基于氧化应激拮抗作用他莫昔芬抗Gal/LPS诱导的急性肝损伤研究

Acute liver injury (ALI) induced endotoxin of G- bacteria is a severe clinical syndrome which account 80-90% mortality. Till now, no other strategy is available for this disease apart from liver transplantation. Therefore, it would be particularly meaningful to find out novel pathway for the prevention or therapy of it. As other research results, in our pioneering investigation it was demonstrated that the early apoptosis induced by TNFα played a pivotal role during the development of some kinds of ALI, particularly ALI given rise by endotoxin. It was reported in some studies that oxidative stress was required for the initiation and maintain of TNFα induced hepatocellular apoptosis. Thus, inhibition of oxidative stress will be a promising direction for the therapy of ALI associated with early hepatocellular apoptosis. Some investigation turned out that Tamoxifen was implicated for the effective protection of another kind of ALI by regulation of the expression of hepatic Mmd2, which antagonize oxidative stress. However, which kind of cell produce this molecule remains to be unknown. Furthermore, the mechanism by which Mmd2 counteracts oxidative stress requires futher study. Based upon these, we suppose that Tamoxifen could prevent or alleviate ALI provoked by the administration of Gal/LPS via the similar action. To this end, in vitro and in vivo assays will be carried out to testify our hypothesis and elucidate the producing cell of Mmd2 after Tamoxifen administration, which followed by clarifying the anti oxidative stress mechanism of this molecule. Hopefully, accomplishment of this programme will not only deep into the mechanistic investigation of ALI induced by invading G- bacteria, but also make some preparations for the coming research(es) about the hepatoprotective effect of Mmd2 during ALI.

G-菌内毒素等引发的急性肝损伤（ALI）是一种严重的临床综合症，病死率达80-90%，除肝移植外目前尚无其它对策。因此，探讨ALI 预防或治疗的新方法具有重要意义。和其它研究相似，本课题组前期研究表明TNFα及其诱导的早期细胞凋亡在内毒素主要成分LPS诱导的ALI发病过程中起关键作用。研究发现氧化应激产物活性氧对于该凋亡的启动和维持具有促进作用。因此，抗氧化应激有望成为ALI治疗的新方向。研究表明他莫昔芬可经调节Mmd2表达而拮抗氧化应激，保护另一种ALI，但Mmd2的肝脏细胞来源和抗氧化应激机制尚未阐明。基于这些，我们推测他莫昔芬可经此作用拮抗Gal/LPS诱发的ALI。为此，本项目拟通过细胞和动物实验探讨他莫昔芬对Gal/LPS诱导ALI的保护作用，明确他莫昔芬给药后肝脏Mmd2的细胞来源，并阐明其抗氧化应激机制，为将来G-菌引起的ALI深入研究及Mmd2的保护性机制研究奠定前期基础。

急性肝损伤是临床常见急危症，原因复杂，后果严重，机制不详。和其他学者的发现一致，我们前期研究也表明D-氨基半乳糖（D-Gal）联合细菌脂多糖（LPS）诱导的小鼠急性肝损伤（ALI）在临床症状及病理生理学特征等方面与临床ALI类似，如转氨酶、血清及肝脏肿瘤坏死因子（TNF）在肝脏急性炎症风暴过程中急剧升高，继而诱发后续的肝细胞早期细胞凋亡及中晚期的细胞坏死甚至近来所说的“坏死性凋亡”，导致肝脏急性衰竭甚至死亡。. 2012年，Y. Yoshikawa等发现他莫昔芬（TAM）在连续三次（1次/天）给药模式下能有效预防、减轻对乙酰基氨基酚（APAP）诱导的小鼠ALI，并提示其作用机制是通过TAM能显著促进肝脏单核细胞--巨噬细胞分化相关蛋白2（mmd-2）表达，进而发挥ALI拮抗作用，但后续机制未能阐明。受此启发以及我们关于ALI方面的研究基础，我们推理该药物（TAM）是否对D-Gal/LPS诱导的ALI也能发挥拮抗作用？尽管这两种ALI动物模型在某些病理生理学机制上有些差异，如APAP诱导的ALI以氧化应激引起的损伤为主，而另一种ALI模型当时认为主要是TNFa诱导的肝细胞凋亡占主导，其常用作模拟临床细菌（尤其G-）引发的ALI或内毒素休克或败血症（sepsis）。因此我们这一科学假说具有一定的实际意义。. 通过为期4年的研究工作，我们不但发现TAM在提前给药模式下能使实验ALI小鼠（BALB/C或KM品系）12小时存活率达30~50%，并有效抑制肝损伤进程，具体表现在血清转氨酶、肝组织病理组织学等方面的明显改善。而且，我们还从mmd-2表达、细胞凋亡、氧化应激等角度分析了TAM的ALI保护机制，结果与我们当初项目申报时所提的科学假说基本一致。本课题的相关研究结果对于后续ALI甚至肝损伤研究的继续深入都将具有一定意义，并为临床ALI治疗提供新的思路。