EGRl通过上调Gadd45在APAP诱导的急性药物性肝损伤中发挥适应性保护作用的机制研究

The adaptive protection mechanism of acute Acetaminophen-induced liver injury (AILI), Which has a crucial function of clinical outcomes, is still unclear, and pend further clarification. Based on our previous results, we hypothesis firstly that, the high expression of EGR1 in the liver cells may induce the transcription and expression of GADD45, inhibiting of the dysfunction of mitochondria, the damage of DNA and cell death, and played a crucial role of adaptive protection of AILI. The present study aims to study the adaptive protection mechanism of EGR1 in AILI from three levels: molecular level, cellular level and individual level. First, at individual level , to analyze the function of EGR1, a model of EGR1;mice would be employed to analyze the degree of liver injury, the expression of GADD45, the function of mitochondria, the damage of DNA and the expression of relevant genes and proteins. Second step from cellular level, in order to investigate how EGR1 affect the expression of Gadd45, the primary hepatocytes isolated from wild type mice and EGR1;mice are expected to be adopted and employ biological technologies of C-horse, western blot, immunohistochemistry /fluorescence and real time PCR to determine the different expression of EGR1 and Gadd45. Finally, from the sight of molecular, we plan to use the technology of chromatin immune coprecipitation and report gene carrier to confirm the function of EGR1 to regulate the transcription of GADD45, then with liver cell lines of Hepa1-6 with Gadd45 knocked out (CRISPR/Cas9 systems), determine the possible mechanism of GADD45 in the protective function of EGR1 in AILI. The project has scientific significance and clinical value for clarifying the adaptive protection mechanism during the process of AILI, and provide new targets for the prevention and treatment of AILI, as well as fresh ideas and scientific evidences.

APAP诱导药物性肝损伤(AILI)过程中同样对临床转归有决定性影响的肝细胞适应性保护机制有待阐明。基于前期研究基础，本项目首次提出：AILI发生时，肝细胞中上调表达的EGR1可能通过转录诱导GADD45表达，抑制线粒体功能障碍、DNA损伤和细胞死亡，发挥适应性保护作用。本项目拟通过整体、细胞和分子三个层面探讨EGR1在AILI发生时的适应性保护作用和机制。利用EGR1-/-小鼠AILI模型和原代EGR1-/-小鼠肝细胞探讨EGR1对肝损伤严重程度、Gadd45表达水平、线粒体功能和DNA损伤及相关基因表达的影响。利用染色质免疫共沉淀和报告基因载体等技术证实EGR1对Gadd45的转录调控作用。本项目将对阐明AILI的发生时肝细胞的适应性保护机制具有重要的科学意义和临床价值，并为防治AILI的发生、探索新的诊断和治疗靶点，提供新的思路和科学依据。

背景：对乙酰氨基酚（APAP）导致的药物性肝损伤（AILI）的发生、进展和临床预后，取决于损伤因素和保护因素的相互作用。目前多数的研究聚焦于损伤机制，鲜有研究关注损伤后的保护机制。因此，深入开展针对肝损伤的适应性保护机制研究，探索新的诊断和治疗靶点，有助于全面了解其机制，并采取相应措施，防止AILI的发生和进展。.主要内容：通过整体、细胞和分子三个层面观察GADD45α和EGR1在APAP诱导的急性药物性肝损伤中的适应性保护作用并探讨它们的适应性保护作用分子机制。.重要结果：本项目通过临床标本、体内和体外三个层面证明GADD45α和EGR1表达水平升高。GADD45α可以和蛋白磷酸酶Ppp2cb相互作用，诱导三羧酸循环（TCA）和糖原分解相关基因，从而对AILI具有保护作用，并减轻了体内和体外小脂质滴的积累。EGR1可以直接结合ACAA2和mTOR并上调其表达，从而上调脂肪酸氧化能力，最终减轻APAP导致的肝损伤和体内外脂质的堆积。.关键数据：本项目通过PCR、WB、IHC验证：GADD45α和EGR1在临床DILI患者、体内和体外三个层面均高表达；敲除GADD45α和EGR1后，APAP所致的肝损伤均加重。① 过表达GADD45α并暴露于APAP后肝细胞中脂肪酸β-氧化，三羧酸循环（TCA）和糖原分解相关基因的表达升高。LC-MS分析表明GADD45α增加了TCA循环代谢产物的水平。Co-IP分析表明，蛋白磷酸酶的催化亚基Ppp2cb可以直接与GADD45α相互作用。② 过表达EGR1后，APAP所致肝损伤减轻。脂质组学显示游离脂肪酸增加。海马实验证明EGR1增强脂肪酸氧化能力。CHIP-SEQ、RNA-SEQ结果显示，EGR1能够直接结合ACAA2和mTOR并且上调其表达从而上调脂肪酸氧化能力,最终使得APAP导诱导的肝损伤减轻。.科学意义：GADD45α、EGR1可能在APAP诱导的药物性肝损伤中起保护作用。