外泌体microRNAs在PPARγ通路介导NASH脂质性炎症中的作用及机制研究

Nonalcoholic steatohepatitis (NASH) is part of a spectrum of nonalcoholic fatty liver disease (NAFLD) and can progress to cirrhosis in 15% of subjects. The mechanisms by which NASH develops and progresses to cirrhosis have not been fully defined. MicroRNAs (miRNA) are non–protein-coding, small single-stranded RNA, typically 21 to 23 nucleotides long, that regulate gene expression via messenger RNA (mRNA) degradation or translational inhibition. Many miRNAs exist in the circulation as well as in tissues, and a large fraction of these are found in exosomes (50–200-nm vesicles that are released from multivesicular bodies). We had analyzed the miRNAs in exosome of NASH and found that miR-122was upregulated and miR-27b was downregulated compared with controls. Our Previous study indicated that the persistent low level expression of PPARγ in visceral adipocytes was closely related to the exacerbation of triglyceride accumulation in adipocytes. PPARγ agonist (pioglitazone) can partially block the accumulation of intracellular fat. The target of miR-122 and miR-27b is PPARγ. So we speculate that exosome secreted from the steatotic hepatocytes carries and translocates miR-122 and miR-27b and target to PPARγ in kupffer cells, which initiates and promotes inflammation by inhibiting the activation of the NF-κB signaling pathway and thereby plays a key role in NASH development. This project intends to construct NASH mice to analyze the effects of miR-122 and miR-27b on PPARγ, lipid metabolism and inflammatory response in liver tissue and to investigate the effect of hepatocyte-specific exosomes on the activation of NF-κB pathway in kupffer cells and the regulatory role of PPARγ by using steatotic hepatocyte model. To explore the mechanism of exosome miRNAs-PPARγ pathway in the pathogenesis of NASH from the level of tissue, cell and molecule will provide scientific clues for further searching for potential intervention targets of NASH.

脂质性炎症是非酒精性脂肪性肝炎（NASH）发生的重要环节，但作用机制尚不明确。我们前期研究发现NASH患者血清外泌体miR-122高表达、miR-27b低表达；并证实miR-122和miR-27b的靶标PPARγ是调控甘油三酯沉积的关键分子。故推测脂肪变性肝细胞分泌外泌体携带并转移miR-122、miR-27b靶向作用于kupffer细胞PPARγ，通过活化NF-kB信号通路而启动并促进炎症，由此在NASH发生发展中起关键作用。本项目拟构建NASH小鼠，研究miR-122、miR-27b表达水平对肝组织PPARγ、脂质代谢及炎症反应的影响；应用脂肪变性肝细胞模型，研究肝细胞特异性外泌体对kupffer细胞NF-kB通路活化的影响及PPARγ的调控作用，从组织、细胞和分子层面探讨外泌体miRNAs-PPARγ途径在NASH发病中的作用机制，为进一步寻找潜在的干预靶点提供科学线索。

肝纤维化是非酒精性脂肪性肝炎（NASH）进展过程中的一个严重病理变化，它与肝星状细胞的激活及巨噬细胞的相互作用密切相关，但具体机制尚未完全阐明。我们的研究结果表明：①在高脂高胆固醇饮食诱导的NASH模型中，肝脏发生轻度纤维化的同时，M2型巨噬细胞标志物的表达显著降低；进一步发现M2型巨噬细胞外泌体可显著抑制肝星状细胞激活，而且这种抑制作用可通过介导miR-411-5p发挥；MiR-411-5p可直接下调其靶基因CAMSAP1的表达，下调CAMSAP1的表达也可以抑制肝星状细胞的激活。②在NAFLD临床样本、NAFLD小鼠模型和细胞模型中，我们发现miR-125b-5p表达减少，而其靶基因ITGA8表达增加；进一步发现miR-125b-5p靶向并下调ITGA8，从而改善NAFLD肝纤维化。③通过对NASH患者肝成纤维细胞的环状RNA（circRNA）表达谱分析，我们观察到线粒体circRNA占NASH成纤维细胞中下调circRNA的相当一部分；进一步发现位于线粒体中的与NASH相关的circRNA（SCAR）可抑制线粒体ROS（mROS）输出和成纤维细胞活化；在体内，靶向SCAR可减轻高脂饮食引起的肝硬化。综上，本项目的研究结果有助于理解肝星状细胞活化和肝纤维化的潜在机制，为NASH肝纤维化的防治提供理论依据和实验基础。

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