1,25-(OH)_2D_3对大鼠特发性肺纤维化的影响及机制

Effects of 1,25-(OH)_2D_3 on idiopathic pulmonary fibrosis of rats and its mechanism

目的观察1,25-(OH)_2D_3对大鼠特发性肺纤维化(IPF)发生、发展的影响,探讨其作用机制。方法 90只雄性SD大鼠随机分为模型组、治疗组和对照组,每组30只。模型组和治疗组经气管内注射博来霉素5 mg/kg建立IPF模型,对照组注射生理盐水200μL/只。自注射后第2天开始,模型组腹腔注射1,25-(OH)_2D_3溶剂(0.1%乙醇、99.9%丙二醇,200μL/只),治疗组腹腔注射1,25-(OH)_2D_32μg/kg,对照组腹腔注射生理盐水200μL/只,均隔天1次。各组于给药后第14、21、28天分别处死10只大鼠,检测肺组织羟脯氨酸含量;分离培养肺成纤维细胞,采用Fluo-3AM荧光负载,激光共聚焦显微镜检测细胞内Ca^(2+)浓度;采用Real-time PCR法检测肺成纤维细胞PI3K、AKT、mTOR mRNA表达。结果给药第14、21、28天,模型组肺组织羟脯氨酸含量、肺成纤维细胞内Ca^(2+)浓度及PI3K、AKT、mTOR mRNA表达均高于对照组,治疗组上述指标在各时间点均低于模型组,组间比较P<0.05或<0.01。模型组和治疗组肺成纤维细胞内Ca^(2+)浓度与PI3K、AKT、mTOR mRNA表达均呈正相关(r分别为0.784、0.647、0.805,P均<0.01)。结论 1,25-(OH)_2D_3可抑制IPF的发生、发展;降低肺成纤维细胞内Ca^(2+)浓度、抑制PI3K-AKT-mTOR通路可能是其作用机制。

Objective To observe the effects of 1,25-（OH）2D3on idiopathic pulmonary fibrosis（ IPF） of rats,and to investigate its mechanism. Methods Ninety male SD rats were randomly divided into the model group,treatment group and control group（ n = 30 each）. Bleomycin（ 5 mg / kg） was injected to the trachea of rats to establish the model of pulmonary fibrosis in the model group and treatment group,while the control group was injected with normal saline（ 200 μL in each）. From the following day,rats in the treatment group and model group received 1,25-（OH）2D3（ 2 μg/kg） and solvant（ 200 μL for each）,respectively and once every other day. The control group was treated with normal saline（ 200 μL for each）. Ten mice in each group were killed randomly on day 14,21 and 28. The hydroxyproline content of lung tissues was measured by basic-hydrolysis method. Lung fibroblasts were separated from lung tissues and labeled by Fluo-3AM,then we used Laser scanning confocal microscope to detect the concentration of Ca^2＋. The mRNA levels of PI3K,AKT and mTOR in the lung fibroblasts were tested by real-time PCR. Results The hydroxyproline content of lung tissues,the Ca^2＋concentration and expression of PI3K,AKT and mTOR in lung fibroblasts were increased in the model group as compared with that of the control group on day 14,21 and 28.The above indicators of treatment group were reduced significantly as compared with those of the model group（ P〈0. 05 or P〈0. 01）. Ca^2＋concentration was positively correlated with mRNA expression levels of three genes in the model group and treatment group（ r = 0. 784,0. 647,0. 805,all P〈0. 01）. Conclusion 1,25-（OH）2D3inhibits the occurrence and development of IPF in rats,and its mechanism may be the reduction of Ca^2＋ concentration and inhibition of PI3K-AKT-mTOR pathway.