AngⅡ-ROS-PKC/Nrf2/HO-1通路调控LX-2细胞增殖的机制研究  

Mechanism of AngⅡ-ROS-PKC/Nrf2/HO-1 Pathway Regulating Proliferation of LX-2 Cells

作　　者：董文珠 曹晓倩 王璐[1] 陈杭萍 董叶娇 方佳敏 张敏[1] 惠慧 姚立[1] DONG Wenzhu;CAO Xiaoqian;WANG Lu;et al(Zhejiang Chinese Medical University, Hangzhou（310053）,China)

机构地区：[1]浙江中医药大学,杭州310053

出　　处：《浙江中医药大学学报》2018年第4期319-325,共7页Journal of Zhejiang Chinese Medical University

基　　金：国家自然科学基金(81173640);浙江省自然科学基金(LY16H280009);浙江中医药大学预研项目(2016ZG11)~~

摘　　要：[目的]研究血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)对体外培养的LX-2细胞增殖的影响,并探讨蛋白激酶C(protein kinase C,PKC)/核因子相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)/血红素氧化酶(hemeoxygenase-1,HO-1)通路在其中的作用。[方法]采用MTT法检测不同浓度AngⅡ作用不同时间对LX-2细胞增殖的影响,流式细胞术检测AngⅡ刺激后不同时间点LX-2细胞的活性氧簇(reactive oxygen species,ROS)表达水平,Western blot技术检测PKC、Nrf2、HO-1蛋白表达水平。[结果]与正常组比较,当AngⅡ浓度为10-5mol·L-1时,LX-2细胞增殖有统计学差异(P<0.01)。与正常组比较,以10-5mol·L-1的AngⅡ刺激LX-2细胞0.5、24h时,细胞增殖有统计学差异(P<0.05),ROS表达显著增加(P<0.05)。Western blot提示,与正常组比较,10-5mol·L-1的AngⅡ刺激LX-2细胞2、24h后,PKC蛋白表达显著增加(P<0.05,P<0.01);刺激细胞2、12、24h后,Nrf2蛋白表达显著增加(P<0.05,P<0.01,P<0.001);刺激细胞24h后,HO-1蛋白表达显著增加(P<0.05)。[结论]AngⅡ刺激LX-2细胞后,早期即可产生ROS,促进细胞增殖,并迅速激活PKC,进而诱导Nrf2从复合物解离并移位入核,激发HO-1的表达,启动了细胞自身潜在的抗氧化作用。[Objective] To investigate the effect of ROS stimulated by angiotensin Ⅱ（AngⅡ） on the proliferation of LX-2 cells and to explore the regulation pathway of the protein kinase C（PKC）/nuclear factor erythroid-2 related factor 2（Nrf2）/hemeoxygenase-1（HO-1）. [Methods] MTT assay was used to detect the proliferation of LX-2 cells induced by Ang Ⅱ. The reactive oxygen species（ROS） of LX-2 cells at various times after being stimulated by Ang Ⅱ were detected by fluorescence probe DCFH-DA and flow cytometry. The protein expression of PKC, Nrf2 and HO-1 was detected by Western blot. [Results]Compared with the normal group, LX-2 cells treated with AngⅡ（10-5 mol·L-1） showed significant difference in cell proliferation（P〈0.01）. Compared with the normal group, when LX-2 cells were stimulated with AngⅡ（10-5 mol·L-1） for 0.5 h and 24 h, there was a significant difference in proliferation（ P〈0.05）, ROS increased significantly（P〈0.05）. Compared with the normal group, Western blot showed that the protein expression of PKC increased significantly with Ang Ⅱ（10-5 mol·L-1） stimulation for 2 h and 24 h（P〈0.05, P〈0.01）; the protein expression of Nrf2 increased significantly for 2 h, 12 h and 24 h（P〈0.05, P〈0.01, P〈0.001）;HO-1 protein expression increased significantly for 24 h（P〈0.05）. [Conclusion] AngⅡ stimulates LX-2 cells to produce ROS at early stage which promotes cell proliferation and activates PKC rapidly, then PKC induces Nrf2 to dissociate from the complex and shift into the nucleus, and Nrf2 stimulates the expression of HO-1 which initiates the potential antioxidant effect.

关 键 词：肝纤维化 机制 血管紧张素Ⅱ 肝星状细胞 增殖 氧化应激 

分 类 号：R331[医药卫生—人体生理学]