氧化应激时血红素加氧合酶1对肝细胞自噬的调控作用及机制研究

Autophagy is regulated elegantly in the cell to prevent its excessive activation which can induce dead signal. However, the mechanism isn't known. Recently studies suggested that heme oxygenase 1 (HO-1) could induce or inhibit autophagy.Through analyzing these data we find a marked difference among these studies in the levels of HO-1. So we put up a hypothesis that defferent levels of HO-1 should determine whether the autophagy is induced or inhibited. Our previous studies have shown hyperbaric oxygen can be used as a tool to induce the protective effect against oxidative stress, including induction of HO-1 and autophagy. In this experiment,firstly we will identify if the levels of HO-1 can regulate autophagy in vivo by using hyperbaric oxygen conditioning with or without HO-1 inhibitor, autophagy inducer and inhibitor. At the same time the p38 MAPK signal pathway will be also detected to identify its role in mediating the regulation effect. Then in a rat model with hepatic ischemia-reperfusion we will further testify the regulation by HO-1 on autophagy and its impact on protective effect provided by hperbaric oxygen pretreatment. At last,in vitro by using virus transfection and RNA interference techniques we will establish primary hepatocytes expressing different levels of HO-1. Test on these cells undergone anoxia/reoxygen will confirm the hypothesis we put up. Through this study we can elucidate the mechanism by which the autophagy is regulated and controled in the cell as well as the internal association among complicated antioxidative signals in vivo.

自噬在细胞内受到精细调控以防过度激活产生促死亡信号，但具体机制还不明确。最新研究提示氧化应激时血红素加氧合酶1（HO-1）对自噬可能有促进和抑制双重作用，进一步分析我们发现不同研究中HO-1表达水平不同。据此我们提出细胞内HO-1水平决定其对自噬的调控方向的假设。我们之前研究证实高压氧是诱导机体抗氧化反应的有效工具，可诱导HO-1表达和激活自噬。本研究首先利用高压氧诱导、HO-1抑制剂、自噬诱导及抑制剂，在体研究大鼠肝脏HO-1表达水平（或活性）对自噬调控的影响，并检测p38 MAPK信号通路在其中的作用；继而在大鼠肝缺血再灌注模型上验证HO-1调控自噬在高压氧预处理保护效应中的作用；通过病毒转染和RNA干扰技术建立HO-1表达差异的原代肝细胞，在体外缺氧/复氧模型上进一步明确HO-1对自噬调控机制。本研究对阐明自噬调控机制以及体内复杂的抗氧化机制之间的联系具有重要意义。

血加氧合酶-1（HO-1）是体内重要的抗氧化损伤分子，诱导HO-1表达可以抑制缺血再灌注、脓毒症以及药物等引起的肝脏炎症反应及细胞凋亡。尽管既往研究显示HO-1酶解血红素产生的代谢产物介导了其保护作用，但是也有研究显示在肝脏缺血再灌注时并无HO-1酶底物的明显增加，提示HO-1可能存在其他保护作用机制。自噬的激活在机体抵抗氧化应激损伤中发挥重要作用，本研究探讨自噬在HO-1保护肝缺血再灌注损伤中的作用。我们建立了大鼠部分肝缺血再灌注模型(I/R)，CoPP预处理可以大大抑制肝缺血再灌注损伤，表现为组织坏死减少，细胞凋亡降低，血清中转氨酶浓度下降。自噬在这一过程中也明显增加。腹腔注射HO-1酶抑制剂ZnPP消除了CoPP预处理的保护作用及对自噬的影响，证实CoPP通过诱导HO-1发挥保护作用，也提示HO-1诱导了自噬。提前给予自噬抑制剂3-MA可抑制CoPP诱导的自噬，而其抑制I/R损伤的作用也消失，提示自噬介导了HO-1的保护作用。我们利用慢病毒转染HO-1的正常基因或siRNA片段至HL7702细胞,获得了HO-1高表达或基因敲减的细胞株，通过体外建立缺氧/复氧模型(A/R)，证明 HO-1高表达可以增加细胞对A/R的耐受，增加自噬表达，抑制细胞凋亡；自噬抑制剂3-MA可消除HO-1高表达对A/R的保护作用。以上体外实验结果进一步证明自噬介导了HO-1在缺血再灌注损伤中的保护作用。以上研究为治疗和预防肝脏缺血再灌注损伤提供了新的靶点和策略。