基于TLR4介导的炎性损伤探究葛根素干预高脂饮食/慢性应激诱导抑郁症的作用及其机制

The high prevalence and high disability rate of depression require researchers to develop safe and effective new antidepressants. Puerarin, an active ingredient isolated from the root of Chinese materia medica Pueraria lobata (Willd.) Ohwi, has strong antidepressant effects, but the mechanism remains unclear. The applicant's previous study found that puerarin significantly improved the depressive-like behaviors of rats induced by high-fat diet/chronic stress. Moreover, puerarin inhibited the expression of inflammatory factors and Toll-like receptor 4 (TLR4) in hippocampus. It is suggested that the anti-depressive effects of puerarin may be related to its anti-inflammatory activities. Based on TLR4-mediated inflammatory injury, this project will further investigate the anti-depressive mechanisms of puerarin. Firstly, the effects of puerarin on the structure and functions of intestine and hippocampus will be determined. Then, TLR-based PCR array, cytokine-based protein chip, and targeted lipidomics techniques will be used to analyze the effects of puerarin on TLR4 and its downstream signal molecules, phospholipid metabolites and metabolic enzymes in intestinal and hippocampal tissues. Finally, a puerarin-regulated molecular network will be constructed at mRNA, protein and metabolite levels to elucidate the anti-depressive mechanisms of puerarin. It would provide new strategies for the treatment of depression.

抑郁症的高患病率和高致残率亟待研究者开发安全有效的新型抗抑郁药物。中药葛根中活性成分葛根素具有较强的抗抑郁作用，但作用机制仍不清楚。申请人前期研究发现，葛根素可显著改善高脂饮食/慢性应激诱导的大鼠抑郁样行为表现，抑制海马组织炎症因子和Toll样受体4（TLR4）的表达，提示葛根素的抗抑郁作用可能与其抗炎活性有关。本项目将基于TLR4介导的炎性损伤，进一步探究葛根素的抗抑郁分子机制。首先，明确葛根素对肠、脑组织海马区结构和功能的影响；然后，运用TLR-based PCR array、cytokine-based蛋白芯片和靶向脂质组学技术，分析葛根素对肠和海马组织TLR4及其下游信号分子、磷脂代谢产物、代谢酶的影响；最后，从mRNA、蛋白质和代谢多水平构建葛根素调控的分子网络，阐明其抗抑郁分子机制，为抑郁症的治疗提供新策略。

本项目通过建立高脂饮食联合慢性应激（HFD/CUMS）诱导的大鼠抑郁模型，以TLR4介导的炎性损伤为切入点，探究葛根素调节肠/脑结构和功能改善HFD/CUMS诱导大鼠抑郁样行为的分子机制。研究发现葛根素干预可显著改善模型大鼠1%糖水偏好、运动活力和探索欲望，该作用与其抑制外周和海马组织炎性损伤有关。TLR4是葛根素修复小肠和脑内炎性损伤的关键靶点，葛根素通过抑制TLR4介导的occludin、claudin-1表达降低修复肠粘膜结构，进而阻断肠内LPS移位入血，抑制TLR4/LPS介导的炎症级联反应；同时，葛根素可抑制TLR4介导的小胶质细胞活化改善海马神经元细胞增殖/凋亡失衡。从分子机制上分析，葛根素通过抑制TLR4/cPLA2/COX-2轴介导的“炎症性磷脂代谢紊乱”发挥抗抑郁作用。此外，我们考察了葛根素对小肠微环境稳态的影响，发现葛根素的抗抑郁作用还与其增加小肠内容物乳酸杆菌、Blautia和双歧杆菌丰度，改善血浆SCFAs含量有关。对其机制研究发现TLR4是葛根素抗抑郁的关键靶点，通过（1）修复小肠微环境稳态阻断TLR4/LPS激活炎症级联反应；（2）抑制TLR4/PPARγ/NF-κB p65介导的胶质细胞活化；（3）抑制TLR4/NLRP3/caspase-1介导的神经元凋亡；（4）促进TLR4/COX-2/AKT/mTOR介导的神经元增殖，发挥多途径抗抑郁作用。本研究初步明确了葛根素通过多重途径调控TLR4信号传导改善HFD/CUMS大鼠抑郁样行为的分子机制，为进一步阐明葛根素的抗抑郁作用机制和拓展葛根素的临床应用提供了实验基础，也为抑郁症的治疗提供了新方向。