Sirt1-Plin1-Fsp27介导的脂滴形成和分解在白藜芦醇防治NAFLD中的作用研究

The incidence of nonalcoholic fatty liver disease (NAFLD) increased worldwide last decade. However, there is still lack of effective methods for the prevention of NAFLD. Hepatic fat accumulation plays an important role in this preceding of NAFLD. The formation and decomposition of lipid droplets is major contributor to the abnormal aggregation of fat in the liver. Previous studies found that resveratrol intervention effectively improved liver fat accumulation in animals. However, the mechanism underlying the effects of resveratrol on the liver fat accumulation or NAFLD is unclear. Resveratrol intervention increased the expression of Sirt1 in several cells. In our preliminary experiment, we found that resveratrol could improve liver lipid accumulation by decreasing the expression of perilinpin (Plin1) and fat specific protein 27 (Fsp27) in the HepG2 cells. Plin1 and Fsp27 play a key role in the formation and decomposition of lipid droplet. Other study found that the expression of Fsp27 significantly decreased in mice with Sirt1 gene knock out. Based on the previous knowledge and our results, the hypothesis in this study is that resveratrol inhibits the lipid droplet formation and promotes the lipid droplet decomposition, and then improves liver lipid accumulation. The study will be conducted on the mouse with or without SIRT1 gene knock down, HepG2 cells and mouse primary hepatocytes. Mouse and cells will be induced by the high fat diet or palmitic acid to perform the liver fat accumulation and NAFLD. This study will elucidate the role of lipid droplets formation and decomposition mediated by Sirt1-Plin1-Fsp27 signaling pathway in the effects of resveratrol on the prevention and treatment of NAFLD. This study will provide a new scientific basis for the prevention of NAFLD through dietary approach.

非酒精性脂肪肝（NAFLD）在全球发病率逐年增高，但缺乏有效预防措施。肝脏脂肪蓄积是NAFLD发生的首要环节，脂滴形成和分解在其中发挥关键作用。白藜芦醇可以有效改善肝脏脂肪蓄积，机制有待完全阐明。研究发现，白藜芦醇可以上调多种细胞的Sirt1表达。本课题组预实验发现，白藜芦醇可以显著降低HepG2细胞内脂滴调控关键分子脂滴包被蛋白（Plin1）和脂肪特异性蛋白27（Fsp27）表达，并抑制脂滴形成。结合文献报道，Sirt1敲除小鼠的Fsp27显著增高。据此提出科学假说：白藜芦醇可能通过上调Sirt1抑制脂滴形成并促进其分解，改善其在肝细胞内聚积。本项目以Sirt1基因敲减小鼠和高脂膳食喂养小鼠、HepG2及小鼠原代肝细胞为对象，深入阐明Sirt1-Plin1-Fsp27信号通路介导的脂滴形成和分解在白藜芦醇防治NAFLD中的作用及机制。本研究将为通过膳食途径预防NAFLD提供新的科学依据。

非酒精性脂肪肝（NAFLD）缺乏有效预防治疗措施，肝脏脂肪蓄积是其发生的首要环节，脂滴形成和分解（脂解和脂噬）在其中发挥关键作用。白藜芦醇（RES）可以有效改善肝脏脂肪蓄积，机制有待完全阐明。本项目以高脂膳食喂养小鼠、Sirt1基因全身敲减小鼠、肝脏靶向Sirt1基因敲减小鼠、C57BL/6小鼠原代肝细胞、HepG2细胞、HHL5细胞（人正常肝细胞）为对象，观察RES调控脂滴蓄积和肝脏脂肪变性的作用，并从脂滴形成和分解途径对其作用机制进行深入研究。动物实验发现，RES明显减轻高脂膳食诱导小鼠肝脏的脂质蓄积，全身或肝脏靶向SIRT1基因敲减后明显减弱RES的作用。高脂膳食可升高野生型小鼠肝脏GRP78表达，激活UPRER且增加PERK、IRE-1、ATF6等相关蛋白表达；而RES干预后SIRT1表达上调，ERS水平及GRP78和ATF6的表达均降低，但不影响PERK和IRE-1的表达；小鼠体内SIRT1基因敲减后显著削弱了RES对GRP78和ATF6表达的抑制作用。高脂膳食导致野生型小鼠肝脏ATGL、CGI58和Rab7表达降低，LC3B表达升高；而RES干预可消除高脂膳食的不良影响，升高ATGL和CGI58表达、并促进二者在细胞内的定位靠近从而促进脂解，升高Rab7表达和降低LC3B表达以促进脂噬；小鼠肝脏靶向SIRT1基因敲减后显著削弱了RES对ATGL和CGI58、Rab7和LC3B的调控作用。细胞实验发现，RES可能通过抑制ATF6与SIRT1结合，促进ATF6的去乙酰化而使其失活，从而削弱ATF6对SIRT1的转录抑制，影响脂滴蓄积相关基因Fsp27的表达，最终抑制脂滴蓄积。RES通过上调Sirt1/ATF6依赖途径抑制脂肪变性的小鼠原代肝细胞和HepG2肝细胞的脂滴形成，减少脂滴蓄积。RES通过上调PKA/PLIN2途径，促进ATGL和CGI-58表达及其相互作用，及ATGL与ATGL在脂滴的共定位增强脂滴脂解，从而减轻脂肪变性HepG2和HHL-5肝细胞的脂质蓄积。本项目明确了RES通过激活Sirt1、上调Sirt1/ATF6、CGI58/ATGL、Rab7/LC3B途径调控脂滴形成和分解，从而抑制肝脏脂肪蓄积的分子机制。本项目对以脂滴调控为靶点治疗NAFLD，以及阐明RES调控脂质代谢的分子机制均提供了重要的实验证据。

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