JNK/p38MAPK信号通路在氧化应激诱导鲤肝细胞凋亡中的作用及机制研究

Oxidative stress-induced apoptosis is a crucial factor for liver injury and dysfunction in fish, however, it remains unclear how the oxidative stress induces hepatocyte apoptosis. Our previous research showed that the mRNA levels of JNK and p38MAPK significantly up-regulated in carp (Cyprinus carpio) apoptotic hepatocyte. Thus, we hypothesize that JNK/p38MAPK signaling pathways may be the key factor which mediates oxidative stress-induced hepatocyte apoptosis in carp. To prove the hypothesis, hydrogen peroxide (H2O2) will be used to establish carp liver injury model (in vivo and in vitro). Based on the established model, we will investigate the correlation between JNK/p38MAPK signaling pathways and apoptosis using molecular and biochemical approaches, such as quantitative real-time PCR (qRT-PCR), western blot, flow cytometry and so on. Subsequently, the effects of activated or inactivated JNK/p38MAPK signaling pathways on apoptosis will be studied via specific inhibitor blocking and RNA-seq. Meanwhile, the molecular network pathway of JNK/p38MAPK regulating apoptosis in oxidative stress-induced hepatocyte will be further find out. Based on above experiments, we will elucidate the role of JNK/p38MAPK signaling pathways in oxidative stress-induced hepatocyte apoptosis both in vivo and in vitro. This study will also provide a new insight to pathology research and prevention of fish liver injury.

氧化应激诱导的细胞凋亡是引起鱼类肝损伤和功能异常的重要因素，但其诱导鱼类肝细胞凋亡的机制尚不明确。项目组前期的研究发现，鲤肝细胞凋亡与JNK和p38MAPK基因表达上调具有密切的关系。因此，我们提出了JNK/p38MAPK信号通路可能是介导鲤肝细胞凋亡关键环节的研究假说。为了论证这一假说，本项目以鲤为研究对象，以过氧化氢（H2O2）为诱导剂构建氧化应激肝损伤模型（体内和体外）。采用荧光定量PCR、Western Blot和流式细胞术等技术分析JNK/p38MAPK信号通路与肝细胞凋亡的相关性。同时采用信号通路阻断剂和转录组测序等技术，研究JNK/p38MAPK信号通路激活或失活对细胞凋亡的影响，并在转录组水平发掘该信号通路调控细胞凋亡的分子网络路径。综合体内、外实验结果，本项目将阐明JNK/p38MAPK信号通路在氧化应激诱导鲤肝细胞凋亡中的作用，为鱼类肝损伤疾病的研究和防治提供新思路。

氧化应激被认为是鱼类多种肝损伤疾病的共同诱发因素，其诱导的细胞凋亡为鱼类肝损伤的重要机制之一。本研究以鲤为研究对象，用H2O2为氧化剂，诱导鲤在体肝组织和离体肝细胞，构建氧化应激损伤模型（体内和体外），观察JNK和p38MAPK以及上下游相关因子mRNA水平、蛋白表达；同时观察细胞凋亡相关因子变化和凋亡程度，分析JNK/p38MAPK 信号通路异常活化与细胞凋亡的相关性。同时用 SP600125（JNK 抑制剂）和 SB203580（p38MAPK抑制剂）阻断JNK和p38MAPK信号通路，明确氧化应激状态下JNK/p38MAPK 信号通路对鲤肝细胞凋亡的调控作用。本研究结果表明，（1）在体外，0.4 mM H2O2处理肝细胞2 h以后，可引起显著的氧化应激、细胞凋亡和损伤；在体内，1 mM H2O2暴露14天可引起显著的氧化应激、细胞凋亡和肝损伤。（2）在氧化应激状态下，JNK/p38MAPK信号通路被活化，同时线粒体介导的细胞凋亡被激活。（3）体外干预研究显示，氧化应激诱导的细胞凋亡中，JNK信号通路起关键调控作用。（4）转录组分析以及延伸研究显示，除细胞凋亡外，蛋白质和脂质代谢异常、内质网应激和炎症反应在氧化应激诱导的肝损伤中具有重要的作用。以上结果初步阐明了JNK/p38MAPK信号通路在鲤肝细胞凋亡中的作用机制，为鱼类肝损伤疾病的研究和保肝药物的开发提供新思路，也对鱼类健康养殖具有重要意义。