脂肝方通过P62泛素化-自噬调控NASH NF-κB/Keap1-Nrf2介导的炎症与抗氧化机制

Oxidative stress and inflammation play a key role in the progression of NASH.The activation of NF-κB pathway mediates the inflammatory response of the liver cells, while the Keap1-Nrf2 pathway activates the antagonistic oxidative damage of the liver cells.Recent studies suggest that autophagy degradation of p62 ubiquitin protein aggregates affects the antioxidant effect of the NF-κB pathway upstream of TRAF6 and the Keap1-Nrf2 pathway.Previous studies have confirmed that ZhiGanFang regulates fatty liver by anti-oxidative, anti-inflammatory and autophagy .We have the hypothesis that ZhiGanFang may block the process of NASH by regulating the autophagy and ubiquitin degradation of p62, which p62 regulates TRAF6 to activate NF-κB pathway and Keap1-Nrf2 oxidative damage. The project intends to replicate the rat model of NASH, ubiquitin aggregates of p62 will be observed by laser confocal microscopy; autophagosomes observed by electron microscopic;the key genes and proteins in the pathway of TRAF6-NF-κB and Keap1-Nrf2 and whose downstream will be detected by RT-PCR, immunohistochemistry and Western Blot ;protein interactions will be detected by CO immunoprecipitation, all that will investigate the molecular mechanism of P62 ubiquitination and autophagy regulation NSAH inflammation and antioxidation, and clarify the effective target of ZhiGanFang.

氧化应激和炎症反应在NASH进展中起关键作用，NF-κB通路激活介导肝细胞炎症反应，而Keap1-Nrf2通路激活拮抗肝细胞的氧化损伤。最新研究表明，p62泛素化蛋白聚集体的自噬降解介导NF-κB通路的上游TRAF6的泛素化和Keap1-Nrf2通路的抗氧化效应。前期研究证实了脂肝方的抗氧化、抗炎及自噬调节作用。我们假说脂肝方可能通过调节p62泛素化聚集体的自噬降解、以及调控TRAF6的泛素化激活NF-κB通路与Keap1-Nrf2抗氧化损伤而阻断NASH的进程。项目拟复制NASH大鼠模型，运用激光共聚焦显微镜观察p62泛素化聚集体，电镜观察自噬体，RT-PCR，免疫组化、Western Blot等检测TRAF6-NF-κB/ Keap1-Nrf2通路及其下游关键基因及其蛋白表达，免疫共沉淀检测蛋白相互作用，探讨P62泛素化-自噬调控NSAH炎症与抗氧化的分子机制，并阐明脂肝方的效应靶点。

氧化应激和炎症反应在NASH进展中起关键作用，NF-κB通路激活介导肝细胞炎症反应，而Keap1-Nrf2通路激活拮抗肝细胞的氧化损伤。最新研究表明，P62泛素化蛋白聚集体的自噬降解介导NF-κB通路的上游TRAF6的泛素化和Keap1-Nrf2通路的抗氧化效应。前期研究已证实了脂肝方的抗氧化、抗炎及自噬调节作用。项目假说脂肝方可能通过调节P62泛素化聚集体的自噬降解、以及调控TRAF6的泛素化调节NF-κB通路与Keap1-Nrf2抗氧化损伤而阻断NASH的进程。项目通过构建NASH大鼠模型，运用激光共聚焦显微镜观察P62泛素化聚集体，电镜观察自噬体，RT-qPCR，Western Blot等检测TRAF6-NF-κB/Keap1-Nrf2通路及其下游关键基因及其蛋白表达，免疫共沉淀检测蛋白相互作用，以探讨P62泛素化-自噬调控NSAH炎症与抗氧化的分子机制，并阐明脂肝方的效应靶点。研究结果显示模型组肝细胞脂肪变、炎症浸润明显，自噬体减少，细胞器损伤，P62表达升高，自噬LC3降低，TRAF6、NF-κBp65及下游炎症因子升高，抗氧化因子表达下降。经脂肝方治疗后肝细胞脂肪变及炎症浸润改善，细胞损伤缓解，自噬水平提高，P62 降低，HDAC6、泛素无明显变化，TRAF6/NF-κBp65炎症反应减弱，Nrf2及HO-1、NQO1抗氧化效应增强，以高剂量脂肝方效果最佳，优于西药阳性对照组。且P62与TRAF6、p-P62与Keap1两组蛋白均存在相互作用。说明脂肝方可能是通过提高肝细胞自噬水平，消耗堆积的P62，导致P62与TRAF6结合减少，从而降低或阻断下游IKKβ/NF-κBp65炎症通路，同时有效促进P62的磷酸化水平，激活Nrf2抗氧化通路，以发挥抗炎和抗氧化的双重效用。