葛根素对高糖环境下H_(2)O_(2)诱导人足细胞氧化应激及线粒体损伤的保护机制  

Protection mechanism of Puerarin on H_(2)O_(2)-induced oxidative stress and mitochondrial damage in human podocytes under high glucose environment

作　　者：朱青青 李雪玲[1] 严佳怡 樊奕琛 刘妮 钟逸斐[1] ZHU Qing-qing;LI Xue-ling;YAN Jia-yi;FAN Yi-chen;LIU Ni;ZHONG Yi-fei(Department of Nephrology,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院肾内科,上海200032

出　　处：《北京中医药》2022年第2期125-131,共7页Beijing Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(81973772);上海市进一步加快中医药事业发展三年行动计划[ZY(2018-2020)-CCCX-2002-02];上海市中医药领军人才计划[ZY(2018-2020)-RCPY-1007];上海市临床重点专科—中医肾病科(shslczdzk04201);上海中医药大学附属龙华医院科研基金面上项目(2018YM01)。

摘　　要：目的 探讨葛根素对高糖环境下H_(2)O_(2)诱导的人永生型足细胞氧化应激及线粒体损伤的影响及其作用机制。方法将足细胞分组给予高糖(30 mmol/L Glucose)及不同浓度H_(2)O_(2)刺激,同时加不同浓度葛根素干预48 h。分组如下:H0组(H_(2)O_(2)0μmol/L)、H100组(H_(2)O_(2)100μmol/L)、H100+P3.3组(H_(2)O_(2)100μmol/L+葛根素3.3μmol/L)、H100+P10组(H_(2)O_(2)100μmol/L+葛根素10μmol/L)、H200组(H_(2)O_(2)200μmol/L)、H200+P3.3组(H_(2)O_(2)200μmol/L+葛根素3.3μmol/L)、H200+P10组(H_(2)O_(2)200μmol/L+葛根素10μmol/L)。CCK-8检测细胞存活率,Western blotting检测TOMM22的蛋白表达;提取基因组DNA,qPCR检测mtDNA的表达;提取RNA,qPCR检测TFAM、NRF1 mRNA表达;测定各组ATP水平、ROS水平,并评价各组足细胞线粒体呼吸功能。结果 与H0组相比,H100、H200组ROS水平升高(P<0.01),H100、H100+P3.3组mtDNA拷贝数均减少(P<0.01),TFAM mRNA表达降低,基础呼吸、质子渗漏、最大耗氧量、非线粒体氧消耗均升高(P<0.05);与H100组相比,H100+P3.3组ROS减少(P<0.05),TFAM mRNA、NRF1 mRNA表达均增加(P<0.05,P<0.01),ATP生成增加(P<0.05),基础呼吸、质子渗漏均降低(P<0.05),而H100+P10组mtDNA拷贝数增加(P<0.05),NRF1 mRNA、基础呼吸、质子渗漏、非线粒体消耗均升高(P<0.05);与H100+P3.3组相比,H100+P10组mtDNA拷贝数明显增加(P<0.01),基础呼吸、质子渗漏、非线粒体氧消耗、ATP水平均升高(P<0.05);与H200组相比,H200+P3.3、H200+P10组ROS水平明显降低(P<0.01),H200+P10组NRF1 mRNA表达增加(P<0.01)、非线粒体氧消耗增加(P<0.05);与H200+P3.3组相比,H200+P10组ROS水平增多(P<0.01),TFAM mRNA表达增加(P<0.05)、NRF1 mRNA表达增加(P<0.01)。各组TOMM22蛋白表达、备用呼吸能力、耦合效率和备用呼吸率比较,差异无统计学意义(P>0.05)。结论 葛根素能部分缓解高糖及H_(2)O_(2)诱导的足细胞氧化应激及线粒体损伤。Objective To explore the effect of puerarin on oxidative stress and mitochondrial damage in human immortal podocytes induced by H_(2)O_(2) under high glucose environment and its mechanism.Methods Human immortal podocytes were stimulated with different concentrations of H_(2)O_(2) in a high-glucose environment,and intervened by different concentrations of puerarin for 48 hours.The groups were divided as follows:group H0(H_(2)O_(2) 0 μmol/L),group H100(H_(2)O_(2) 100 μmol/L),group H100+P3.3(H_(2)O_(2) 100 μmol/L+puerarin 3.3 μmol/L),group H100+ P10(H_(2)O_(2) 100 μmol/L+ puerarin 10 μmol/L),group H200(H_(2)O_(2) 200 μmol/L),group H200+P3.3(H_(2)O_(2) 200 μmol/L+ puerarin 3.3 μmol/L),group H200+P10(H_(2)O_(2) 200 μmol/L+puerarin 10 μmol/L).CCK-8 was used to detect cell viability.Western blotting was used to detect the protein expression of TOMM22;Genomic DNA was extracted and qPCR was used to detect the expression of mtDNA;RNA was extracted and qPCR was used to detect the mRNA expression of TFAM and NRF1;the levels of ATP and ROS in each group were measured and the mitochondrial respiratory function were evaluated.Results Compared with H0 group,ROS level was increased in H100 and H200 groups(P<0.01),mtDNA copy number was decreased in H100 and H100+P3.3 groups(P<0.01),TFAM mRNA expression was decreased,basal respiration,proton leakage,maximum oxygen consumption and nonmitochondrial oxygen consumption were increased(P<0.05).Compared with H100 group,ROS in H100+P3.3 group was decreased(P<0.05),TFAM mRNA and NRF1 mRNA expression were increased(P<0.05,P<0.01),ATP production was increased(P<0.05),basal respiration and proton leakage were decreased(P<0.05) in group H100+P3.3.In H100+P10 group,mtDNA copy number was increased(P<0.05),NRF1 mRNA,basal respiration,proton leakage and non-mitochondrial consumption were increased(P<0.05).Compared with H100+P3.3 group,the mtDNA copy number of H100+P10 group was significantly increased(P<0.01),basal respiration,proton leakage,non-mitochondrial oxygen consumption and ATP l

关 键 词：葛根素 糖尿病性肾脏疾病 氧化应激 线粒体损伤 

分 类 号：R285[医药卫生—中药学]