大黄煎剂作用于肠道菌群调节LPS/TLR4通路对MHE神经保护作用的机制研究

In recent years, patients with cirrhosis and Minimal Hepatic Encephalopathy (MHE) increase year by year. If not intervened and treated in time, it can further develop into symptomatic HE, which is more difficult to treat, and the mortality is increased. The pathogenesis of MHE is extremely complex. At present, the understanding is not perfect. Its mechanism has not been fully elucidated until now. There is no standard MHE diagnosis and treatment guideline at home and abroad. Previous study found that MHE serum metabolites and intestinal flora imbalance, lipid metabolism and so on, but whether the rhubarb decoction by LPS/TLR4 pathway regulation related proteins, which play anti apoptotic neuroprotective effects, as well as the specific regulatory mechanism is unknown? This project firstly established MHE rat model, and then using the Morris water maze test, BAEP assay, immunofluorescence technique, TUNEL in situ technique combined with confocal laser scanning microscope imaging and WesternBlot technology, to observe whether it can pass the neuroprotective effect of LPS/TLR4 pathway in MHE rats, thus explaining the pathogenesis of MHE, may provide potential treatment options for prevention and treatment of MHE.

近年来，肝硬化并发轻微型肝性脑病(Minimal Hepatic Encephalopathy，MHE)患者逐年增多，若不及时干预和治疗，可进一步发展为症状型HE，救治难度增加，且死亡率增高。MHE发病机制极其复杂，目前研究认识还不完善，其机制至今未完全阐明，国内外都未形成标准的MHE诊断与治疗指南。课题组前期研究发现MHE患者血清中代谢物与肠道菌群失衡、脂质代谢紊乱等有关，但大黄煎剂是否通过LPS/TLR4通路调控相关蛋白分子，从而发挥抗凋亡神经保护的作用，以及具体调控机制尚不明确。本项目首先建立MHE大鼠模型，然后采用morris水迷宫实验，BAEP检测、免疫荧光双标技术，TUNEL原位凋亡技术，结合激光共聚焦显微镜成像、WesternBlot等技术，观察其是否能够通过LPS/TLR4通路影响MHE大鼠神经保护作用，从而阐释MHE的发病机制，可能为MHE防治提供潜在的治疗方案。

肝硬化并发轻微型肝性脑病患者逐年增多，若不及时干预和治疗，可进一步发展为症状型HE，救治难度增加，且死亡率增高。MHE发病机制极其复杂，目前研究认识还不完善，其机制至今未完全阐明，国内外都未形成标准的MHE诊断与治疗指南。课题组前期研究发现MHE患者血清中代谢物与肠道菌群失衡、脂质代谢紊乱等有关，但大黄煎剂是否通过LPS/TLR4通路调控相关蛋白分子，从而发挥抗凋亡神经保护的作用，以及具体调控机制尚不明确。研究采用TAA造模方法制备MHE大鼠模型，然后采用Morris水迷宫实验、免疫荧光双标技术，TUNEL原位凋亡技术，结合扫描电镜、qRT-PCR和WesternBlot等技术，观察大黄煎剂对MHE大鼠认知功能的作用，大黄煎剂对MHE大鼠海马区组织形态的影响及其神经保护作用机制。研究发现通过大黄煎剂对MHE大鼠的干预发现，大黄煎剂组干预后与模型组相比可改善学习记忆功能。大黄煎剂可以改善MHE大鼠海马胶质细胞炎性程度，抑制大鼠海马IL6表达。大黄煎剂可减轻肝纤维化的程度，改善肝功能，减少血清LPS和血氨含量，调节肠道菌群的多样性和丰富度，并抑制肝组织中TLR4、MyD88、p65和Iκb-a蛋白表达，调控p65、Iκb-a磷酸化减少NFκB激活，通过调节神经炎症和全身炎症起到MHE神经保护作用。阐明了大黄煎剂抑制肝硬化轻微型肝性脑病可能的新的分子机制，为这种传统中药抗MHE的广泛使用奠定了理论基础，可能为MHE防治提供潜在的治疗方案。