基于PPARγ激动作用和MAPK信号通路的甜茶素脂代谢紊乱调节机制研究

PARγ agonists and MAPK signaling pathways are one of the targets for the development of new drugs for regulating glycolipid metabolism. In the early stage, we found that rubusoside can significantly improve the blood lipid levels of high-fat animals: recent experiments have found that it can promote the differentiation of 3T3-L1 cell fat, significantly increased the mRNA transcription levels of PPARγ and its downstream genes LPL, Glut4 and aP2, as well as C/EBPα and Adiponectin; transcriptome analysis revealed that the MAPK signaling pathway and related genes were affected, suggesting that its mechanism of action to improve lipid metabolism disorder is related to PPARγ agonism and MAPK signaling pathway. In this project, 3T3-L1 cells were induced by rubusoside, and the PPARγ agonist effect of rubusoside was determined by detecting the phosphorylation level of PPARγ protein. The transcription and protein expression levels of related genes were detected by RNA interference and WB on the basis of transcriptomics. At the same time, a high-fat animal model was established, using PPARγ and MAPK pathway inhibitors to detect lipid metabolism-related physiological indicators and mRNA transcription and protein expression, and verifying PPARγ and MAPK signaling pathway at the cell, molecular and animal Levels in the regulation of rubusoside on lipid metabolic disorders.

PPARγ激动剂和MAPK信号通路是糖脂代谢调节新药研发的作用靶点之一。我们前期研究发现甜茶素能明显改善高脂动物血脂水平；同时还可促进3T3-L1细胞脂肪的分化，显著增加PPARγ及其下游基因LPL、Glut4和aP2，以及C/EBPα和Adiponectin mRNA的转录；转录组分析揭示MAPK信号通路及相关基因受影响。上述结果表明甜茶素改善脂代谢紊乱的作用机制与PPARγ激动作用和MAPK信号通路有关。本项目拟以甜茶素诱导3T3-L1细胞，通过检测PPARγ蛋白磷酸化水平确定甜茶素的PPARγ激动剂作用；在转录组学基础上采用RNA干扰和WB等方法检测相关基因mRNA转录和蛋白表达水平；同时建立高脂动物模型，采用PPARγ和MAPK通路抑制剂，检测脂代谢相关生理指标和mRNA转录及蛋白表达，从细胞、分子和整体动物水平验证PPARγ和MAPK信号通路在甜茶素对脂质代谢紊乱调节的作用。

PPARγ激动剂和MAPK信号通路是糖脂代谢调节新药研发的作用靶点之一。课题组前期研究发现甜茶素能明显改善高脂动物血脂水平；同时还可促进3T3-L1细胞脂肪的分化，显著增加PPARγ及其下游基因LPL、Glut4和aP2，以及C/EBPα和Adiponectin mRNA的转录；转录组分析揭示MAPK信号通路及相关基因受影响。上述结果表明甜茶素改善脂代谢紊乱的作用机制与PPARγ激动作用和MAPK信号通路有关。本项目以甜茶素诱导3T3-L1细胞，通过检测PPARγ蛋白磷酸化水平确定甜茶素的PPARγ激动剂作用；在转录组学基础上采用RNA干扰和WB等方法检测相关基因mRNA转录和蛋白表达水平；同时建立高脂动物模型，采用PPARγ和MAPK通路抑制剂，检测脂代谢相关生理指标和mRNA转录及蛋白表达，从细胞、分子和整体动物水平验证了PPARγ和MAPK信号通路在甜茶素对脂质代谢紊乱调节的作用。结果表明：甜茶素能够促进3T3-L1前脂肪细胞分化并增加细胞内TC、TG含量，PPARγ抑制剂则拮抗了甜茶素促进3T3-L1细胞分化及细胞脂质积累的作用。转录组学富集结果显示MAPK信号通路为甜茶素调控前脂肪细胞分化潜在作用通路，Mknk2及Lgasl3为其调节脂质代谢紊乱潜在作用靶点。体外RNA干扰实验证明甜茶素通过提高Mknk2及Lgasl3转录活性、上调p38MAPK及ERK1/2蛋白磷酸化水平激活MAPK/ERK1/2信号通路，进而促进PPARγ磷酸化，增强PPARγ激动活性。体内实验表明甜茶素能够有效降低高脂饮C57BL/6J小鼠体重、肝脏系数、血清脂质含量升高，并减轻小鼠肝脏脂肪变性的情况，具有调节脂代谢紊乱的作用。甜茶素通过激活PPARγ-ERK1/2-p38MAPK信号传导，改善高脂饮食小鼠脂质代谢紊乱，缓解肝脂肪变。本研究分别从体内、体外水平阐明了甜茶改善肝脏脂肪沉积、调节脂质代谢的作用机理，为甜茶素进一步应用于食品添加或临床使用提供了理论依据。