【作者】 张勇；

【导师】 张乾勇；

【作者基本信息】 第三军医大学 ， 营养与食品卫生学， 2015， 硕士

【摘要】 非酒精性脂肪性肝病(Non-alcoholic fatty liver disease,NAFLD)目前已成为全球主要公共卫生问题之一,我国成年人患病率约占20%,且有逐步发展成肝硬化甚至肝癌的趋势,然而,至今尚未发现有效防治NAFLD的理想药物。白藜芦醇(Resveratrol,RSV)是一种天然多酚类植物化合物,已有报道称其具有缓解NAFLD的生物学作用。但是其潜在的作用机制尚不清楚。自噬最近被认为在防止NAFLD发展过程中有关键的保护作用。研究人员已经发现,自噬可将细胞内部分或全部脂滴包裹在由自噬双层膜结构形成的囊泡内,并运送至溶酶体,使之降解为脂肪酸。自噬的增加能促进脂肪酸的β氧化,进而减少肝脏脂质的积累;同时,当自噬相关基因7(ATG7)敲除后自噬被沉默,进一步加剧了NAFLD的进展。有研究表明,RSV可通过激活内皮细胞自噬,从而抑制内皮细胞炎性反应。RSV能否通过诱导细胞自噬而抑制肝脏脂肪变性及RSV在细胞内的确切作用靶点和机制还需要进一步的研究。本课题使用PA处理HepG2细胞建立肝细胞脂肪变性实验模型,并用高脂肪饮食诱导小鼠建立NAFLD模型。首先,应用油红O染色法、HE染色法、甘油三酯(triglyceride,TG)含量检测观察RSV对肝细胞脂肪变性的影响;然后,应用激光共聚焦、透射电镜等技术检测肝细胞自噬,并结合使用自噬激动剂、自噬抑制剂、RNAi和质粒转染,研究诱导肝细胞自噬在RSV改善肝脂肪变性中的作用。最后,应用免疫印迹检测了RSV对体内外肝细胞上调沉默信息调节因子1(Silent information regulator1,SIRT1),蛋白激酶A(Protein kinase A,PRKA)和磷酸化腺苷酸活化蛋白激酶(phosphorylated Adenosine 5’-monophosphate activated protein kinase,pAMPK)的表达的影响,并应用腺苷酸环化酶(Adenylyl cyclase,AC)抑制剂KH7,PRKA抑制剂H-89,AMPK抑制剂Compoud C或SIRT1抑制剂EX-527,或者使用PRKA,AMPK,SIRT1的siRNA处理细胞,深入研究cAMP/PRKA/AMPK/SIRT1信号通路在RSV诱导肝细胞自噬改善肝细胞脂肪变性中的作用。主要结果和结论如下:(1)RSV能明显改善肝细胞脂肪变性,主要表现为能减少肝细胞中TG的含量。(2)自噬在RSV改善肝细胞脂肪变性中有关键作用。当延长RSV处理时间,及增加RSV的处理剂量,肝细胞的自噬表达水平都上升,而当抑制自噬,RSV抑制肝细胞脂肪变性的作用减弱。(3)RSV调控肝细胞自噬过程中,SIRT1信号通路扮演着重要角色。RSV能显著增加肝细胞cAMP的含量,促进PRKA的表达,进而激活AMPK的表达,并促进SIRT1的表达;而当使用RNAi沉默干扰PRKA、AC、AMPK、SIRT1的表达或抑制PRKA、AC、AMPK、SIRT1的活性后,经RSV处理,肝细胞的自噬表达水平明显下降。实验证实,在RSV促进肝细胞自噬表达过程中,信号通路c AMP/PRKA/AMPK/SIRT1起着关键的调控作用。以上研究结果提示,RSV能通过调控SIRT1相关信号通路而激活自噬抑制肝细胞脂肪变性,从而实现其改善NAFLD的健康效应。更多还原

【Abstract】 Non-alcoholic fatty liver disease(NAFLD) has become the most common cause of chronic liver injury worldwide. However, there is currently no satisfactory therapeutic strategy for NAFLD. Resveratrol(RSV) is a natural polyphenol that has been reported to attenuate NAFLD, though its underlying mechanism is unclear. Recently, autophagy has been identified as a critical protective mechanism during the development of NAFLD. It has been reported that droplets could been partly or completely wrapped by the doublemembrane-structured autolipophagosome vesicles, with which are transported to lysosomes and then degraded into fatty acids. Increasing autophagy induces β-oxidation, thus attenuating hepatic steatosis; meanwhile, lipid accumulates in the liver of autophagy-related gene 7(ATG7) knockout mice.Studies have shown that, RSV can inhibit the inflammatory response of endothelial cells by activating autophagy.Therefore, we propose that RSV inhibits hepatic steatosis by inducing autophagy in hepatic cells.We established hepatic steatosis cell model using palmitic acid(PA), and NAFLD mouse model using high-fat diet. First, we observed the protective effects of RSV on hepatic steatosis with oil red O staining, HE staining, triglycerides(TG) content detection. Second, we studied the mechanism of the protective effects of RSV on hepatic steatosis. Here, we treated cells with autophagy agonist, autophagy inhibitors, siRNA, and then detected autophagy using confocal laser scanning, transmission electron microscopy. At last, we further studied c AMP/PRKA/AMPK/SIRT1 signaling pathway in RSV inducing autophagy in hepatic cells. We treated cells with inhibitors and siRNAs of adenylate cyclase(Adenylyl cyclase, AC),protein kinase A(Protein kinase A,PRKA),AMP-activated protein kinase(Adenosine 5’-monophosphate activated protein kinase,AMPK),sirtuin 1(Silent information regulator 1, SIRT1) and then detected the expression of AC,PRKA, AMPK, SIRT1 using western blot technique.The main results and conclusions are as follows:(1) RSV decreases TG content in PA-treated HepG2 cells, suggesting its potential in inhibiting hepatic steatosis.(2) Autophagy plays an important role in inhibiting hepatic steatosis.RSV induces autophagy in Hep G2 cells in dose-and time-dependent way. RSV’s effects on hepatic steatosis significantly decrease when autophagy is inhibited.(3) Sirt1 signaling plays a key role in the regulation of RSV-induced autophagy.RSV prominently increases the content of cAMP in HepG2 cells, upregulate the expression of PRKA, activates AMPK and thus induces the expression of SIRT1; After using siRNA to knock down PRKA, AC, AMPK and SIRT1 gene, or inhibitors to inhibit the activities of PRKA,AC,AMPK and SIRT1, RSV-induced autophagy is remarkably inhibited. All of the above show that, c AMP/PRKA/AMPK/SIRT1 signaling plays a key role in the regulation of RSV-induced autophagy.In conclusion, RSV inhibits hepatic steatosis by activating SIRT1-related signaling pathway to induce autophagy, thus preventing NAFLD.更多还原