TLR2介导的高脂高胆固醇诱导型NASH的发病机理

The present Studies suggest that elevated TLR2 is closely related to the development of NASH induced by high-fat-and-high-cholesterol diet（HFHC-NASH）, but the regulational mechanism is unknown. This project firstly plan to get the differential expression gene and miRNA profile of primary kupffer's cells from HFHC-NASH rat livers by using the gene expression microarray and the miRNA sequencing technology. Combining the prediction information of TLR2 transcriptional factors (TFs) with the differential expression genes, we will screen out the differentially expressed TFs, regulating TLR2 mRNA expression, and custom chip which is named as the primary screening TF array. Again, combining the prediction information of miRNAs which can regualte TLR2 via binding the TLR2 3'UTR with the differential expression miRNA spectrum, we will screen out differentially expressed miRNAs, regulating TLR2 mRNA expression , and then custom chip which is named as the primary screening miRNA array. Secondly，The rat NR8383 cells is treated with the affecting factors such as lipopolysaccharide (LPS) and cholesterol, which can upregualte TLR2 expression. The key TFs and miRNAs which are closely related to TLR2 upregulation is screened out by using the primary screening TF array and the primary screening miRNA array respectively. Then, the function of regulated TLR2 will be verified through the overexpression or inhibiting expression of the selected TF and miRNA. Lastly, the molecular mechanism of the TLR2 expression regulated by the key TFs will be definited by using CHIP, report gene system and EMSA, and the molecular mechanism of the TLR2 expression regulated by the key miRNAs will be definited by using dual Luciferase Reporter system for miRNA target-gene studying. Finally，the molecular mechanism of "affecting factors- key transcription factors/key miRNAs-upregulated TLR2" will be explicit， and TLR2 mediated etiopathogenesis of HFHC-NASH can be finally clarified, which will provide new drug targets and strategies for prevention and treatments in clinic NASH.

研究提示，TLR2上调与HFHC-NASH发生密切相关，但其上调机理不明。本课题拟用基因表达谱芯片和miRNA测序技术获得HFHC-NASH模型大鼠原代肝枯否氏细胞基因和miRNA差异表达谱，结合预测信息、初筛出调节TLR2且差异表达的转录因子和miRNA、并分别定制芯片；用能使TLR2表达上调的LPS、胆固醇等影响因素处理NR8383巨噬细胞，用定制芯片筛出与TLR2表达上调相关的转录因子和miRNA，将其在NR8383细胞中过表达或抑制表达验证其上调TLR2的作用，确定上调TLR2的关键转录因子和miRNA；通过CHIP、启动子报告基因、EMSA、3'UTR报告基因等分析关键转录因子和miRNA调控TLR2表达的分子机制，明晰"影响因素-关键转录因子/ miRNA-TLR2上调"的分子机制，阐明TLR2介导的HFHC-NASH发病机理，为其防治提供新的药物靶点和策略。

课题组前期用含2%胆固醇的高脂饲料饲喂近交系E3大鼠6个月成功构建非酒精性脂肪性肝炎模型（HFD-NASH），出现明显的血脂紊乱、糖耐量受损、胰岛素抵抗及NASH表型；Toll样受体2（TLR2）在该模型肝中表达升高、且主要表达于肝枯否氏细胞；但其在HFD-NASH中作用及机制尚不清楚。.项目批准后，课题组利用RT-qPCR、Western blotting、启动子荧光素酶报告基因、CHIP、基因过表达/低表达慢病毒载体等技术，主要研究了模型大鼠肝枯否氏细胞中上调TLR2的影响因素以及“影响因素-关键转录因子或miRNA- TLR2上调”的分子机制。结果发现：在HFD-NASH模型中，低表达的miR-144通过靶向结合TLR2 mRNA分子3’UTR区的减少而促进TLR2表达上调以及促炎因子TNF-α、INF-γ等的生成、进而引起NASH的发生；高表达的TGF-β、IFN-γ分别通过Smad3途径、STAT1途径显著上调TLR2表达及促炎因子生成、进而引起NASH的发生；饮食因素高糖、高胆固醇分别通过CHREBP、CEBPα/β上调TLR2并引起下游促炎因子的生成；这些结果说明促炎因子、高糖、高胆固醇等多种因素通过关键转录因子或miRNA上调肝枯否氏细胞中TLR2表达并触发炎症的发生。另外还发现，模型中上调的miR-497通过靶向胰岛素受体导致肝胰岛素抵抗的发生；胆固醇通过固醇感受器LXRα 及转录因子C/EBPβ上调lnc-HC（从用抑制性消减杂交成功构建的模型肝文库中获得并鉴定的一个新长链非编码RNA）的表达，lnc-HC通过与hnRNPA2B1、靶基因Cyp7a1 和Abca1 的mRNA结合而降低靶基因的表达，最终逆向调控肝脏胆固醇代谢；这表明miR-497、lnc-HC参与了肝糖、胆固醇代谢调节，与HFD-NASH模型高血糖、高胆固醇血症密切相关，进而参与了肝TLR2表达上调，炎症激活及NASH的发生。.上述结果表明TLR2在HFD-NASH触发过程中发挥着非常关键的作用，是防治NASH的潜在靶点。课题组针对TLR2胞外段制备的抗TLR2的单克隆抗体在体内外具有明显的抗炎抗损伤作用；还可明显降低HFD-NASH大鼠体重、肝重与空腹血糖，减少巨噬细胞的浸润和肝脏促炎因子的表达，改善肝纤维化水平，而达到治疗作用，为NASH的预防与靶向治疗提供了新思路和新策略。

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