TGF-β1与Notch信号通路交叉对话在哮喘气道重构中的调控机制研究

Airway remodeling is one of the main pathological features of severe asthma, and the proliferation of airway smooth muscle cells (ASMCs) plays a key role in its occurrence and development. Recent research has shown that HES-1 is an intersection point downstream of TGF-β1 and Notch signaling pathway. Our previous study showed that TGF-β1 was involved in the increase of Notch-1 production in ASMCs culture supernatants and intracell. In this regard, the expression of Notch-1 was thought to be positively correlated with the proliferation of ASMCs. In addition, pyrin recombinant protein（PRP）inhibited the expression of both TGF-β1 and Notch-1, and exerted a significant inhibitory effect on airway remodeling. However, the effects of cross-talk described above and the effect of PRP on airway remodeling have not been completely evaluated. In order to elucidate the effect of the cross-talk between TGF-β1 and Notch-1 signaling pathway on airway remodeling, we investigated the effects of an agonist and an inhibitor of TGF-β1 and Notch-1, and the effect of PRP on TGF-β1 and Notch-1 signaling pathway. In addition, to clarify the underlying mechanism of PRP in mediating the molecular cross-talk between TGF-β1 and Notch-1 signal transduction, we evaluated the mechanisms of the cross-talk between TGF-β1 and Notch-1 signaling pathway on airway remodeling, and the effects of an agonist and an inhibitor of TGF-β1 and Notch-1 on TGF-β1 and Notch-1 signaling pathway in both in vivo and in vitro experiments. This study will clarify the theoretical basis for the effective prevention or treatment of the airway remodeling in asthma and for the development of new drugs.

气道重构是难治性哮喘的主要病理特征，其中气道平滑肌细胞(ASMCs)的增殖起关键性作用。研究表明，TGF-β1和Notch信号通路在下游HES-1进行交叉。前期研究发现：TGF-β1干预ASMCs培养上清液和细胞内Notch-1水平明显增高，ASMCs增殖与Notch-1呈正相关；而且Pyrin重组蛋白(PRP)减少TGF-β1和Notch-1蛋白的表达，明显抑制气道重构的进展。但上述信号通路交叉与气道重构以及PRP干预的相关机制尚不清楚。本课题拟通过ASMCs和哮喘气道重构模型，探讨TGF-β1和Notch激动剂、抑制剂以及PRP对TGF-β1、Notch信号通路及靶基因的影响，从体内、体外实验证实上述两种信号通路交叉对话在哮喘气道重构中的作用，明确PRP介导TGF-β1和Notch信号通路交叉改善气道重构的作用机制，为防治哮喘气道重构及新药开发提供有效的理论依据。

为阐明Pyrin重组蛋白通过TGF-β1，Notch1信号通路缓解哮喘气道重塑的免疫调节机制，项目组圆满完成了计划研究内容。（1）建立以卵清蛋白（OVA）诱导的慢性支气管哮喘小鼠气道重塑模型，给予Pyrin重组蛋白及阳性对照药物地塞米松后，通过检测支气管肺泡灌洗液的细胞、肺组织HE观察气道炎症改善情况、PAS染色明确杯状细胞增生、Masson染色观察胶原沉积，免疫组化观察α-SMA，TGF-β1，Notch1蛋白在肺内表达情况、ELISA测定肺泡灌洗液中TNF-α,IL-1β,IL-4,IL-13表达情况、Westernblote及RT-qPCR验证TGFβ1，p-Smad3,Smad3,Jagged1,Notch1,NICD,Hes1,CSL,α-SMA,E-cadherin蛋白在肺内表达变化等，明确了Pyrin重组蛋白在体内模型中的抗过敏及抗气道重塑机制。（2）建立以TGFβ1诱导上皮细胞构建出上皮间质转化(EMT），给予Pyrin重组蛋白进行干预治疗,利用信号通路及各种分子生物学检测方法来检测其对于哮喘上皮细胞中EMT相关指标α-SMA,E-cadherin的调控作用，同时通过TGFβ1抑制剂SB431542,NOTCH1抑制剂DAPT观察TGFβ1及NOTCH1通路在EMT中转录因子HES1的调控作用，最终阐明TGFβ1及NOTCH1在诱导EMT过程中均是通过调控HES1的表达来实现的，后续使用TGFβ1下游信号smad3siRNA及NOTCH1下游信号NICD过表达明确smad3和NICD在EMT转录因子Hes1表达过程中起到相互促进，共同促进Hes1表达，既TGFβ1和NOTCH1在Hes1表达中存在信号交互。