PDCD4在肺纤维化模型中的表达及意义

Expression of programmed cell death factor 4 in pulmonary fibrosis model

目的:检测程序性细胞死亡蛋白4(PDCD4)在肺纤维化模型中的表达水平及对细胞活力和肺纤维化指标的影响及其机制。方法:Western blot和RT-qPCR检测PDCD4、α-平滑肌肌动蛋白(α-SMA)和I型胶原蛋白(COL-I)在人胚肺成纤维细胞系HFL-1组和HFL-1+TGF-β1组的表达水平。将质粒pEZ-M03-PDCD4和空质粒pEZ-M03转染进肌成纤维细胞(MB),应用Western blot检测PDCD4的蛋白表达水平;应用Western blot检测空白对照组、pEZ-M03-PDCD4组、pEZ-M03组和LY294002组PI3K/AKT信号通路相关分子p-AKT和AKT的蛋白水平及周期相关蛋白c-Myc和cyclin D1的表达情况; CCK-8法检测PDCD4对MB活力的影响;羟脯氨酸消化法检测HFL-1细胞和转染后MB上清液羟脯氨酸含量。Western blot检测小鼠模型组与对照组中肺组织PDCD4的表达水平。结果:与HFL-1组比较,HFL-1+TGF-β1组α-SMA和COL-I的mRNA及蛋白表达明显上调(P <0. 01),PDCD4mRNA表达无明显变化,PDCD4的蛋白表达明显下调(P <0. 01)。与空白对照组及pEZ-M03组比较,pEZ-M03-PDCD4组的PDCD4蛋白表达水平明显上调(P <0. 05),c-Myc和cyclin D1的蛋白表达都显著减少(P <0. 05),细胞活力明显受到抑制(P <0. 01),上清液中羟脯氨酸的含量显著减少(P <0. 05)。与空白对照组相比,pEZ-M03-PDCD4组及LY294002组中的p-AKT蛋白水平显著减少(P <0. 05),pEZ-M03组的差异无统计学显著性。在小鼠体内模型实验中,与对照组相比,模型组的PDCD4表达减少(P <0. 01)。结论:PDCD4在肺纤维化过程中低表达。过表达PDCD4可抑制MB活力,减少羟脯氨酸含量,抑制PI3K/AKT信号通路。

AIM:To detect the expression of programmed cell death protein4(PDCD4)in pulmonary fibrosis model and its effect on cell viability and pulmonary fibrosis indicators,and to explore its mechanism.METHODS:The expression level of PDCD4,α-smooth muscle actin(α-SMA)and collagen type I(COL-I)in human embryonic lung fibroblasts cell line HFL-1group and HFL-1+TGF-β1group were detected by Western blot and RT-qPCR.The plasmid pEZ-M03-PDCD4and empty vector pEZ-M03were transfected into myofibroblasts(MB),and the protein expression level of PDCD4was detected by Western blot.The protein levels of p-AKT and AKT in blank control group,pEZ-M03-PDCD4group,pEZ-M03group and LY294002group and the expression of cell cycle-related proteins c-Myc and cyclin D1were determined by Western blot.The effect of PDCD4on the viability of MB was measured by CCK-8assay.The hydroxyproline content in the culture supernatant of HFL-1cells and transfected MB was detected by hydroxyproline digestion method.The expression of PDCD4in the lung tissues of the mice in model group and control group was detected by Western blot.RESULTS:Compared with HFL-1group,the expression ofα-SMA and COL-I at mRNA and protein levels in HFL-1+TGF-β1group was significantly increased(P<0.01),the mRNA expression of PDCD4was not significantly changed,while PDCD4protein was significantly down-regulated(P<0.01).Compared with blank control group and pEZ-M03group,the protein expression of PDCD4in pEZ-M03-PDCD4group was significantly increased(P<0.05),the protein expression of c-Myc and cyclin D1was significantly decreased(P<0.05),the cell viability was also significantly inhibited(P<0.01),and the content of hydroxyproline in the culture supernatant was significantly reduced(P<0.05).Compared with blank control group,the protein levels of p-AKT were significantly decreased in pEZ-M03-PDCD4group and LY294002group,and no significant difference between blank control group and pEZ-M03control group was observed.Compared with control group,PDCD4expression was decreased in model group(P<0.01).CONCLUSION:PDCD4is low expressed in the process of pulmonary fibrosis.Over-expression of PDCD4inhibits the viability of MB,decreases the content of hydroxyproline,and inhibits the PI3K/AKT signaling pathway.