TLR4 介导的mTOR与Nrf2信号通路在哮喘气道重塑中的作用及山茱萸新苷的干预机制

Airway smooth muscle cell (ASMC) proliferation is a major factor in airway remodeling. As immune cells ASMC increase inflammatory reaction by synthesize and secrete inflammatory cytokines. Notch-1 and HO-1 have been known as therapeutic targets of airway remodeling. They were contributed with regulating of TLR4/mTOR and TLR4/Nrf2 signaling pathways, which produces the network of TLR4/mTOR/Notch-1 and TLR4/Nrf2/HO-1.In the previous study, we demonstrated that cornuside suppressed the Notch1 and HO-1 expression in the lung, improved the antioxidant enzymes activity and prevented airway remodeling. But there were no reports on the roles of TLR4/mTOR/Notch-1 and TLR4/Nrf2/HO-1 signal pathway on airway remodeling or on its protective mechanism of cirnuside. In this study, we evaluate TLR4 mediated mTOR and Nrf2 activation in cultured ASMC and mice airway remodeling model. In addition, we examine suppressive effects on TLR4 mediated mTOR and Nrf2 in cultured ASMC and also on airway remodeling in mice model. Furthermore we evaluate the mechanism of suppressive effects of cirnuside on TLR4/mTOR/Notch-1 and TLR4/Nrf2/HO-1 signaling pathways. Through these molecular mechanisms, cirnuside may control the expression of Notch-1 and HO-1. This study may provide us new tools, such as cirnuside, contributing to prevention and therapy for airway remodeling and development of new kinds of drugs.

气道平滑肌细胞(ASMC)增殖是气道重塑发生、发展的关键环节，而且作为免疫细胞可合成和分泌炎症细胞因子加重炎症反应。研究认为Notch-1和HO-1是气道重塑的重要治疗靶点，可能受TLR4/mTOR以及TLR4/Nrf2信号通路的调控，形成TLR4/mTOR/Notch-1以及TLR4/Nrf2/HO-1信号网络。前期研究表明山茱萸新苷可调控肺组织中Notch-1和HO-1表达、提高抗氧化酶活性，防止气道重塑。但是上述信号通路与气道重塑以及山茱萸新苷干预的相关研究尚未见报道。 本课题拟通过ASMC和小鼠哮喘气道重塑模型，从分子、细胞和整体水平，以TLR4为切入点并延伸到下游mTOR和Nrf2途径，探讨山茱萸新苷介导TLR4/mTOR 及TLR4/Nrf2信号通路调节Notch-1和HO-1的作用机制，明确山茱萸新苷对此信号通路中的作用靶点，为寻找防治气道重塑以及新药开发提供有效的理论依据。

为阐明山茱萸新苷通过TLR4介导的mTOR和Nrf2信号通路缓解哮喘气道重塑的免疫调节机制，项目组圆满完成了4项计划研究内容，并进行了拓展研究。（1）建立支气管哮喘小鼠模型，给予山茱萸新苷，通过检测支气管肺泡灌洗液的细胞分类及上清液中炎症细胞因子的含量、血清IgE含量、肺组织组化和免疫组化、Western blot等指标，阐明和确认山茱萸新苷通过TLR4、mTOR、STAT3以及Notch1等信号通路抗哮喘的作用机制；实验研究进一步拓展建立支气管哮喘小鼠模型，给予红景天苷、芝麻素、羟基红花黄色素A、人参皂苷Rh2等天然药物的有效成分，通过检测Western blot等指标，阐明和确认其抗哮喘作用及机制；（2）建立Anti-DNP IgE介导的肥大细胞过敏模型，给予山茱萸新苷, 通过检测组胺等炎症介质和Western blot等指标，阐明和确认山茱萸新苷通过MAPK和NF-κB信号通路抗过敏反应及作用机制。实验研究进一步拓展建立Anti-DNP IgE介导的肥大细胞过敏模型，给予红景天苷、芝麻素、羟基红花黄色素A、人参皂苷Rh2等天然药物的有效成分, 通过检测Western blot等指标，阐明其抗过敏作用及机制。（3）在体内建立支气管哮喘小鼠模型，之后利用PKC-δ抑制剂、PI3K抑制剂、mTOR抑制剂、HIF-1a抑制剂、VEGF抑制剂不同途径给予小鼠治疗，用HE、PAS染色观察肺部炎症和气道粘液分泌，以及免疫组织化学染色观察肺组织mTOR、VEGF蛋白的表达；用ELISA检测支气管肺泡灌洗液中IL-4、ICAM-1等蛋白水平；用Western blot等方法检测肺组织IL-4、ICAM-1以及PKC-δ、PI3K、mTOR、HIF-1a、VEGF等蛋白的表达；在体外进行BEAS-2B 细胞培养，予以TNF-a/IL-4进行激发，之后利用siRNA和抑制剂进行干预，用Western blote等方法检测肺组织PKC-δ、PI3K、mTOR、HIF-1a、VEGF等蛋白的表达。最终阐明PKC-δ分别通过上调PI3K/Akt/mTOR/HIF-1a/VEGF和mTOR/NF-κB信号通路加重支气管哮喘气道重塑；（4）研究内容进一步拓展, 发现NQO1和Tiam1蛋白分别在非小细胞癌和卵巢癌中高表达，可能是评估非小细胞肺癌和卵巢癌患者不良预后的一个重要指标和潜在治疗靶点