旁观者效应在镉致大鼠肝细胞自噬中的作用机制研究

Gap junctional intercellular communication (GJIC) is an important communication pathway, involved in various mammalian cellular functions ranging from cell growth to cell death. One typical example is the “bystander” damaging effect of healthy cells by injured cells. We found that cadmium induce apoptotic hepatocytes could induce apoptosis in an adjacent healthy hepatocytes by bystander effect. Recently, we also discovered that cadmium can induce autophagy in rat hepatocytes. So, we next hypothesize that cadmium induced autophagic cells could induce autophagy in adjacent cells via GJIC. In relation to this central hypothesis, we asked whether autophagic cells could stimulate autophagy in adjacent healthy hepatocytes and whether GJIC is involved in this induction process. In this project, experimental model to analyze in vitro bystander effect has been already established for cadmium-induced rat hepatic injury. Gap junction channels expression levels, their morphological structure, communication function, and autophagy will be detected by western blot, real-time quantitative PCR, immunofluorescence microscopy, flow cytometry, and other routine assays. Overall, this study will establish and elucidate the existence of the bystander effect of rat hepatocytes autophagy induced by cadmium, and hence facilitate the understanding of cadmium mediated liver damage.

细胞缝隙连接通讯（GJIC）是哺乳动物细胞间普遍存在的一种通讯方式，在细胞增殖、分化、凋亡、死亡等生理过程中具有重要作用。受损细胞可以通过GJIC引起相邻细胞损伤，该作用称为“旁观者效应”。前期研究表明，镉暴露可引起大鼠肝细胞发生凋亡和自噬，且诱导的凋亡具有旁观者效应。镉诱导的自噬是否也存在旁观者效应，以及自噬与GJIC内在关系尚不清楚。本项目拟在Transwell共培养体系中建立镉损伤大鼠肝细胞旁观者效应模型，通过流式细胞术、Western Blot、RT-PCR、免疫电镜等方法，检测细胞缝隙连接通道相关蛋白的表达水平和通讯功能，以及自噬水平、自噬体、和自噬相关基因和蛋白的表达水平。目的是阐明镉致大鼠肝脏自噬过程中是否存在旁观者效应、GJIC在镉致大鼠肝细胞自噬旁观者效应中的调控作用，以及自噬对旁观者细胞GJIC的影响机理，为进一步揭示镉的肝脏毒性损伤机理提供理论依据。

镉（Cd）是一种重要的工业和环境污染物，可通过食物链影响人类的健康。缝隙连接是相邻细胞之间由缝隙连接蛋白组成的一种特殊膜结构。小分子物质可通过细胞缝隙连接通讯（GJIC）在相邻细胞间进行物质和能量的直接交换。自噬是通过溶酶体降解细胞内受损的细胞器及大分子蛋白等的过程。自噬对维持细胞内稳态、促进细胞存活方面至关重要，并参与多种生理和病理过程。本项目研究发现：1. Cd致大鼠肝细胞BRL 3A的毒性损伤呈时间效应关系；2. Cd暴露过程中，BRL 3A细胞自噬水平呈先上升后下降的趋势；Cd在促进细胞自噬的同时引起自噬流受阻；3. Cd暴露可抑制BRL 3A细胞GJIC功能及Cx43和p-Cx43表达水平；4. GJIC的功能与胞膜上Cx43的分布及p-Cx43的表达量呈正相关；抑制GJIC可减轻Cd对肝细胞的毒性损伤，促进GJIC则增强Cd对肝细胞的毒性损伤；5. 在Cd暴露过程中，抑制GJIC能够促进肝细胞自噬的发生，促进GJIC能够抑制自噬的发生。6. Cd对镉暴露受损细胞的毒性作用呈旁观者效应，Cd暴露抑制肝细胞GJIC，GJIC的抑制具有双重作用，即保护正常细胞免受Cd暴露细胞的毒性损伤作用以及进一步加强Cd对Cd暴露受损细胞的毒性作用。. 本项目阐明镉致大鼠肝脏自噬过程中存在旁观者效应、GJIC在镉致大鼠肝细胞自噬旁观者效应中的调控作用，以及自噬对旁观者细胞GJIC的影响机理，为进一步揭示镉的肝脏毒性损伤机理及防控镉中毒提供了理论基础。