生物活性分子淫羊藿素对哮喘气道重塑的抑制作用及其机制研究

黄酮类化合物可以通过抑制气道重塑起到抗哮喘作用，但是作为淫羊藿黄酮肠代谢产物淫羊藿素ICT在哮喘气道重塑中的作用尚未有人研究。平滑肌细胞（ASMCs）增生是气道重塑的重要病理学特征，我们拟通过体外培养大鼠ASMCs，研究ICT是否影响ASMCs的增殖、凋亡，进一步探索ICT是否通过影响TLR4和NF-κB 信号通路实现对ASMCs增殖、凋亡调控。拟联合TNF-α和TLR4抗体处理，检测ICT对NF-κB活性调节是否特异依赖TLR4，联合TNF-α和和NF-κB抑制剂处理，验证ICT对TLR4激活的ASMCs增殖调控是否依赖NF-κB活性。体内实验拟用卵清蛋白致敏激发建立大鼠哮喘模型，通过组织学病理学和免疫组化检测ICT对气道重塑及ASMCs增殖、凋亡的影响。本课题力图阐明ICT调节哮喘气道重塑的作用机制，探索ICT成为更经济高效的哮喘候选药物的可能性。

The application of Flavonoids in asthma has been a hot research topic. Flavonoids could reduce airway inflammation and hyperresponsiveness, inhibit airway smooth muscle cells (ASMCs) proliferation. The study on intestinal metabolism of Epimedium Flavonoid (EF) has shown that a small molecule Icaritin is involved in intestinal metabolism of those seven flavonoid compounds with parent ring in EF, as a single equal for acting on pharmacological targets. But whether ICT can affect airway remodeling in asthma has not been studied. Airway smooth muscle cells (ASMCs) hyperplasia is one of the most important pathological features of airway remodeling in asthma. ICT suppressed human prostatic smooth muscle cells proliferation and promoted apoptosis, and another EFs compound Icariin inhibited the proliferation of vascular smooth muscle cells, but whether ICT can inhibit ASMCs proliferation in asthma and the potential mechanism has not been studied. Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) involved ASMCs proliferation and apoptosis in asthma. ICT reduced the TLR4 expression in human peripheral blood mononuclear cells and NF-κB activity in cancer cells. Whether ICT involved airway remodeling through regulating TLR4 /N F-κB signal pathway in ASMCs proliferation and apoptosis will be deep studied in this project. Rat ASMCs will be isolated and cultured for In vitro study, the cultured cells will be treated with ICT, then the proliferation and apoptosis will be detect to check whether Icariin can affect ASMCs proliferation and apoptosis. The effect of ICT on the activity of NF-κB p65 and its phosphorylation will be evaluated, and then the phosphorylation of NF-κB inhibitory protein IκB p32, 36 and its ubiquitination and proteasomal degradation will be further detected. Combined treatment with TLR4 antibody and TLR4/NF-κB agonist tumor necrosis factor- α (TNF-α) to inhibit TLR4 activity while activate NF-κB activity to confirm whether the inhibitory effect of Icariin on NF-κB signal activity is TLR4 dependent. Combined treatment with TNF-α and NF-κB inhibitor Pyrrolidinedithiocarbamic acid,ammonium salt (PDTC) to confirm whether inhibitory effect of ICT on AMSCs proliferation activated by TLR4 is NF-κB activity dependent. Eventually we try to elucidate whether ICT regulates ASMCs proliferation and apoptosis in asthamic rat specifically depending on TLR4/NF-κB signal pathway. For in vivo study, we will establish ovalbumin (OVA) induced rat asthmatic model, evaluation the proliferation changes of ASMCs and detection the expression of TLR4 and the activity of NF-κB in asthmatic airway tissues. Through systemically in vitro and in vivo study, we try to elucidate the mechanism of small molecule of ICT in regulation of asthma airway remodeling, exploring the clinical application possibility of ICT as a new and efficient therapeutic agent for asthma treatment in further.