商陆皂苷甲防治对乙酰氨基酚诱导的急性肝损伤的作用及机制

发布时间：2018-05-20 09:17

本文选题：商陆皂苷甲 + 急性肝损伤　；参考：《吉林大学》2017年硕士论文

【摘要】：目的:对乙酰氨基酚(Acetaminophen,APAP)过量是引发急性肝衰竭的主要原因。氧化应激是APAP诱导肝毒性发生的重要因素,因此抗氧化作用将成为预防和治疗APAP肝损伤的新手段。商陆皂苷甲具有抗炎、抗凋亡、抗肿瘤细胞增殖及抗氧化的活性,但其抑制APAP诱导的急性肝损伤的作用仍需进一步研究。方法:本实验通过构建体内体外两种模型,对商陆皂苷甲的保肝作用及机制进行研究。在体外实验中,我们利用APAP及H2O2诱导Hep G2细胞损伤及氧化应激模型。MTT法检测细胞活力;GSH、H2O2及O2-的测定反应细胞氧化应激的情况;免疫印迹法(Western Blot)及CRISPR/Cas9系统探究商陆皂苷甲体外作用机制。在体内,我们利用APAP诱导急性肝损伤的模型,并利用HE染色等技术对商陆皂苷甲的保肝作用及其相关分子机制进行进一步的研究。结果:1.在体外,陆皂苷甲抑制APAP或H2O2诱导的细胞毒性、H2O2及O2-的产生、GSH的耗竭以及凋亡。说明,商陆皂苷甲具有抗氧化的作用并抑制细胞损伤。2.商陆皂苷甲上调Nrf2的表达及核转录,同时增加其下游抗氧化基因HO-1的表达。在利用CRISPR/Cas9系统构建Nrf2敲除的Hep G2细胞系中,商陆皂苷抑制APAP肝毒性的作用被抑制。说明,商陆皂苷甲通过激活抗氧化基因Nrf2从而发挥细胞保护作用。3.商陆皂苷甲可显著上调AMPK、AKT及GSK3β的磷酸化。使用AKT抑制剂预处理之后可显著抑制商陆皂苷甲诱导的GSK3β的磷酸化、Nrf2核转录及细胞保护作用。此外,使用AMPK抑制剂后下调商陆皂苷甲诱导的AKT、GSK3β的磷酸化、Nrf2核转录及细胞保护作用。说明商陆皂苷甲通过激活AMPK/AKT/GSK3β信号通路激活Nrf2从而发挥细胞保护作用。4.在体内,商陆皂苷甲可显著抑制APAP诱导的死亡率、ALT及AST的增高以及病理组织学变化。说明商陆皂苷甲可以抑制APAP诱导的小鼠急性肝损伤。5.商陆皂苷甲可上调肝组织中GSH、SOD的水平,同时下调MPO、MDA、GSSG及GSSG/GSH的比率;商陆皂苷甲抑制APAP诱导JNK的磷酸化及线粒体易位、Bax线粒体易位、AIF及细胞色素c的分泌以及Caspase-3的活化。说明,商陆皂苷甲通过抑制氧化应激、线粒体功能紊乱及凋亡发挥保肝作用。6.与体外结果相似,商陆皂苷甲上调Nrf2核转录、AMPK、AKT及GSK3β的磷酸化。结论:综上所述:商陆皂苷甲通过AMPK/AKT/GSK3β/Nrf2抗氧化通路抑制氧化应激及线粒体功能紊乱进而发挥保肝作用。
[Abstract]:Objective: excessive acetaminophen (Acetaminophen, APAP) is the main cause of acute liver failure. Oxidative stress is an important factor in the pathogenesis of hepatotoxicity induced by APAP. Therefore, the antioxidative effect will be a new part of the prevention and treatment of APAP liver injury. However, the effect of its inhibition of APAP induced acute liver injury still needs further study. Methods: in this experiment, two models in vitro and in vivo were constructed to study the liver preservation and mechanism of Phytolacca saponins. In vitro, we used APAP and H2O2 to induce Hep G2 cell damage and oxidative stress model.MTT method to detect cell viability, GSH, H2O. 2 and O2- were used to determine the oxidative stress of reactive cells; Western Blot and CRISPR/Cas9 system were used to explore the mechanism of the effect of saponins in vitro. In vivo, we use APAP to induce acute liver injury model, and use HE staining techniques to further protect the liver and related molecular mechanism of phytolaccin. Results: 1. in vitro, 1. in vitro, saponins a inhibited the cytotoxicity induced by APAP or H2O2, the production of H2O2 and O2-, the depletion of GSH and the apoptosis. It indicated that phytolaccin a had antioxidant effect and inhibited cell damage.2., the expression and nuclear transcription of Nrf2 was up to be up-regulated, and the expression of the downstream antioxidant gene HO-1 was increased at the same time. RISPR/Cas9 system constructs Nrf2 knockout Hep G2 cell lines. The effect of phytolaccin on the inhibition of APAP hepatotoxicity is inhibited. It is indicated that phytolaccin a by activating antioxidant gene Nrf2 and thus exerting cytoprotective effect,.3. saponins a can significantly up-regulation the phosphorylation of AMPK, AKT and GSK3 beta, which can be significantly inhibited with AKT inhibitor pretreatment. The phosphorylation of GSK3 beta induced by saponin A, Nrf2 nuclear transcription and cell protection. In addition, after using AMPK inhibitors, AKT, GSK3 beta phosphorylation, Nrf2 nuclear transcription and cell protection are downregulated by AMPK inhibitors. It shows that Phytolacca saponin activates Nrf2 by activating AMPK/AKT/GSK3 beta signaling pathway and thus plays a cellular protective effect. In vivo, saponins a can significantly inhibit the mortality induced by APAP, the increase of ALT and AST and histopathological changes. It shows that saponins a can inhibit the acute liver injury induced by APAP,.5.,.5., to up regulate the level of GSH and SOD in liver tissue, and reduce the ratio of MPO, MDA, GSSG and GSSG/GSH, and the inhibition of A by Phytolacca saponins a A PAP induced the phosphorylation of JNK and mitochondrial translocation, Bax mitochondrial translocation, the secretion of AIF and cytochrome c and the activation of Caspase-3. It shows that phytolaccin A is similar to the results of.6. in vitro by inhibiting oxidative stress, mitochondrial dysfunction and apoptosis,.6. is similar to the in vitro results, and the phosphorylation of Nrf2 nuclear, AMPK, AKT and GSK3 beta is up-regulated by phytolaccin a Conclusion: In conclusion, phytolaccin a can inhibit oxidative stress and mitochondrial dysfunction through AMPK/AKT/GSK3 beta /Nrf2 antioxidant pathway, and thus play a protective role in liver.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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