| Energy excess leading to obesity that causes non-alcoholic fatty liver disease (NAFLD) is increasingly the focus of healthy question in all the worldwide. But so far the pathogenesis of NAFLD is not fully understood. Many of theories are in the hypothesis stage, need to be confirmed, including the more convincing "two-hit hypothesis". It proposed that insulin resistance leading to hepatic lipid deposition becomes a first hit in the development of NAFLD, which accelerates oxidative stress and peroxidative injury of lipid are that become the second hit. The second hit increases apoptosis and necrosis susceptibility of hepatocytes, further promotes the occurrence and development of hepatic fibrosis and cirrhosis, finally leads to the NAFLD. It can be seen that dysregulated hepatic lipid metabolism induces hepatic steatosis and fatty liver. Fatty acids uptake and synthesis beyond its oxidation secretion of hepatocyte, promotes the accumulation of fatty acids in liver. We cure NAFLD on the clinical through controlling body weight, taking insulin sensitizers, hypolipidemic drugs, antioxidant and liver protectant. But meta-analysis of large samples indicated that above parts of drugs may not effectively attenuate liver histological inflammatory.Silence information regulator (SIR) gene family, known as NAD+-dependent histone and non-histone protein deacetylase, which is highly conserved structure. Silencing information regulator2(SIRT2) is characterized by unique biological properties and function, which plays a critical role in energy metabolism, oxidative coupling and regulation of genes expression that becomes a hot topic of regulating longevity genes. SIRT1is a member of the sirtuin family of proteins, homologs of the SIRT2gene. Recent studies suggested that SIRT1not only involves in cellular energy metabolism, but also improves insulin sensitivity, and regulates lipid metabolism and attenuates inflammatory response and oxidative stress damage through alternative approaches. Hence, it has become a new target for the treatment of NAFLD, and provided a crucial way to novel drugs of looking for NAFLD prevention.AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism, which includes feeling the changes of AMP/ATP in the cytoplasm, and the activation regulated by upstream LKB1, CaMKK and TAK1, further affects cells metabolism to maintain the balance between supple and demand of cells energy. Studies have reported that resveratrol is able to enhance SIRT1deacetylase activity, and promotes the expression and activation of LKB1and phopho-AMPK, and regulates lipid metabolism through SIRT1/LKB1/AMPK pathway. Involving in the activation of SIRT1/LKB1/AMPK pathway exerts a central role on hepatical lipid metabolism, which modifies transcription factors, such as p53, FoxO, NF-κB, PGC-α and SREBP-1by deacetylase and phosphorylation, accelerates fatty acid oxidation and suppresses the de novo synthesis of fatty acids.Nuclear factor E2-related factor2(Nrf2) is extremely the protection of preventing hepatic oxidative stress, regulation of hepatic homeostasis and hepatic steatosis. Previous studies suggested that SIRT1, AMPK and Nrf2have an important role on regulation of energy and lipid metabolism, respectively. Lipoic acid (alpha-lipoic acid, ALA) is an endogenous antioxidant, involved in energy metabolism in the form of coenzyme. The present study demonstrated that ALA directly regulates hepatic SIRT1expression and activity in vivo and in vitro experiments. ALA activates SIRT1/AMPK and SIRT1/Nrf2signaling pathway by SIRT1siRNA and SIRT1inhibitor or activator, regulates hepatic lipid metabolism and improves NAFLD. We hope to provide a theoretical guidance of clinical drugs through clarifying the mechanisms of ALA on hepatic lipid metabolism.Research Methods:1. Our study showed that ALA dose-dependently pretreated HepG2cells, that ALA might regulate SIRT1expression and activity by measurement of NAD+/NADH ratio and SIRT1deacetylase activity. We defined the interaction of molecule SIRT1and LKB1with co-immunoprecipitation method in vitro. We found that ALA regulated the phosphorylation of AMPK and ACC by immunoblotting. In addition, our study showed that SIRT1can directly regulate phospho-AMPK/-ACC and affect synthesis and storage of hepatic triglyceride with using SIRT1inhibitor and AMPK inhibitor or activator in vitro experiment. Moreover, we demonstrated that SIRT played a directly role on FAS and ATGL expression and synthesis and lipolysis of hepatic triglyceride by using RNAi technology.2. HepG2cells were pretreated with ALA, and incubated with NA, CC and AICAR, respectively. We explored FoxO1and SREBP-1expression and nuclear translocation by western blot and immunofluorescence.3. HepG2cells were pretreated with SIRT1inhibitor-NA and SIRT1siRNA, and treated with ALA. We examined SIRT1, nuclear Nrf2, antioxidatase SOD2, catalase and HO-1expression by western blot.4. The weight, blood glucose and lipids in high-fat diet-induced NAFLD in C57BL/6J mice. The measurement of food intake, weights and hepatic triglyceride suggested the basic conditions in different groups mice. We measured the accumulation of lipid droplets in the liver tissue by histochemical methods, and examined SIRT1/AMPK signaling pathway by western blot. We measured reactive oxygen content in the liver tissue of mice by superoxide anion fluorescent probe DHE.Research Results:1. ALA dose-dependently increased SIRT1activity and NAD+/NADH ratio in HepG2cells. ALA activated AMPK phosphorylation via interaction of SIRT1and LKB1. We demonstrated that ALA regulated hepatic lipid metabolism through activation of SIRT1/LKB1/AMPK signaling pathway.2. When HepG2cells were pretreated with SIRT1and AMPK inhibitor or activator, and treated with ALA, ALA significantly reduced phospho-FoxO1nuclear translocation, and increased ATGL expression, and also increased phospho-SREBP-1nuclear translocation and reduced FAS expression.3. SIRT1inhibitor-NA and SIRT1siRNA can significantly block SIRT1, nuclear Nrf2, SOD2, catalase and HO-1expression. Long-term ALA dietary significantly reduced fat weights in liver tissue in HFD-induced mice, and also suppressed weights and food intake (P<0.05). Despite hepatic index in groups was no significant difference, but ALA lowed hepatic triglyceride compared to HFD-fed mice (P<0.05). Oil O staining indicated that ALA can significantly improve lipid droplets in the liver tissue of HFD-fed mice. ALA also improved blood glucose and lipids (P<0.05). ALA obviously reduced ROS levels in the liver tissue in HFD-induced mice. In addition, ALA significantly up-regulated SIRT1, phospho-AMPK, phospho-ACC, phospho-SREBP-1and ATGL expression, and down-regulated phospho-FoxO1and FAS expression.Research Discussion:1. ALA regulates hepatic lipid metabolism through activation of SIRT1/LKB1/AMPK pathway and its downstream FoxO1/ATGL and SREBP-1/FAS.2. ALA regulates hepatic antioxidant system and improves hepatic lipid metabolism through elevation of SIRT1and Nrf2interaction and activation of Nrf2/ARE pathway. |
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