NADPH氧化酶在瘦素诱导肝星状细胞株HSC—T6产生活性氧中的作用及其机制  

The role and mechanism of NADPH oxidase in leptin-induced reactive oxygen species production in hepatic stellate cells

作　　者：何文华[1] 李博[1] 朱萱[1] 张煜和[1] 李弼民[1] 刘志坚[1] 刘戈云[1] 王剑[1] 

机构地区：[1]南昌大学第一附属医院消化内科,330006

出　　处：《中华肝脏病杂志》2010年第11期849-854,共6页Chinese Journal of Hepatology

摘　　要：目的 研究NADPH氧化酶（NOX）是否参与瘦素诱导肝星状细胞（HSC）产生活性氧（ROS）,并探讨其机制. 方法取对数生长期的HSC-T6细胞,随机分为9组：瘦素组、瘦素＋ROS生成系统抑制剂[包括氯化二亚苯基碘嗡（DPI）、鱼藤酮、甲双吡丙酮、别嘌呤醇和吲哚美辛]组成的干预组、瘦素＋JAK抑制剂AG490组成的干预组、正常对照组和阴性对照组.HSC-T6细胞经药物作用1、12、24 h后,荧光显微镜和（或）流式细胞术观察细胞内二氯荧光素强度来反应ROS水平;分光光度计检测NADPH的吸光度,以NADPH的消耗量表示NOX活性;逆转录聚合酶链反应检测Rac1和p22Phox的mRNA相对表达量.数据分析用单因素方差分析、SNK-q检验或秩和检验,P＜0.05为差异有统计学意义.结果 HSC-T6细胞加入瘦素（100 ng/ml）作用1 h后,荧光强度较正常对照组明显升高（92.91±4.19比27.56±6.27,P＜0.01）;DPI（20 μmol/L）和AG490（50 μ mol/L）能抑制ROS的产生,荧光强度值分别为37.35±4.66和53.12±6.63,均低于瘦素组的92.91±4.19（q值分别为31.78和22.76,P值均＜0.01）.瘦素作用HSC-T6细胞1 h后,NADPH的消耗量较正常对照组明显升高[（1.90±0.22）pmol·min-1·mg1比（0.76±0.06）pmol·min-1·mg-1,P＜0.05）],而作用12、24 h后的消耗量明显高于1 h时的消耗量（x2值均为7.54,P值均＜0.05）;DPI及AG490干预能抑制瘦素诱导NOX活性的上调（q值分别为16.58和16.23,P值均＜0.05）.瘦素作用12 h后,HSC-T6细胞内Racl和p22Phox的mRNA相对表达量明显高于正常对照组（分别为0.41±0.13比0.14±0.08和0.45±0.12比0.20±0.08,P值均＜0.05）.结论 瘦素诱导HSC产生的ROS主要来源于NOX,其机制可能是瘦素通过JAK信号通路直接激活NOX,并通过上调NOX的亚基表达使NOX活性持续增高而产生大量ROS.Objective To investigate whether or not NADPH oxidase （NOX） participates in leptininduced reactive oxygen species （ROS） production in hepatic stellate cells （HSC） and to explore the possible mechanism. Methods HSC-T6 cells （rat hepatic stellate cells line） were divided into nine groups： Groupl：leptin （100 ng/ml） treated; Group2-6： leptin treated together with inhibitors that block different ROS-producing systems： diphenylene-iodonium （DPI） （20 μmol/L）, Rotenone （20 μmol/L）, Metyrapone （250 μ mol/L）,Allopurinol （100 μmol/L） and Indomethacin（100 μmol/L）; Group7： leptin treated together with Janus kinase （JAK） inhibitor AG490 50 μmol/L; Group8： normal control group（treated DMEM with 0.1% DMSO）; Group9：negative control group （untreated）. Intracellular ROS levels were measured with dichlorodihydrofluorescein diacetate （DCFH-DA） dye assay by Fluorescence microscope and/or flow cytometry. NOX activity was analyzed by using spectrophotometer to calculate the absorbance of NADPH. The mRNA levels of Rac1 and p22Phox were evaluated by RT-PCR. Results （1） Leptin increased significantly the ROS production as compared to normal control group （92.91 ± 4.19 vs.27.56 ± 6.27, P 〈 0.01） in HSC-T6 cells. Both the NADPH oxidase inhibitor DPI and AG490 （50 μmol/L） blocked the ROS production, inhibitors of other ROS producing systems had no significant effect on ROS production induced by lepin （P〉0.05）. （2） Leptin treated HSC-T6 cells for 1 hour up-regulated the NOX activity significantly compared with that in normal control group activity increased after being treated with leptin for 12 hours and 24 hours than being treated for 1 hour.Leptin-induced up-regulation of NOX activity was inhibited by pretreatment with DPI or AG490. （3） The RTPCR results indicated that mRNA expressions of Rac1 and p22Phox in HSC-T6 cells with 12 hours of leptin stimulation increased significantly as compared with normal control group （0.41 ± 0.1

关 键 词：NADPH氧化酶 瘦素 活性氧组分 肝星状细胞 

分 类 号：R686[医药卫生—骨科学]