MiR-19b-3p调控PPARα表达并减少NAFLD小鼠肝脏脂肪沉积的机制研究

Non-alcoholic fatty liver disease (NAFLD) is usually co-morbid with type 2 diabetes. Effective treatments are non-available for NAFLD now. Recently, some studies reported that microRNAs (miRs) could regulate the development of NAFLD. Moreover, it has also been discovered that glucagon-like peptide-1 receptor agonist (GLP-1RA) could ameliorate the progress of NAFLD. But, the mechanisms of miRs involving in the remission of NAFLD treated by GLP-1RA remains to elucidate. We previously found that GLP-1RA could reverse the downregulation of miR-19b-3p induced by high-fat, ameliorate lipid accumulation and inflammation simultaneously in hepatocytes. Target gene prediction online and our preliminary experiments showed that miR-19b-3p could regulate the expression of PPARα. Accordingly, we presume that miR-19b-3p involves in the remission of lipid accumulation treated by GLP-1RA through regulating PPARα. Using the technology of gene overexpression and gene silence, we will treat the high-fat fed mice or hepatocytes cultured by high fat with GLP-1RA or GLP-1(9-36) and explore: 1) Is PPARα the target gene of miR-19b-3p? 2) Does miR-19b-3p mediate the remission of lipid accumulation and inflammation that originated from GLP-1RA treatment by regulating the expression of PPARα, and retard the development of NAFLD?

非酒精性脂肪性肝病（NAFLD）常与2型糖尿病并存，目前尚缺乏安全有效的治疗手段。研究发现：miRs可调控NAFLD发生发展，而抗糖尿病药胰高糖素样肽-1受体激动剂（GLP-1RA）可延缓NAFLD的进展。但miRs是否参与了GLP-1RA改善NAFLD的机制未见报道。我们前期发现GLP-1RA可逆转高脂培养引起的肝细胞miR-19b-3p表达改变，减轻肝细胞脂肪积聚；在线靶基因预测和细胞预实验表明：miR-19b-3p可影响PPARα表达。据此推测：miR-19b-3p可调控PPARα表达、缓解肝脏脂肪积聚。我们拟用GLP-1RA或GLP-1(9-36)处理高脂喂养的小鼠及高脂培养的肝细胞，结合基因过表达或沉默，探讨：1.PPARα基因是miR-19b-3p的靶基因？2.miR-19b-3p通过调控PPARα表达介导GLP-1RA减少肝脏脂肪积聚和局部炎症反应，从而延缓NAFLD的进展？

肝纤维化是非酒精性脂肪性肝病(NAFLD)的严重阶段，与肝星状细胞(HSC)的激活密切相关，其中涉及microRNAs (miRNAs)。钠-葡萄糖共转运蛋白2抑制剂(SGLT2i)可改善NAFLD患者的肝纤维化，但其潜在机制尚不清楚。.我们用高脂饮食诱导的C57BL/6小鼠和ob/ob小鼠构建了两种非酒精性脂肪性肝炎(NASH)相关纤维化模型。通过对多种miRNAs的筛选和检测，我们发现miR-34a-5p在NASH肝脏中高表达。在分离的小鼠肝实质、间充质细胞和细胞系中，我们通过RT-qPCR证实miR-34a-5p主要在HSC中高表达。此外，在empagliflozin (EMPA)的作用下，NASH小鼠的肝纤维化得到改善，miR-34a-5p在肝脏中的表达减少，TGFβ信号通路也被抑制。我们通过RT-qPCR、western blot和免疫荧光染色进一步证明，过表达miR-34a-5p促进了HSC细胞的激活，而下调miR-34a-5p则抑制了HSC细胞的激活。然后，通过数据库预测和双荧光素酶报告基因实验，我们发现GREM2是miR-34a-5p的靶基因。最后，以蛋氨酸和胆碱缺乏(MCD)饲料喂养ob/ob小鼠，构建NASH相关纤维化模型。组织病理学和免疫组化染色显示，EMPA不仅能改善肝纤维化，促进GREM2的表达，还能抑制BMP2的表达和TGFβ信号通路的激活。.SGLT-2抑制剂可通过调控miR-34a-5p/GREM2/BMP2-TGFβ信号通路抑制HSC活化，改善肝纤维化。.本研究揭示了NASH肝纤维化的部分分子机制，为研发NASH治疗药物提供了理论和实验基础。