VEGFR2介导的p38MAPK、PI3K/Akt信号通路在肺纤维化中的作用及Pyrin重组蛋白干预机制的研究

Pathogenesis of pulmonary fibrosis is still not clear and its prognosis is very poor because of no effective pharmacological treatments. Therefore, prevention and early intervention of pulmonary fibrosis are important. MMP-9/2 and HO-1 have been known as therapeutic targets of early pulmonary fibrosis. They are thought to be under control of VEGFR2/p38MAPK & PI3K/NF-kappa B and VEGFR2/p38MAPK & PI3K/Nrf2 signaling pathways. In the previous study, we demonstrated that Pyrin recombinant protein suppressed the NF-kappa B expression in the lung, improved the antioxidant enzymes activity, and prevented pulmonary fibrosis. But there were no reports on the roles of VEGFR2/p38MAPK & PI3K/NF-kappa B/MMP-9/2 and VEGFR2/p38MAPK (PI3K) / Nrf2 / HO-1 signal pathway in pulmonary fibrosis or on its protective mechanism of Pyrin recombinant protein. In this study, we evaluate VEGFR2 mediated p38MAPK and PI3K/Akt activation in cultured fibroblasts and mice pulmonary fibrosis model. In addition, we examine suppressive effects on VEGF mediated p38MAPK and PI3K/Akt in cultured fibroblasts and also on pulmonary fibrosis in mice model. Furthermore we evaluate the mechanism of suppressive effects of Pyrin recombinant protein on VEGFR2/p38MAPK & PI3K/NF-kappa B and VEGFR2/p38MAPK & PI3K/Nrf2 signaling pathways. Through these molecular mechanisms, Pyrin recombinant protein may control the expression of MMP-9/2 and HO-1. This study may provide us new tools, such as Pyrin recombinant protein, contributing to prevention and therapy for pulmonary fibrosis.

肺纤维化的发病机制尚不清楚，现无有效的治疗药物，因此早期防治致关重要。研究认为MMP-9/2和HO-1可能是早期肺纤维化的重要治疗靶点，可能受VEGFR2/p38MAPK&PI3K/NF-κB以及VEGFR2/p38MAPK&PI3K/Nrf2信号通路的调控。我们的前期研究表明Pyrin重组蛋白可调控肺组织中MMP-9/2和HO-1表达、提高抗氧化酶活性，而对肺纤维化起保护作用。但是到目前为止尚无有关上述信号通路与肺纤维化以及Pyrin的相关研究报道。本课题拟通过成纤维细胞和小鼠肺纤维化模型，从分子、细胞和整体水平，探讨Pyrin重组蛋白介导VEGFR2/p38MAPK&PI3K/NF-κB 及VEGFR2/p38MAPK&PI3K/Nrf2信号通路调节MMP-9/2和HO-1的作用机制，阐明Pyrin重组蛋白在此信号通路中的作用靶点，为寻找防治肺纤维化以及新药开发提供有效的理论依据。

为阐明Pyrin重组蛋白通过VEGFR2/介导的p38MAPK和PI3K/Akt信号通路缓解肺纤维化的免疫调节机制，项目组圆满完成了4项计划研究内容，并进行了拓展研究。（1）建立肺纤维化模型，给予Pyrin重组蛋白和VEGF抑制剂SU5416、PI3K/Akt 抑制剂LY-294002、NF-KB 抑制剂PDTC不同途径给予小鼠治疗，通过检测支气管肺泡灌洗液的细胞分类及上清液中炎症细胞因子的含量、肺组织HE染色，三色染色及免疫组化、Western blot等指标阐明Pyrin重组蛋白通过VEGF/VEGFR2/MMP-9信号通路抗肺纤维化的作用机制；（2）建立肺纤维化小鼠模型，给予Pyrin重组蛋白和VEGF抑制剂SU5416、PI3K/Akt 抑制剂LY-294002、NF-KB 抑制剂PDTC不同途径给予小鼠治疗，用HE染色观察肺泡炎程度、用MASSON染色观察肺纤维化程度、免疫组化、RT-PCR 及Western blot等方法检测NF-κB、TGF-β1、CTGF蛋白表达，研究发现Pyrin重组蛋白抗肺纤维化作用可能与抑制NF-κB、TGF-β1、CTGF蛋白的表达有关。（3）建立支气管哮喘小鼠气道重构模型，给予芝麻素、羟基红花黄色素A、人参皂苷Rh2，通过检测支气管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、肺组织组化和免疫组化、Western blot等指标，阐明和确认芝麻素、羟基红花黄色素A、人参皂苷Rh2通过MAPK和NF-KB等信号通路抗哮喘的作用机制；（4）建立Anti-DNP IgE介导的肥大细胞过敏模型，给予芝麻素、羟基红花黄色素A、人参皂苷Rh2, 通过检测组胺等炎症介质和Western blot等指标，阐明和确认芝麻素、羟基红花黄色素A、人参皂苷Rh2通过MAPK和NF-κB信号通路抗过敏反应及作用机制。