基于“温病理论”从PI3K/Akt/mTOR自噬通路探讨五加减正气散抗轮状病毒的作用机制

The incidence of Rotavirus enteritis remains high, and the autophagy mechanism of host cells and effective intervention virus replication have become the focus of research. This disease belongs to the category of "Febrile disease" in Traditional Chinese Medicine, and the treatment should be determined by lifting medium coke. Early studies have confirmed that the Wujiajiajianzhengqi Powder can inhibit rotavirus biosynthesis, regulate key proteins and downstream factors in the TLR3 signal pathway, and play a significant antiviral role. In this paper, Caco-2 cells were infected with RV WA strain by in vitro cell culture method to verify the effect of Wujiajiajianzhengqi Powder on autophagy of infected cells. Based on the PI3K/AKT/mTOR autophagy pathway, inflammatory factors such as IL-1 β, IL-6, TNF-α were detected by using autophagy inhibitors and autophagy activators. autophagy related genes PI3K, AKT, mTOR, P62 and other proteins and mRNA expression; It further reveals the antiviral mechanism and possible target of Wujiajiajianzhengqi Powder. The results of this study will form a key technique based on the clinical evaluation of the antiviral efficacy of Chinese herbal medicine compound, provide experimental basis for the treatment of rotavirus enteritis by Chinese medicine, and truly realize the organic combination of thermology and virology.

轮状病毒性肠炎发病率居高不下，宿主细胞的自噬机制及有效干预病毒复制成为研究重点。本病属中医“温病”范畴，治疗当以升降中焦为定法。前期研究已证实，五加减正气散可抑制轮状病毒生物合成，调控TLR3信号通路关键蛋白及下游因子，发挥显著的抗病毒作用。本课题进一步采用体外细胞培养法，以RVWA株感染Caco-2细胞，验证五加减正气散对染毒细胞自噬的影响；基于PI3K/AKT/mTOR自噬通路，通过应用自噬抑制剂、自噬激活剂分别设组对照，检测IL-1β、IL-6、TNF-α等炎性因子表达，自噬相关基因PI3K、AKT、mTOR、P62等蛋白和mRNA表达；进一步揭示五加减正气散的抗病毒机制和可能的作用靶点。研究结果将形成基于临床的中药复方抗病毒疗效评价的关键技术，为中医药治疗轮状病毒性肠炎提供实验依据，真正实现温病学与病毒学的有机结合。

轮状病毒性肠炎发病率居高不下，宿主细胞的自噬机制及有效干预病毒复制成为研究重点。本病属中医“温病”范畴，治疗当以升降中焦为定法。前期研究已证实，五加减正气散可抑制轮状病毒生物合成，调控TLR3信号通路关键蛋白及下游因子，发挥显著的抗病毒作用。本课题进一步采用体外细胞培养法，以RV感染Caco-2细胞，验证五加减正气散对染毒细胞自噬的影响；基于PI3K/AKT/mTOR自噬通路，通过应用自噬抑制剂、自噬激活剂分别设组对照，检测IL-1β、IL-6、TNF-α等炎性因子表达，自噬相关基因PI3K、AKT、mTOR、P62等蛋白和mRNA表达；进一步揭示五加减正气散的抗病毒机制和可能的作用靶点。通过自噬流实验和透射电镜观察到在轮状病毒感染早期阶段染毒细胞的自噬水平不断增强，到4h时自噬水平最强。加入含药血清后，五加减正气散高、中、低剂量组的自噬水平显著低于RV组，其中以五加减正气散高剂量组最显著，自噬相关蛋白Beclin1的水平显著下降，P62的水平显著升高，同时ATG5、ATG12mRNA的表达亦显著降低（P＜0.01），PI3K/AKT/mTOR信号通路相关PI3K、p-AKT、p-mTOR的蛋白和PI3K、AKT、mTORmRNA水平显著降低（P＜0.01），相关炎性因子IL-1β、IL-6、TNF-α的表达亦不同程度降低（P＜0.05）。结果证实，五加减正气散对抗RV的作用机制可能与其能够干预PI3K/AKT/mTOR信号通路从而调控Caco-2细胞的自噬相关。该结果形成了基于临床的中药复方抗病毒疗效评价的关键技术，为中医药治疗轮状病毒性肠炎提供实验依据，实现了一定程度的温病学与病毒学的有机结合。