CGI-58和PAT蛋白协同作用调节脂滴稳态及对脂肪肝影响的机制研究

Excessive lipid droplet accumulation is a hallmark of nonalcoholic fatty liver diseases (NAFLDs). The full spectrum of liver pathologies in NAFLD includes simple steatosis, steatohepatitis, fibrosis, and cirrhosis. CGI-58 as a lipid-droplet-associated protein plays an important role in intracellular triglyceride(TG) breakdown by activating Adipose Triglyceride Lipase (ATGL), the rate-limiting enzyme of TG hydrolysis. Loss-of-function mutations in CGI-58 cause lipid droplet deposition in almost all cell types including hepatocytes. We observed that liver-specific CGI-58 knockout (LivKO) mice develop the full spectrum of NAFLD pathologies even on chow diet, which was associated with increases in PAT family proteins including Perilipin 2 (Plin2), Plin3, and Plin4. PAT proteins coat lipid droplets and play a critical role in lipid droplet dynamics. Interestingly, the expression pattern of PAT proteins in LivKO mice is different from that in other NAFLD models that hardly develop liver fibrosis such as liver-specific ATGL knockout mice and mice on high fat diet. It was reported that CGI-58 can interact with Plin2 and Plin3. Thus, we hypothesize that CGI-58 deficiency-induced changes in hepatic PAT protein expression patterns may be responsible for NAFLD progression in LivKO mice. To provide preliminary evidence in support of this hypothesis, we knocked down Plin2 in the liver of LivKO mice using adeno-associated virus (AAV)-shRNAs and found that their lipid droplet accumulation and fibrosis were significantly reduced in liver. However, Plin2 knockdown in liver upregulated expression of other PAT proteins. In this proposal, we will test whether simultaneously inhibition of several PAT proteins will work better than single knockdown. We will first identify the best combination of PAT protein inhibition in Huh7 hepatic cells using the CRISPR/Cas9 gene editing technology. Based on in vitro results, we then determine whether a specific combination of PAT protein inhibition will prevent NAFLD development and progression in LivKO mice. We will further identify potential mechanisms. The outcomes of these studies will establish PAT-CGI58 protein interactions as a novel therapeutic target for NAFLD.

脂滴过度沉积是非酒精性脂肪肝(NAFLD)的重要病理特征。脂滴相关蛋白CGI-58在促进甘油三酯分解中起重要作用，其功能缺失导致细胞脂滴异常沉积。我们发现肝细胞CGI-58敲除小鼠(LivKO)会出现脂滴沉积、脂性肝炎、到肝纤维化的系列改变，且伴主要脂滴包被蛋白Plin2、Plin3、Plin4等PAT家族成员显著增高。单个PAT蛋白表达异常常难引起伴有肝纤维化的NAFLD模型，结合文献报道提示CGI-58与PAT蛋白存在协同作用，推测该协同作用失衡可能与NAFLD进展及纤维化相关。我们发现敲低Plin2后，LivKO小鼠脂质沉积和肝纤维化明显减轻，但其他PAT蛋白上调，提示联合改变多个PAT蛋白水平可能效果更好。我们将在Huh7肝细胞系中验证多种PAT蛋白编辑方式效果，用LivKO小鼠进行验证该PAT蛋白表达模式的防治效果，并探讨其分子、细胞和整体机制，为NAFLD防治提供新靶点。

脂滴作为新兴研究的细胞器，其在肝细胞内的动态平衡调节是肝脏脂质代谢平衡调节的关键。肝细胞内过量的脂滴蓄积会导致脂质沉积，引发脂肪肝，并进一步诱发肝脏炎症、纤维化发生，从而导致肝炎、肝硬化甚至肝癌。本研究主要以CGI-58Flox/Flox实验小鼠为研究对象，以嗜肝细胞的腺相关病毒AAV2/8为载体，插入肝脏特异性启动子hTBG，通过靶向表达Cre蛋白和Plin2/Plin3/Plin4基因的miRNA，构建了肝细胞特异性敲除CGI-58和敲低Plin2/Plin3/Plin4的小鼠模型。实验以单纯CGI-58敲除和CGI-58敲除合并Plin2/Plin3/Plin4敲低为对照组和实验组，在高脂饮食诱导下检测小鼠的葡萄糖耐量试验、胰岛素耐量实验、肝脏的质量、血生化指标，同时运用RNA-Seq、脂质组学、肝脏免疫组化、qPCR、WB等实验探究协同作用的具体分子机制。同时我们以AML-12细胞为细胞模型，通过构建靶向SiRNA实现AML-12细胞内Plin2/Plin3的敲低，在油酸诱导的条件下，运用qPCR、WB和免疫荧光探究Plin2/Plin3在AML-12细胞脂滴形成的作用。我们研究发现，小鼠肝脏Plin2/Plin3/Plin4基因的敲低均可缓解CGI-58基因敲除所致的非酒精性脂肪肝，主要表现为肝脏体积减小、ALT/AST改善、肝脏TG含量降低；且Plin4基因敲低的防治作用强于Plin3强于Plin2。在机制上，Plin2基因的敲低抑制了肝细胞内微泡型脂滴的形成，Plin3的敲低通过降低炎症反应并缓解肝细胞的坏死性凋亡，Plin4的敲低显著抑制PPARα通路的激活。同时细胞实验证实Plin2/Plin3均参与细胞内脂滴的形成。研究阐明了PAT蛋白家族Plin2/Plin3/Plin4在CGI-58缺失所致肝脏脂肪变的作用，为Chanarin–Dorfman Syndrome患者提供了新的治疗策略，具有临床意义。通过本基金支持，先后培养了博士研究生2名，硕士研究生2名，发表文章5篇，其中SCI4篇。

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