TGF-β1与TWEAK信号通路交叉对话在哮喘气道重塑中的作用及隐丹参酮的干预机制研究

Airway smooth muscle cells (ASMCs) proliferation is well-known to be a major factor in asthmatic airway remodeling. It has been reported that STAT3 was an intersection point under TGF-β1 and TWEAK signaling pathway downstream in previous research. Our previous study showed TGF-β1 expression increasing in ASMCs and culture supernatant treatment with TWEAK had positive correlation with ASMCs proliferation. In addition, Cryptotanshinone inhibited the proteins expression of TGF-β1 and TWEAK to play a significant inhibitory effect on airway remodeling. In all of the research detailed evaluation of the effects of cross-talk above and cryptotanshinone on airway remodeling is still missing. Here we use ASMCs and airway remodeling model mice to investigate the effects of agonist and inhibitor of TGF-β1 and TWEAK and cryptotanshinone on TGF-β1 & TWEAK signaling pathway and target gene, and elucidate the effect of the cross-talk between TGF-β1 and TWEAK signaling pathway on airway remodeling. Furthermore, we will clarify the underlying mechanism of cryptotanshinone on mediating the molecular cross-talk between TGF-β1 and TWEAK signal transduction. This study will obtain the effective theoretical basis for prevention or treatment of airway remodeling in asthma and the development of new drugs.

气道平滑肌细胞(ASMCs)增殖是哮喘气道重塑发生、发展的关键环节。研究表明，TGF-β1和TWEAK信号通路在下游STAT3进行交叉。我们的前期研究发现：TWEAK干预ASMCs培养上清液和细胞内TGF-β1 的水平明显增高，ASMCs增殖与TGF-β1 呈正相关；而且隐丹参酮通过减少TGF-β1和TWEAK蛋白的表达，明显抑制气道重塑的演进。但上述信号通路交叉与气道重塑以及隐丹参酮干预的相关机制尚不清楚。本课题拟通过ASMCs和哮喘气道重塑模型，从分子、细胞和整体水平，探讨TGF-β1和TWEAK激动剂、抑制剂以及隐丹参酮对TGF-β1、TWEAK信号通路及靶基因的影响，阐明TGF-β1和TWEAK 信号通路交叉对话在哮喘气道重塑中的作用，明确隐丹参酮介导TGF-β1和TWEAK 信号通路交叉改善气道重塑的作用机制。为寻找防治哮喘气道重塑及新药开发提供有效的理论依据。

为阐明隐丹参酮(CTS)通过TWEAK和TGF -β1信号交互缓解哮喘气道重塑的免疫调节机制，项目组圆满完成了3项计划研究内容，并进行了拓展研究。（1）建立气道平滑肌细胞和气道重塑模型，给予CTS，通过检测支气管肺泡灌洗液的细胞及炎症细胞因子的含量、血清IgE含量、肺组织组化和免疫组化、ELISA、Western blot等分子生物学指标和信号通路，结果发现：CTS能明显抑制OVA诱导的支气管肺泡灌洗液(BALF)中嗜酸性粒细胞和中性粒细胞浸润，降低哮喘小鼠气道阻力，减少哮喘小鼠肺组织炎症细胞聚集、杯状细胞增生和胶原沉积，显著减轻BALF中炎性细胞渗漏和OVA特异性IgE水平；CTS抑制α-平滑肌肌动蛋白、TWEAK、Fn14、TGF-Smad1、β-4的表达，抑制Smad2/3、STAT3的磷酸化(Tyr705)；与TWEAK抑制剂或TWEAK小干扰RNA(SiRNA)相比，CTS对气道重塑的影响与它们相似；CTS还在TGF-β1/β3信号通路中与TGF-STAT1抑制剂或TGF -β1siRNA在气道重塑中发挥相似的作用。得出CTS抗炎和抗气道重塑作用机制可能是通过 TWEAK和TGF-β-1在哮喘中的信号交互进而抑制STAT3信号通路而实现的。（2）建立支气管哮喘小鼠模型，给予苦豆碱和积雪草苷。检测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、肺组织染色和免疫组化、Western blot等指标。最终得出苦豆碱是通过调控NF-KB、MAPK、Nrf2/HO-1信号通路参与抗哮喘过程；（3）建立Anti-DNP IgE介导的肥大细胞过敏模型，给予积雪草苷和虎杖苷及山茱萸新苷等, 通过检测组胺等炎症介质和Western blot等指标。确认积雪草苷和虎杖苷及山茱萸新苷等通过MAPK和NF-κB信号通路抗过敏反应及作用机制。