AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UI...AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucoseoxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species(ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase(JNK), insulin signal essential molecules such as insulin receptor substrate-1(IRS-1), protein kinase B(Akt), glycogen synthase kinase-3β(GSK-3β), and glucose transporter-2(Glut 2), and NADPH oxidase subunits such as gp91 phox, p67 phox, p47 phox, p40 phox, and p22 phox were evaluated by Western blot.RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption(P < 0.05)and glycogen content(P < 0.01) in Hep G2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression(P < 0.01) and phosphorylation of IRS-1(P < 0.05), associated with down-regulation of Akt(P < 0.05) and GSK-3β(P < 0.05) phosphorylation levels, and the expression of Glut 2(P < 0.001), indicating an insulin-resistance state in Hep G2 cells. Furthermore, UII enhanced the phosphorylation of JNK(P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1(P < 0.001), phosphorylation of IRS-1(P < 0.001) and GSK-3β(P < 0.05), and glycogen synthesis(P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation(P < 0.05) and NADPH oxidase subunit expression(P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production(P < 0.05), JNK phosphorylation(P < 0.05), and insulin resistance(P < 0.05) in HepG 2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG 2 cells.展开更多
目的研究贝那普利对肝纤维化进程中肝组织还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)及血管紧张素Ⅱ(AngⅡ)表达的影响,探讨贝那普利可能的抗纤维化机制。方法将22只SD大鼠随机分为3组:正常对照组(n=6)、模型组(n=8)、贝那普利治疗组...目的研究贝那普利对肝纤维化进程中肝组织还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)及血管紧张素Ⅱ(AngⅡ)表达的影响,探讨贝那普利可能的抗纤维化机制。方法将22只SD大鼠随机分为3组:正常对照组(n=6)、模型组(n=8)、贝那普利治疗组(n=8)。制备40%四氯化碳(CCl_4)诱导的大鼠肝纤维化模型,贝那普利治疗组同时应用贝那普利灌胃,共8周。对肝组织行常规HE及Masson染色,观察其病理变化;利用双抗体夹心ELISA(ABC-ELISA)法测定肝匀浆血管紧张素Ⅱ(AngⅡ)浓度;逆转录-聚合酶链反应(RT-PCR)检测肝组织NOX mRNA的表达量。结果模型组肝匀浆AngⅡ浓度较正常对照组明显升高[(48.625±1.357)ng/ml vs(23.330±1.606)ng/ml,P<0.05],模型组肝组织NOX4 mRNA表达较对照组明显升高(31.78±3.96 vs 2.03±0.31,P<0.05)。治疗组肝匀浆AngⅡ浓度较模型组有所下降[(45.518±3.072)ng/ml vs(48.625±1.357)ng/ml,P<0.05],治疗组肝组织NOX4 mRNA的表达较模型组明显下降(15.93±5.01 vs 31.78±3.96,P<0.05)。三组大鼠肝组织中AngⅡ与NOX4表达变化呈正相关(r=0.472,P<0.05)。结论贝那普利可能通过抑制肝纤维化过程中AngⅡ的表达并进一步抑制NOX4介导的氧化应激反应发挥其抗纤维化作用。展开更多
基金Supported by National Natural Science Foundation of China,No.81272757the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality,No.IDHT20150502
文摘AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucoseoxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species(ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase(JNK), insulin signal essential molecules such as insulin receptor substrate-1(IRS-1), protein kinase B(Akt), glycogen synthase kinase-3β(GSK-3β), and glucose transporter-2(Glut 2), and NADPH oxidase subunits such as gp91 phox, p67 phox, p47 phox, p40 phox, and p22 phox were evaluated by Western blot.RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption(P < 0.05)and glycogen content(P < 0.01) in Hep G2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression(P < 0.01) and phosphorylation of IRS-1(P < 0.05), associated with down-regulation of Akt(P < 0.05) and GSK-3β(P < 0.05) phosphorylation levels, and the expression of Glut 2(P < 0.001), indicating an insulin-resistance state in Hep G2 cells. Furthermore, UII enhanced the phosphorylation of JNK(P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1(P < 0.001), phosphorylation of IRS-1(P < 0.001) and GSK-3β(P < 0.05), and glycogen synthesis(P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation(P < 0.05) and NADPH oxidase subunit expression(P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production(P < 0.05), JNK phosphorylation(P < 0.05), and insulin resistance(P < 0.05) in HepG 2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG 2 cells.
文摘目的研究贝那普利对肝纤维化进程中肝组织还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)及血管紧张素Ⅱ(AngⅡ)表达的影响,探讨贝那普利可能的抗纤维化机制。方法将22只SD大鼠随机分为3组:正常对照组(n=6)、模型组(n=8)、贝那普利治疗组(n=8)。制备40%四氯化碳(CCl_4)诱导的大鼠肝纤维化模型,贝那普利治疗组同时应用贝那普利灌胃,共8周。对肝组织行常规HE及Masson染色,观察其病理变化;利用双抗体夹心ELISA(ABC-ELISA)法测定肝匀浆血管紧张素Ⅱ(AngⅡ)浓度;逆转录-聚合酶链反应(RT-PCR)检测肝组织NOX mRNA的表达量。结果模型组肝匀浆AngⅡ浓度较正常对照组明显升高[(48.625±1.357)ng/ml vs(23.330±1.606)ng/ml,P<0.05],模型组肝组织NOX4 mRNA表达较对照组明显升高(31.78±3.96 vs 2.03±0.31,P<0.05)。治疗组肝匀浆AngⅡ浓度较模型组有所下降[(45.518±3.072)ng/ml vs(48.625±1.357)ng/ml,P<0.05],治疗组肝组织NOX4 mRNA的表达较模型组明显下降(15.93±5.01 vs 31.78±3.96,P<0.05)。三组大鼠肝组织中AngⅡ与NOX4表达变化呈正相关(r=0.472,P<0.05)。结论贝那普利可能通过抑制肝纤维化过程中AngⅡ的表达并进一步抑制NOX4介导的氧化应激反应发挥其抗纤维化作用。
