目的探讨PI3K/Akt/HIF-1α信号通路在博来霉素(BLM)诱导的小鼠肺纤维化中的作用机制。方法 56只C57BL/6小鼠随机分为对照组和实验组,实验组通过气管内滴注BLM(2.5 mg/kg)建立肺纤维化模型,对照组在相同条件下气管内滴注等量0.9%氯化钠溶液。在造模第21 d取小鼠肺组织标本行HE和Masson染色分析肺组织形态学变化;运用Ashcroft评分以及检测羟脯氨酸含量评估肺纤维化程度;Western blot方法检测PI3K/Akt/HIF-1α信号通路的变化以及肺泡表面活性物质(Pro-SPC)蛋白含量;实时定量PCR(RT-PCR)法检测胶原蛋白3(Collagen3)mRNA表达水平;免疫组织化学法检测肺组织内Collagen3蛋白和细胞凋亡数的变化。结果实验组与对照组相比,肺泡炎和肺纤维化程度明显加重,肺组织内充填大量炎细胞及纤维病灶。试验组Ashcroft评分和羟脯氨酸含量均较对照组显著升高(P<0.05)。同时,PI3K/Akt/HIF-1α信号通路在实验组肺内明显活化,并伴有Pro-SPC蛋白生成减少,Collagen3蛋白含量及mRNA水平增加,以及肺内细胞凋亡数明显增加。结论 PI3K/Akt/HIF-1α信号通路的异常活化促进了肺纤维化形成。Objective To investigate the role of PI3K/Akt/HIF-1α signaling pathway in bleomycininduced pulmonary fibrosis in mice.Methods Fifty-six C57 BL/6 mice were randomly divided into a control group and a bleomycin(BLM)group.The pulmonary fibrosis model was induced by single intratracheal instillation of BLM(2.5 mg/kg)in the BLM group.Similarly,0.9% saline was instilled directly into the trachea in the control group.Then all mice were sacrificed on 21 stday.The lungs were collected for morphometric analysis with HE and Masson staining.The degree of pulmonary fibrosis was evaluated with Ashcroft score and content of hydroxyproline.The activity of PI3 K/Akt/HIF-1α signaling pathway and prosurfactant protein C(Pro-SPC)were measured by Western blot.The level of collagen3 mRNA was assessed with quantitative real time PCR analysis.Collagen3 protein and numbers of apoptosis cells were observed with immuno-histochemistry.Results It was exhibited that the thickening alveolar septa,accumulation of inflammatory cells,and fibrous obliteration in the BLM group but not in the control group.There was a significant difference in Ashcroft score and hydryoproline content in the BLM group.Meanwhile,the activity of PI3 K/Akt/HIF-1α signaling pathway was up-regulated and the protein of Pro-SPC was decreased in the BLM group.It was revealed that the numbers of apoptosis cells,expressions of Collagen3 protein and mRNA were increased in the BLM group.Conclusion Aberrant activity of PI3 K/Akt/HIF-1α signaling pathway may aggravate the pulmonary fibrogenesis.福建省科技局课题(编号:J00162
