本文選題：商陸皂苷甲 + 急性肝損傷　； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：目的:對乙酰氨基酚(Acetaminophen,APAP)過量是引發(fā)急性肝衰竭的主要原因。氧化應(yīng)激是APAP誘導(dǎo)肝毒性發(fā)生的重要因素,因此抗氧化作用將成為預(yù)防和治療APAP肝損傷的新手段。商陸皂苷甲具有抗炎、抗凋亡、抗腫瘤細胞增殖及抗氧化的活性,但其抑制APAP誘導(dǎo)的急性肝損傷的作用仍需進一步研究。方法:本實驗通過構(gòu)建體內(nèi)體外兩種模型,對商陸皂苷甲的保肝作用及機制進行研究。在體外實驗中,我們利用APAP及H2O2誘導(dǎo)Hep G2細胞損傷及氧化應(yīng)激模型。MTT法檢測細胞活力;GSH、H2O2及O2-的測定反應(yīng)細胞氧化應(yīng)激的情況;免疫印跡法(Western Blot)及CRISPR/Cas9系統(tǒng)探究商陸皂苷甲體外作用機制。在體內(nèi),我們利用APAP誘導(dǎo)急性肝損傷的模型,并利用HE染色等技術(shù)對商陸皂苷甲的保肝作用及其相關(guān)分子機制進行進一步的研究。結(jié)果:1.在體外,陸皂苷甲抑制APAP或H2O2誘導(dǎo)的細胞毒性、H2O2及O2-的產(chǎn)生、GSH的耗竭以及凋亡。說明,商陸皂苷甲具有抗氧化的作用并抑制細胞損傷。2.商陸皂苷甲上調(diào)Nrf2的表達及核轉(zhuǎn)錄,同時增加其下游抗氧化基因HO-1的表達。在利用CRISPR/Cas9系統(tǒng)構(gòu)建Nrf2敲除的Hep G2細胞系中,商陸皂苷抑制APAP肝毒性的作用被抑制。說明,商陸皂苷甲通過激活抗氧化基因Nrf2從而發(fā)揮細胞保護作用。3.商陸皂苷甲可顯著上調(diào)AMPK、AKT及GSK3β的磷酸化。使用AKT抑制劑預(yù)處理之后可顯著抑制商陸皂苷甲誘導(dǎo)的GSK3β的磷酸化、Nrf2核轉(zhuǎn)錄及細胞保護作用。此外,使用AMPK抑制劑后下調(diào)商陸皂苷甲誘導(dǎo)的AKT、GSK3β的磷酸化、Nrf2核轉(zhuǎn)錄及細胞保護作用。說明商陸皂苷甲通過激活A(yù)MPK/AKT/GSK3β信號通路激活Nrf2從而發(fā)揮細胞保護作用。4.在體內(nèi),商陸皂苷甲可顯著抑制APAP誘導(dǎo)的死亡率、ALT及AST的增高以及病理組織學(xué)變化。說明商陸皂苷甲可以抑制APAP誘導(dǎo)的小鼠急性肝損傷。5.商陸皂苷甲可上調(diào)肝組織中GSH、SOD的水平,同時下調(diào)MPO、MDA、GSSG及GSSG/GSH的比率;商陸皂苷甲抑制APAP誘導(dǎo)JNK的磷酸化及線粒體易位、Bax線粒體易位、AIF及細胞色素c的分泌以及Caspase-3的活化。說明,商陸皂苷甲通過抑制氧化應(yīng)激、線粒體功能紊亂及凋亡發(fā)揮保肝作用。6.與體外結(jié)果相似,商陸皂苷甲上調(diào)Nrf2核轉(zhuǎn)錄、AMPK、AKT及GSK3β的磷酸化。結(jié)論:綜上所述:商陸皂苷甲通過AMPK/AKT/GSK3β/Nrf2抗氧化通路抑制氧化應(yīng)激及線粒體功能紊亂進而發(fā)揮保肝作用。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285

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相關(guān)期刊論文 前2條

1 Gareeballa Osman Adam;Md.Mahbubur Rahman;Sei-Jin Lee;Gi-Beum Kim;Hyung-Sub Kang;Jin-Shang Kim;Shang-Jin Kim;;Hepatoprotective effects of Nigella sativa seed extract against acetaminophen-induced oxidative stress[J];Asian Pacific Journal of Tropical Medicine;2016年03期

2 ;Effects of esculentoside A on production of interleukin-1, 2, and prostaglandin E_2[J];Acta Pharmacologica Sinica;2004年06期

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本文編號：1914037