基于RIPK1/RIPK3信号探讨金欣口服液调控上皮细胞程序性坏死治疗RSV肺炎机理

Respiratory syncytial virus (RSV) pneumonia is the most commonviral pneumonia in children, which is a serious threat to children's health.What an important molecular pathological mechanism of viral pneumonia is excessive immune activation of immune cells leads to inflammation and target cell injury. The focus of our previous research is on the overactivation of immune signals regulated by drugs. However, it is also very important that infected target cells-lung epithelial cells are protected and repaired directly by drug.Recent research has provedthat the key to tissue damage is the programmed necrosis of host cells induced by RSV which is regulated by RIPK1/RIPK3/MLKL system , and our preliminary experiments suggested that Jin Xin oral liquid could inhibit the excessive activation of RIPK1 and RIPK3.According to the literature and previous studies, we put forward the scientific hypothesis that Jinxin oral liquid could directly protect the target cells, by which mechanisms probably related to its intervention in RIPK1/RIPK3-mediated programmed necrosis of cells. In this study, we aim to explore whether Jinxin oral liquid has direct target cell protection and the mechanism of regulating programmed necrosis through RIPK1/RIPK3 using the in vitro and in vivo models of RSV infection and combining with modern medical research methods. Finally, it will reveal the internal mechanism of the prescription in the treatment of RSV pneumonia in children and the protection of target cells, and provides scientific basis and new ideas for the treatment of viral pneumonia with traditional Chinese medicine.

呼吸道合胞病毒（RSV）肺炎是小儿最常见病毒性肺炎，严重威胁儿童健康。免疫细胞过度激活导致炎症损伤靶细胞是病毒性肺炎重要病理机制。课题组前期研究焦点均在调节免疫细胞免疫信号过度活化上，而感染的靶细胞-肺上皮细胞直接保护修复也非常关键。最新研究显示，受体相互作用蛋白激酶（RIPK）1/RIPK3/MLKL系统调控RSV等病毒诱导的宿主细胞程序性坏死是引起组织损伤的关键，且预实验提示金欣口服液抑制RIPK1、RIPK3过度活化。根据文献和前期研究提出科学假说：金欣口服液可直接保护靶细胞，机制可能与其干预RIPK1/RIPK3介导的细胞程序性坏死有关。本研究建立了RSV感染体内外模型，并结合现代医学研究方法，探讨金欣口服液是否有直接靶细胞保护作用及其通过RIPK1/RIPK3调节程序性坏死机制。最终揭示该方治疗小儿RSV肺炎、保护靶细胞的科学内涵，为中医药治疗病毒性肺炎提供科学依据及新思路。

呼吸道合胞病毒肺炎是最常见的小儿病毒性肺炎，严重的会引起死亡，死亡率呈逐年增加的趋势，临床上不可忽视。目前尚无有效的RSV疫苗和理想药物，金欣口服液临床上治疗小儿RSV肺炎痰热闭肺证有显著疗效，临床使用十多年。基于中医药复方在小儿病毒性肺炎治疗中的独特优势，阐明临床小儿病毒性肺炎有效经典方药的作用机制，寻找开发更安全，有效，机制明确的复方及中成药，这些问题值得我们进一步探索。.课题组对于金欣口服液前期实验和临床研究发现该方可以明显改善RSV肺炎导致的肺损伤，通过调节免疫信号，抑制炎症因子如释放，减少过度免疫激活，减轻肺炎程度。近年来发现一种新的细胞死亡方式：程序性坏死（Necroptosis），该过程在呼吸道病毒感染引起的肺部炎症及组织损伤坏死中起关键作用。本课题聚焦于该点，建立了RSV感染的体内外模型，体内动物实验中观察到金欣口服液对RSV肺炎整体改善效果显著，电镜形态学观察到RSV可引起组织程序性坏死包括内质网及线粒体严重损伤，还发现有自噬现象，金欣口服液能起到明显改善作用。对RSV引起的过度炎症及损伤坏死相关细胞因子的检测，发现金欣口服有很好的回调作用。通过免疫组化、免疫荧光、mRNA水平和蛋白水平观察，金欣口服液治疗RSV肺炎是通过调控程序性坏死关键途径RIPK1/RIPK3/MLKL信号来实现，另外通过查阅文献和研究中发现，PI3K/AKT信号与程序性坏死的信号调控密切相关，金欣口服液对该信号也有较好的调控作用，具体机制有待后续研究。建立了小鼠巨噬细胞及肺上皮A549细胞模型，金欣口服液作用在体外细胞水平上也得到了验证。另外，该方有效成分大黄酸也可以明显干预程序性坏死RIPK1/RIPK3途径，并影响线粒体功能，具体机制有待后续系统深入的研究。.本课题提示金欣口服液有直接细胞保护作用及其通过RIPK1/RIPK3调节程序性坏死的机制，一定程度揭示该方治疗小儿RSV肺炎、保护靶细胞的内在机制，为该复方的配伍优化、物质基础研究以及新药开发提供基础及依据，使其更好的应用于临床。同时也为中医药治疗伴有严重组织损伤的感染性疾病提供一个新的策略。