从TLR4/caveolin-1/eNOS信号通路探讨芪冬活血饮治疗急性肺损伤的分子机制

Acute lung injury (ALI) is a serious respiratory disease caused by accumulation of pyretic toxicity and deficiency of both vital energy and yin based on theory of traditional Chinese medicine. However, so far few active drugs to cure the disease can be available. Recent studies found that caveolin-1 indirectly activated inflammation-associated signaling pathways by inhibition of eNOS to evoke the over expression of proinflammatory cytokines, the unbalance of proinflammatory and anti-inflammatory cytokines that causing the pathogenesis of ALI. Our recent study revealed that QiDongHuoxue decoction, a traditional Chinese medicine, had the notable effect to cure ALI patients with anti-inflammatory action and control of proinflammatory and anti-inflammatory cytokine balance, but its mechanism of action remains unknown. In the present project, we plan to determine the mechanism of QiDongHuoxue decoction to cure ALI by directly inhibiting NF-κB, p38MAPK and JNK signaling pathways or indirectly inhibiting the inflammatory signaling through activation of eNOS. Firstly, LPS-induced neutrophil, macrophage and pulmonary vascular endothelial cell inflammatory models as well as mouse ALI model will be established. Using the methods such as siRNA-based silence and over expression of caveolin-1 and eNOS encoding genes, phosphorylation detection and specific inhibitor blockage of critical signaling kinases, and cytokine quatitative microarry, the major signaling pathways and mechanism of anti-inflammatory effect and cytokine balance control of QiDongHuoxue decoction will be determined according to the quantitative alteration of proinflammatory cytokines such as IL-1 and TNF-α, and anti-inflammatory cytokines such as IL-4 and IL-10 as well as the pulmonary histopathological changes in the cell and animal models. The results of this project will provide the theoretical and experimental evidences for QiDongHuoxue decoction to cure ALI patients in clinic.

急性肺损伤（ALI）为热毒瘀结、气阴两虚危重呼吸道病症，但治疗ALI有效药物甚少。新近发现caveolin-1能抑制eNOS间接激活炎症相关信号通路，导致促炎因子过表达及促炎与抗炎因子失衡而发生ALI。我们前期研究证实芪冬活血饮对ALI疗效显著并有抗炎及调控细胞因子平衡的作用，但其机制不明。本项目拟从芪冬活血饮直接抑制炎症相关NF-κB、p38MAPK和JNK通路或活化eNOS间接抑制炎症信号的思路出发，采用LPS诱导的中性粒细胞、巨噬细胞和肺血管内皮细胞炎症模型及小鼠ALI模型以及caveolin-1和eNOS等靶基因沉默及过表达、信号激酶磷酸化检测及阻断、细胞因子定量芯片等方法，了解芪冬活血饮作用前后IL-1和TNF-α等促炎因子、IL-4和IL-10等抗炎因子水平及肺组织病理变化，确定芪冬活血饮抗炎及调控细胞因子平衡的信号通路及其作用机制，为芪冬活血饮治疗ALI提供理论和实验依据。

急性肺损伤（ALI）为热毒瘀结、气阴两虚危重呼吸道病症，但治疗ALI有效药物甚少。项目组前期研究证实芪冬活血饮对ALI疗效显著，因此该项目拟从芪冬活血饮抑制炎症相关信号NF-κB、p38MAPK和JNK通路或活化eNOS间接抑制炎症信号的思路出发，采用LPS诱导建立ALI小鼠模型，通过对小鼠肺泡灌洗液、血清的炎症因子谱25种炎症因子的检测，以及肺组织炎症因子mRNA的测定、TLR4/caveolin/eNOS的病理、免疫组化测定，明确芪冬活血饮对急性肺损伤的治疗作用，以及对TLR4/caveolin/eNOS的抑制效果。然后从细胞层面出发，用LPS诱导巨噬细胞和肺微血管内皮细胞，建立细胞ALI模型。用芪冬活血饮药物血清培养细胞的方法，通过对细胞CXCL-10、selectin的mRNA和蛋白测定确定芪冬活血饮的作用。在巨噬细胞ALI模型上，我们采用大黄素和白藜芦醇两种中药单体代表芪冬活血饮，对细胞因子谱、信号通路磷酸化及关键位点caveolin-1的检测，确定芪冬活血饮的抗炎机制，再用信号激酶磷酸化阻断实验，验证芪冬活血饮的抗炎机制。. 该项目我们采用多种手段、多个方面、多个角度阐述了芪冬活血饮抗炎机制，为中医“清热解毒、祛瘀通腑、益气养阴”提供理论和实验基础，为抗炎药物开发提供新靶点和依据。