大鼠肺纤维化和活性维生素D3干预后P32和PKD1的表达及作用研究

摘要

Objective To explore the expressions and interaction of P32 and PKD1 during the genesis and development of pulmonary fibrosis in rats, and expression variation of the two factors after the intervention of active vitamin D3. Methods Ninety male SD rats were randomly divided into model group, treatment group and control group (30 in each group). Bleomycin (BLM, 5 mg/kg) was injected into the trachea of rats to establish the model of pulmonary fibrosis in the model group and the treatment group, while the control group was intratracheally injected with isopyknic saline (500 μl/kg). From the next day of BLM injection, the rats of the treatment group were intraperitoneally injected with 1,25-(OH)2D3(2 μg/kg) every other day, while the rats of the model group and the control group were intraperitoneally injected with isopyknic VD3solvent (99.9% propylene glycol and 0.1% ethanol) and saline respectively at the same time. On the 14th, 21st and 28th day after BLM injection, 10 rats in each group were sacrificed. The pulmonary fibrosis was verified by measuring the content of hydroxyproline in the lungs of the rats. The mRNA and protein expression levels of P32 and PKD1 were detected by RT-PCR and immunohistochemistry respectively. Results At the early stage (the 14th day) of pulmonary fibrosis, the expressions of P32 and PKD1 in the treatment group were higher than those in the model group at both mRNA and protein levels (P < 0.05). Correlation analysis showed that the expressions of these two genes had significant correlations at both mRNA and protein levels. Conclusions Active VD3may promote the the expressions of P32 and PKD1 at early stage during pulmonary fibrosis in rats. Active VD3can achieve antioxidant effect through the interaction between P32 and PKD1 in the mitochondria, thereby inhibit the development of pulmonary fibrosis.%目的 探讨大鼠肺纤维化发生发展中透明质酸结合蛋白(P32)和蛋白激酶D1(PKD1)的表达及相互作用,以及活性维生素D3(VD3)干预后这2种因子表达的变化.方法 90只清洁级雄性SD大鼠随机分成3组:对照组、模型组和治疗组.模型组和治疗组通过气管内注射博来霉素(BLM,5 mg/kg)的方法复制大鼠肺纤维化模型,对照组经气管注射等体积的生理盐水(500μl/kg).术后第2天,治疗组腹腔注射VD3(2μg/kg),模型组腹腔注射等量的VD3的溶剂(99.9%丙二醇和0.1%乙醇),对照组腹腔注射等量的生理盐水,各组均注射1次/2 d.各组分别于术后第14、21、28天处死10只大鼠.通过检测大鼠肺组织羟脯氨酸含量来验证肺纤维化,应用实时聚合酶链反应(real-time PCR)和免疫组织化学技术分别检测大鼠肺组织中P32和PKD1的表达水平.结果 纤维化早期(14 d),治疗组中P32和PKD1 mRNA和蛋白质表达量与模型组比较,差异有统计学意义(P <0.05),治疗组均高于模型组;相关性分析发现这2种基因的mRNA和蛋白质表达量具有相关性.结论 在大鼠肺纤维化过程中,活性VD3可能在纤维化早期促进P32和PKD1的表达,通过P32和PKD1在线粒体内的相互作用来实现抗氧化作用,进而抑制肺纤维化的发生发展.