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目的:探讨活性氧(ROS)在血管紧张素Ⅱ(AngⅡ)诱导的c-Jun氨基末端激酶(JNK)/活化蛋白(AP-1)信号通路活化及系膜细胞增殖中的作用并探讨其来源。方法:体外培养人肾小球系膜细胞,应用3H-胸腺嘧啶(3H-TdR)掺入法和细胞计数测定系膜细胞增殖;荧光探针2,7-二氯二氢荧光素乙酰乙酸(DCFDA)检测细胞内ROS的产生;化学发光法检测尼克酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性;WesternBlot检测JNK活化。结果:AngⅡ呈时间依赖性和剂量依赖性促进肾小球系膜细胞ROS产生。AngⅡ刺激3min,系膜细胞内ROS产生明显增加,至60min达到高峰。AT1R血管紧张素Ⅱ-1型受体(AT1R)拮抗剂氯沙坦完全阻断了AngⅡ诱导的ROS产生。NADPH氧化酶抑制剂夹竹桃麻素和二联苯碘(DPI)几乎完全阻断AngⅡ诱导的ROS产生,而线粒体复合体Ⅰ抑制剂鱼藤酮、黄嘌呤氧化酶抑制剂别嘌醇、环氧化酶抑制剂吲哚美辛、脂氧化酶抑制剂去甲二氢化愈创木酸、细胞色素P450氧化酶抑制剂酮康唑以及一氧化氮合成酶抑制剂N-硝基-L-精氨酸甲酯(L-NAME)对AngⅡ诱导的ROS产生均无明显影响。AngⅡ显著刺激NADPH氧化酶活化及p47phox和p67phox膜转位。氯沙坦、乙酰半胱氨酸(NAC)、夹竹桃麻素和DPI阻断AngⅡ诱导的JNK/AP-1活化和系膜细胞增殖。结论:NADPH氧化酶来源的ROS调控AngⅡ诱导的JNK/AP-1信号通路活化及系膜细胞增殖。NADPH氧化酶抑制剂能显著抑制AngⅡ诱导的系膜细胞增殖,可能具有一定的治疗作用。
Objective: To investigate the role of reactive oxygen species (ROS) in activation of c-Jun N-terminal kinase (JNK) / activator protein (AP-1) signaling pathway and mesangial cell proliferation induced by angiotensin Ⅱ . Methods: Human glomerular mesangial cells were cultured in vitro. The proliferation of mesangial cells was determined by 3H-thymidine incorporation and cell counting. The fluorescence intensity of 2,7-dichlorodihydrofluorescein acetoacetate DCFDA). The chemiluminescence method was used to detect nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Western Blot was used to detect the activation of JNK. Results: Ang Ⅱ promoted the production of ROS in mesangial cells in a time - and dose - dependent manner. Ang Ⅱ stimulated 3min, mesangial cells ROS production increased significantly, reaching a peak at 60min. Losartan, an AT1R angiotensin II-1 receptor (AT1R) antagonist, completely blocked Ang II-induced ROS production. NADPH oxidase inhibitors apocynin and diphenyliodide (DPI) almost completely blocked the production of ROS induced by Ang II, whereas the inhibitors of mitochondrial complex I, rotenone, xanthine oxidase inhibitor allopurinol, epoxidation Indomethacin, an enzyme inhibitor, nordihydratable acid, a lipoxygenase inhibitor, and ketoconazole, a cytochrome P450 oxidase inhibitor, and a nitric oxide synthase inhibitor, N-nitro-L-arginine Methyl ester (L-NAME) had no significant effect on Ang II-induced ROS production. AngⅡ significantly stimulated NADPH oxidase activation and p47phox and p67phox membrane translocation. Losartan, acetylcysteine (NAC), apocynin and DPI blocked AngⅡ-induced JNK / AP-1 activation and mesangial cell proliferation. Conclusion: NADPH oxidase - derived ROS regulates Ang Ⅱ - induced JNK / AP-1 signaling pathway and mesangial cell proliferation. NADPH oxidase inhibitor can significantly inhibit Ang Ⅱ induced mesangial cell proliferation, may have a therapeutic effect.