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

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.

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。

背景：黄酮类化合物具有抑制或改善气道炎症，降低气道高反应性，抗气道重塑，改善免疫失衡，增强抗氧化能力和清除自由基的能力等功能。但是作为淫羊藿黄酮肠代谢产物淫羊藿素（Icaritin，ICT）在哮喘气道重塑中的作用尚未有人研究。内容：本课题通过建立哮喘小鼠模型，探索ICT对哮喘气道炎症的抑制作用，进一步探索其能否通过调节Th1/Th2细胞分化，干预气道炎症；同时探索其对气道重塑的抑制作用，观察能否影响气道平滑肌细胞（Airway Smooth Muscle Cells，ASMCs）增生；体外机制方面，通过分离培养大鼠ASMCs，进行ICT干预处理，检测ASMCs增殖、凋亡变化，进一步探索ICT对ASMCs增殖、凋亡的调控是否通过TLR4信号通路进行，最终阐明ICT抑制哮喘气道炎症，并能通过TLR4信号通路，调节ASMCs增殖和凋亡，从而抑制哮喘气道重塑。重要结果：（1）ICT抑制气道炎症浸润和粘液分泌；（2）ICT抑制嗜酸性粒细胞；（3）ICT抑制炎症细胞因子分泌；（4）ICT抑制GATA-3但是促进T-bet基因和蛋白表达；（5）ICT抑制胶原沉积和ASMCs增生；（6）ICT抑制ASMCs增殖；（7）ICT导致ASMCs周期S阻滞和调节周期蛋白Cyclin A和Cyclin E表达；（8）ICT促进ASMCs凋亡；（9）ICT激活FADD/Caspase-8/Caspase-3依赖的凋亡通路。关键数据：淫羊藿素能够通过调节Th1/Th2细胞分化抑制哮喘炎症反应，能够通过造成周期S阻滞抑制ASMCs增殖，通过FADD/Caspase信号通路促进ASMCs凋亡，从而达到抑制哮喘炎症和气道重塑的效果。科学意义：本研究发现小分子药物淫羊藿素能够抑制哮喘炎症和气道重塑，表明其可能成为哮喘治疗新的、经济、高效的候选药物。