration groups(5,25,125μmol·L^(-1)),and cell survival rate was measured by CCK-8 method after 24-hour cell culture.(3)HGMCs were cultured in vitro,and the cells were divided into blank control group,model group(60 mmol·L^(-1)glucose),crocetin groups(60 mmol·L^(-1)glucose+5,25,125μmol·L^(-1)crocetin)and positive control groups[60 mmol·L^(-1)glucose+1μmol·L^(-1)Ferroptosis inhibitor(Ferrostatin-1,Fer-1)].After 48-hour cell culture,cell viability was measured by CCK-8 method;intracellular reactive oxygen species(ROS) level was detected by flow cytometry;protein expressions of GPX4,heme oxygenase-1(HO-1)and transferrin receptor(TFRC)were detected by Western Blot;the protein expression of nuclear factor E2-related factor 2(Nrf2)in the nucleoprotein of HGMCs was measured by immunofluorescence and Western Blot.Results (1)Compared with the blank control group,the cell survival rate and mRNA expression level of GPX4 in the negative control group were not significantly different(P>0.05);the cell survival rate and mRNA expression levels of GPX4 in the 30,60 and 90 mmol·L^(-1)glucose experimental groups were significantly reduced(P<0.01),and the effect was most pronounced in the 60 mmol·L^(-1)concentration group.(2)Compared with the blank control group,the cell survival rate of HGMCs was not significantly affected in the 5μmol·L^(-1)crocetin group(P>0.05),and the cell survival rat

关 键 词：西红花酸 高糖诱导 人肾小球系膜细胞 铁死亡 谷胱甘肽过氧化物酶4 Nrf2/HO-1通路 

分 类 号：R285.5[医药卫生—中药学]