NRF2在脂肪肝细胞衰老及其激活肝星状细胞促纤维化进程中的作用机制研究

Nonalcoholic fatty liver cells have a tendency to senescence, and can induce liver fibrosis. The objective of this study is to find a more effective clinical treatment strategy by elaborating the mechanisms of hepatocyte senensence on liver fibrosis process in NAFLD. Previous studies had showed that NRF2 was closely related to cell senescence and liver fibrosis induced by hepatic stellate cells. This topic will focus on the role and regulatory mechanisms of NRF2 in the process of activating hepatic stellate cells and promoting liver fibrosis. In our study, we will detect the activity of Keap1-NRF2 - ARE signaling pathway and the expression level of NRF2 protein under cell senescence induced by nonalcoholic fatty liver model in vitro. We will detect the effects of cell senescence on activation of HSC and the process of fibrosis in cell co-culture model with fatty liver cells：HSC (5:1). In fatty liver cells，we will detect the effects of cell senescence on activation of HSC and the process of fibrosis when we alter the expression of NRF2 in cell co-culture model with fatty liver cells：HSC (5:1). The effect of NRF2 in hepatocyte senescence and liver fibrosis will be validated in the mouse model of NAFLD induced by high-fat +CCL4. The significance of this study is whether NRF2 intervention in liver cells will be helpful to slow down the process of liver fibrosis in NAFLD.

非酒精性脂肪性肝病（NAFLD）肝细胞出现早期衰老现象，且易于进展为肝纤维化阐述脂肪肝细胞衰老及肝纤维化的机制，寻找更有效的临床治疗策略是本项目关注点。NRF2被报道与细胞衰老和肝星状细胞（HSC）活化密切相关，本课题重点阐述NRF2通过促脂肪肝细胞衰老，进而激活HSC促肝纤维化的作用机制。通过体外诱导正常肝细胞脂肪化发生细胞衰老，检测Keap1-NRF2-ARE信号通路活性及NRF2的表达；采用细胞共培养方式，脂肪肝细胞：HSC=5:1模拟体内肝环境，检测细胞衰老对HSC活化和纤维化进程的影响；在脂肪肝细胞中人为改变NRF2的表达水平，检测细胞衰老的改变及细胞衰老改变后对HSC活化和纤维化进程的影响。NRF2在脂肪肝细胞衰老及激活HSC促纤维进程中的作用机制在高脂+CCL4诱导的NAFLD小鼠模型中进行验证。本项目意义在于对NRF2进行干预是否更有利于减缓NAFLD肝纤维化进程。

非酒精性脂肪性肝病（NAFLD）肝细胞可出现衰老现象，易进展为肝纤维化，研究脂肪肝细胞衰老及肝纤维化机制，将对NAFLD相关性肝纤维化的诊治具有重要意义。据报道，NRF2与细胞衰老和肝星状细胞（HSC）活化密切相关，故本课题拟研究NRF2通过影响脂肪肝细胞衰老，激活HSC促肝纤维化的机制。本项目通过体外诱导AML12细胞脂肪化，检测衰老标记物、衰老基因及细胞周期蛋白的表达，检测Keap1-NRF2-ARE信号通路活性；采用细胞共培养方式，脂肪肝细胞：HSC=5:1模拟体内肝环境，检测脂肪肝细胞衰老对HSC活化和纤维化进程的影响；在脂肪肝细胞中干扰及过表达NRF2，检测细胞衰老的改变及其对HSC活化和纤维化进程的影响；通过高脂高糖诱导建立NAFLD肝纤维化小鼠模型，抑制NRF2的表达，检测肝脏衰老、HSC活化和纤维化相关指标的表达。结果提示亚油酸和棕榈酸混合物诱导AML12细胞72小时可构建较好的脂肪肝细胞模型，细胞呈现明显的衰老现象，表现为细胞衰老-β-半乳糖苷酶和衰老基因p21表达上调；G1期细胞百分比增加，S期、G2期细胞百分比降低；AML12细胞脂肪化后NRF2表达上调，NRF2上游多个抑制性调控基因表达下调，但Keap1-NRF2-ARE信号通路活性并未相应增强， AML12细胞脂肪化后细胞衰老；敲除NRF2可以抑制Keap1-NRF2-ARE信号通路活性，过表达NRF2可以激活该信号通路活性；脂肪肝细胞与肝星状细胞共培养后，HSC活化因子（α-SMA、Desmin）和肝纤维化相关因子（Collagen I、Collagen III、Collagen IV、Fibronectin、MMP2、MMP9、ICAM-1）的表达量明显高于对照组；过表达NRF2可激活Keap1-NRF2-ARE信号通路活性，抑制HSC的激活，降低纤维化相关因子的表达；敲除NRF2可抑制Keap1-NRF2-ARE信号通路活性，促进纤维化相关因子的表达；以高糖高脂喂养小鼠16周可以建立NAFLD小鼠相关性肝纤维化动物模型；以包裹敲减Nrf2的腺病毒载体干预NAFLD，可降低抗氧化酶的表达，增加氧化应激产物的表达，增加HSC活化因子和肝纤维化相关因子的表达。本项目结果提示肝细胞脂肪化引起细胞衰老后可激活HSC，促进肝纤维化进程；干扰NRF2的表达可以影响NAFLD相关性肝纤维化进程。