【作者】 陈炯；

【导师】 施敏；

【作者基本信息】 上海交通大学 ， 消化内科， 2020， 硕士

【摘要】 背景:急性肝衰竭(Acute liver failure,ALF)病死率高,病情凶险,目前尚无有效的治疗方法,因此探索并寻找急性肝衰竭潜在的治疗靶点以及药物显得尤为重要。单核-巨噬细胞系统是肝脏中宿主防御系统的重要组成部分。巨噬细胞的促炎反应与ALF的病理生理有关,其释放过量的炎性细胞因子是导致肝损伤的重要原因,因此我们旨在寻找能够抑制巨噬细胞炎性细胞因子的释放、减轻肝损伤的新型小分子药物。目的:1)筛选出能够抑制巨噬细胞炎性细胞因子释放的小分子化合物;2)探讨PH797804是否对LPS/D-Gal诱导的小鼠ALF具有保护作用;3)探讨PH797804治疗小鼠ALF的作用机制。方法:1)建立两种巨噬细胞炎症模型,以细胞培养上清中TNF-α含量为评价标准,对1200种生物活性小分子化合物进行筛选,找到能够有效抑制巨噬细胞促炎细胞因子释放的小分子化合物;2)将实验小鼠随机分成四组,每组16只。腹腔注射LPS/D-Gal诱导建立小鼠ALF模型,设立生理盐水对照组(a)、LPS/D-Gal诱导模型组(b)、LPS/D-Gal诱导和PH797804干预组(c)及PH797804处理对照组(d)。观察小鼠48h内的存活率,检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的含量,HE染色组织切片观察肝脏病理变化,Elisa检测血清中TNF-α和IL-6的表达水平;3)对各组小鼠肝组织进行高通量转录组测序,采用Real-time PCR法验证测序结果。分析测序结果,筛选差异表达基因,并对差异基因进行GO及KEGG富集分析,探寻PH797804作用机制。结果:1)从生物活性库中筛选出小分子化合物-PH797804,体外实验证实它对细胞毒性低,可呈剂量依赖性显著抑制LPS诱导的RAW 264.7和THP-1细胞培养上清中TNF-α的表达。2)相较于ALF模型组小鼠,PH797804处理后c组小鼠48h存活率从20%提高至80%,小鼠肝脏组织病理学异常显著改善,血清中ALT、AST含量较ALF模型组相比显著降低(P<0.01),血清中TNF-α和IL-6等促炎细胞因子含量较ALF模型组相比显著降低(P<0.01)。3)RNA测序结果提示PH797804对LPS/D-Gal诱导小鼠ALF的保护作用主要与抑制炎症反应相关。GO功能聚类和KEGG pathway富集分析结果表明:差异基因主要富集于炎症、趋化、细胞粘附等生物学过程中;主要与白细胞经内皮细胞迁移、细胞因子受体间相互作用以及FcγR-介导的吞噬作用等信号通路有关。4)PH797804处理能够降低肝脏总的CD14表达以及巨噬细胞表面CD14的表达。结论:1)小分子化合物PH797804在体外具有较强的抗炎活性;2)PH797804对LPS/D-Gal诱导的ALF具有显著的保护作用;3)PH797804发挥肝脏保护作用可能与其抑制小鼠肝脏炎症,降低CD14的表达有关,这为治疗急性肝衰竭提供了新的思路和方法。更多还原

【Abstract】 Background:At present,there is no effective treatment for acute liver failure because of its high mortality and dangerous condition.Therefore,it is very important to explore the potential therapeutic targets and drugs for acute liver failure.Monocyte macrophage lineage cells constitute an important part of the host defense system in the liver.The pro-inflammatory response of macrophages is related to the pathophysiology of ALF,and the release of excessive inflammatory cytokines is an important cause of liver injury.Therefore,we are looking for new small molecular drugs that can inhibit the release of macrophage inflammatory cytokines and reduce liver injury.Objective:1)High-throughput screening of small molecular compounds that inhibit the release of inflammatory cytokines in macrophages;2)To investigate whether PH797804 has protective effect on ALF induced by LPS/D-Gal in mice;3)To investigate the mechanism of treating ALF with PH797804 in mice.Methods:1)Two models of macrophage inflammation were established.Based on the TNF-αlevels in cell culture supernatant as the evaluation standard,1200bioactive compounds were screened to find small molecular compounds that could effectively inhibit the release of pro-inflammatory cytokines in macrophages.2)Sixty-four mice were randomly divided into four groups,16 in each group.ALF model of mice induced by intraperitoneal injection of LPS/D-Gal,the four groups were normal saline control group(a),LPS/D-Gal induction model group(b),LPS/D-Gal induction and PH797804 pretreatment group(c),PH797804 control group(d).The survival rate of mice was observed within 48 hours,and the contents of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum were measured,the pathological changes of liver were observed by HE staining tissue sections,and the expression levels of TNF-αand IL-6 were detected by Elisa.3)The high-throughput transcriptome sequencing was performed on the liver tissue of each group to explore the mechanism of action,and the sequencing results were verified by Real-time PCR.The transcriptome sequencing results were analyzed and the differentially expressed genes were screened,and the GO and KEGG enrichment of differentially expressed genes were analyzed to explore the mechanism of action of PH797804.Results:1)PH797804,a small molecular compound,was screened from the Selleck bioactive chemical library.The results showed that ph797804 had low cytotoxicity on the cells in vitro,and could significantly inhibit the expression of TNF-αin the supernatants of raw 264.7 and THP-1 cells induced by LPS in a dose-dependent manner;2)Compared with the ALF model group,the 48-hour survival rate of mice in group c after PH797804 treatment increased from 20%to 80%,the liver histopathological abnormalities of mice were significantly improved,the content of ALT and AST in serum were obviously lower than that in ALF model group(P<0.01).and the content of proinflammatory cytokines such as TNF-αand IL-6 in serum were significantly lower than that in ALF model group(P<0.01).3)RNA sequencing results suggested that the protective effect of PH797804 on LPS/D-Gal-induced ALF in mice is mainly related to inhibiting the inflammatory response.The results of GO function clustering and KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly enriched in the biological processes of inflammation,chemotaxis and cell adhesion.It is mainly related to leukocyte migration through endothelial cells,interaction between cytokine receptors and phagocytosis mediated by Fc gamma R.4)Treatment with PH797804 reduced the expression of total CD14 in the liver and on the surface of macrophages.Conclusions:1)Small molecule compound PH797804 has strong anti-inflammatory activity in vitro.2)PH797804 has significant protective effect on ALF induced by LPS/d-gal;3)The protective effect of PH797804 on the liver may be related to the inhibition of liver inflammation and the decrease of CD14 expression in mice,which provides a new idea and method for the treatment of ALF.更多还原