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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 ± 0.13 vs 0.14 ± 0.08,0.45 ± 0.12 vs 0.20 ± 0.08, all P ＜ 0.05), while the DPI and AG490 had no effect on the mRNA expressions of Racl and p22Phox. Conclusion NOX is the main cellular source of the reactive oxygen species (ROS)generated by HSCs in response to leptin stimulation. The mechanism is probably that leptin can directly activate NOX through JAK signal transduction and hence induce the expression of NOX subunit to promote the activity of NOX which generates considerable ROS in HSC.