【作者】 邵荟竹；

【导师】 姚继红；

【作者基本信息】 大连医科大学 ， 药理学， 2010， 硕士

【摘要】 目的：研究鼠尾草酸通过二相酶的诱导对HepG2细胞H202损伤的保护作用与PI3K/Akt-Nrf2转录途径之间的关系。方法：1.鼠尾草酸对HepG2细胞二相酶的诱导作用HepG2细胞随机分为四组：对照组；低、中、高浓度鼠尾草酸组(5μM、10μM,20μM)。药物与细胞共同孵育不同时间后分别测定以下指标：采用化学分光光度法测定醌氧化还原酶(NQO 1)的活性和还原型谷胱甘肽(GSH)的活性；用免疫印迹法(Western blotting)测定转录因子Nrf2、NQO1和血红素氧合酶-1(HO-1)的蛋白表达。2.鼠尾草酸对HepG2细胞二相酶诱导的信号转导通路研究(1)HepG2细胞随机分为：对照组;20μM鼠尾草酸组；20μM鼠尾草酸+阻断剂组(P13K通路阻断剂LY294002组、NQO1抑制剂Dicoumarol组、HO-1抑制剂ZNPPIX组)。Western blotting法测定转录因子Nrf2、NQO1和HO-1的蛋白表达。(2)HepG2细胞随机分为：对照组；PI3K阻断剂组；20μM鼠尾草酸组;20μM鼠尾草酸+PI3K阻断剂组Western blotting法测定Akt和P-Akt的蛋白表达。3.鼠尾草酸对H202损伤的HepG2细胞保护作用研究(1)HepG2细胞随机分为：对照组；H202损伤组(终浓度为2.4 mM)；低、中、高浓度鼠尾草酸治疗组(5μM、10μM、20μM)。采用MTT法测定细胞存活率；采用化学分光光度法测定细胞上清液中LDH活性。(2)HepG2细胞随机分为：对照组；H202损伤组；20μM鼠尾草酸治疗组；20μM鼠尾草酸+阻断剂组。采用MTT法测定细胞存活率；采用化学分光光度法测定细胞中LDH活性。结果：1.鼠尾草酸与HepG2细胞共同孵育8h后,与对照组相比,药物组的细胞内二相酶GSH和NQO1活性均明显提高(P<0.01,P<0.01),在5-20μM呈浓度依赖性。16h时,上述两种酶的活性均有不同程度的降低,呈一定的时间相关性。2.鼠尾草酸与HepG2细胞共同孵育8h后,与对照组相比,中、高浓度药物组细胞内Nrf2、NQO1和HO-1的蛋白表达显著提高(P<0.01,P<0.01,P<0.01),并呈一定的浓度依赖性。3.PI3K通路的特异性阻断剂LY294002可以明显抑制鼠尾草酸诱导的HepG2细胞Nrf2、NQO1和HO-1的蛋白表达(P<0.01,P<0.01,P<0.01)。鼠尾草酸诱导的NQO1和HO-1的蛋白表达还可分别被特异的NQO1抑制剂Dicoumarol、HO-1抑制剂ZNPPIX所抑制(P<0.01,P<0.01)。4.与对照组相比,鼠尾草酸不影响Akt的水平,但可明显增加P-Akt的蛋白表达水平。在鼠尾草酸同时存在或不存在时,PI3K通路的特异性阻断剂LY294002可明显减少P-Akt的蛋白表达。5.H2O2刺激HepG2细胞后,与对照组相比,损伤组细胞生存率明显下降,LDH活性明显增高(P<0.01,P<0.01),而中、高浓度鼠尾草酸处理组细胞生存率显著提高,LDH活性显著下降(P<0.01,P<0.01),呈浓度依赖性。而且,特异性PI3K通路阻断剂LY294002、NQO1抑制剂Dicoumarol以及HO-1抑制剂ZNPPIX可以部分取消鼠尾草酸的细胞保护作用。结论：1.鼠尾草酸通过激活HepG2细胞的转录途径PI3K/Akt-Nrf2上调二相代谢酶HO-1、NQO1、GSH的活性和蛋白表达,并且呈浓度依赖性和时间相关性。2.鼠尾草酸对H2O2损伤的HepG2细胞具有保护作用,其机制主要与鼠尾草酸激活PI3K/Akt-Nrf2通路,显著提高HepG2细胞二相代谢酶NQO1、HO-1等的表达有关。更多还原

【Abstract】 Objective:To investigated whether Carnosic acid (CA) could induce antioxidant and phase 2 enzymes and confer protection against oxidative damage in HepG2 cells.Methods:HepG2 cells were randomly divided into four groups (n=3): control group; carnosic acid (CA) group (5μM、10μM、20μM). The activity of NAD(P)H:quinone oxidore-ductase (NQO1) in HepG2 cells were measured by 2,6-dichloroindophenol reduction method. The activities of Glutathione Reductase (GSH) were determined with the method of chemical colorimetry. The protein expressions of nuclear factor E2-related factor2 (Nrf2), NQO1 and Heme oxygenase-1 (HO-1) in HepG2 cells were detected by Western blotting. HepG2 cells were randomly divided into five groups (n=3):control group; 20μM CA group; 20μM CA+inhibitor group (LY294002; Dicoumarol; ZNPPIX). The protein expressions of Nrf2, NQO1 and HO-1 in HepG2 cells were detected by Western blotting.HepG2 cells were randomly divided into four groups (n=3):control group; PI3K inhibitor group; 20μM CA group; 20μM CA group+PI3K inhibitor group. The protein expressions of Akt and P-Akt in HepG2 cells were detected by Western blotting. HepG2 cells were randomly divided into five groups and damaged by H2O2 (n=4):control group; H2O2 groups; CA treated group (5μM、10μM、20μM). MTT method was used to examine cell survival rate. The activities of lactate dehydrogenase (LDH) were determined with the method of chemical colorimetry. Then HepG2 cells were randomly divided into six groups (n=4):control group; 20μM CA treated group; 20μM CA group+inhibitor (LY294002, Dicoumarol, ZNPPIX) treated group. MTT method was used to examine cell survival rate. The activities of lactate dehydrogenase (LDH) were determined with the method of chemical colorimetry.Results:The results showed that CA could induce Heme Oxygenasel (HO-1), quinone oxidoreductase 1 (NQO1) and glutathione reductase (GSH) expression in HepG2 cells in a time-and dose-dependent manner and protect them against hydrogen peroxide caused oxidative damage. The induction of HO-1 and NQO1 by CA was accompanied with the accumulation of nuclear factor-E2-related factor-2 (Nrf2) in nucleus and the activation of phosphatidylinositol 3-kinase PI3K/Akt signal pathway. Additionally, the specific inhibitor of PI3K/Akt could obviously decrease not only the induced expression of HO-1, NQO1 and GSH but also the antioxidant effect of CA. In conclusion, this study proved that CA exerts antioxidant effect by inducing HO-1, NQO1 and GSH expression mediated by PI3K/Akt and Nrf2.Conclusion:The result indicated that CA increased the antioxidant and phase 2 enzymes through an activation of the PI3K-Akt/Nrf2 pathway. This may be a primary mechanism of antioxidative action of CA concerned with its therapeutic effectiveness in HepG2 cells.更多还原