过表达线粒体融合蛋白2对ARDS肺纤维化的抑制作用及其机制研究

Overexpression of mitofusion 2 inhibits acute respiratory distress syndrome pulmonary fibrosis and its mechanism

目的探讨过表达线粒体融合蛋白2(Mfn2)对急性呼吸窘迫综合征(ARDS)肺纤维化的抑制作用及其机制。方法离体培养人胚肺成纤维细胞(HELF),待细胞生长贴壁率达80%以上进行消化传代,选取状态良好的细胞进行实验。给予5 mg/L脂多糖(LPS)刺激制备ARDS细胞模型(LPS组);并将75 mol/L携带线粒体融合蛋白2腺病毒载体(Adv-Mfn2)转染到HELF中(Adv-Mfn2+LPS组);同时设空白对照组(完全培养基培养)和空载腺病毒载体(Adv-vector)+LPS组作为对照。采用磺基罗丹明B(SRB)法观察各组细胞培养0、12、24、36、48 h的增殖情况;Hoechst 33342染色后,共聚焦显微镜下观察细胞形态学改变;采用蛋白质免疫印迹试验(Western blotting)检测抗凋亡基因Bcl-2和天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)的蛋白表达;采用实时荧光定量聚合酶链反应(RT-qPCR)检测Bcl-2和caspase-3的基因表达。结果经LPS刺激12~48 h,LPS组细胞增殖率随时间延长而逐渐增加,而且明显高于空白对照组〔12 h:(10.75±1.51)%比(0.73±1.22)%,24 h:(20.09±1.71)%比(1.15±1.12)%,36 h:(20.58±1.55)%比(1.20±1.12)%,48 h:(21.30±1.51)%比(1.23±1.10)%,均P<0.01〕;LPS组与Adv-vector+LPS组各时间点细胞增殖率比较差异则均无统计学意义;而Adv-Mfn2+LPS组过表达Mfn2后12、24、36、48 h的细胞增殖率分别为(8.93±1.14)%、(10.52±1.24)%、(10.72±1.30)%、(10.91±1.20)%,均较LPS组明显降低(均P<0.05)。共聚焦显微镜下显示,空白对照组细胞核大小不一,部分细胞核碎裂或核固缩,形成凋亡小体;LPS组和Adv-vector+LPS组细胞核呈圆形或椭圆形,大小均匀,仅出现个别凋亡细胞;而Adv-Mfn2+LPS组过表达Mfn2后,凋亡细胞较LPS组增多。Western blotting和RT-qPCR检测结果显示,给予LPS刺激后,LPS组Bcl-2表达较空白对照组明显上调〔Bcl-2蛋白(Bcl-2/GAPDH):0.68±0.01比0.29±0.01,Bcl-2 mRNA(2-ΔΔCT):2.23±0.34比1.00±0.00,均P<0.01〕,而caspase-3的表达较空白对照组明显下调〔caspase-3蛋白(caspase-3/GAPDH):0.37±0.02比0.66±0.02,caspase-3 mRNA(2-ΔΔCT):0.31±0.05比1.00±0.00,均P<0.01〕;与LPS组比较,Adv-Mfn2+LPS组过表达Mfn2蛋白后,Bcl-2表达明显下调〔Bcl-2蛋白(Bcl-2/GAPDH):0.46±0.01比0.68±0.01,Bcl-2 mRNA(2-ΔΔCT):1.45±0.14比2.23±0.34,均P<0.01〕,caspase-3表达明显上调〔caspase-3蛋白(caspase-3/GAPDH):0.54±0.02比0.37±0.02,caspase-3 mRNA(2-ΔΔCT):0.88±0.10比0.31±0.05,均P<0.01〕。结论Mfn2蛋白参与ARDS肺纤维化,可能与线粒体介导的抑制细胞增殖途径有关。

Objective To study the inhibitory effect of overexpression of mitofusion 2(Mfn2)protein on acute respiratory distress syndrome(ARDS)pulmonary fibrosis and its mechanism.Methods Human embryo lung fibroblasts(HELF)were cultured in vitro,and digested and passaged when the adherent rate of HELF reached 80%,and then the cells in good condition were selected for experiment.The ARDS cell model was reproduced by 5 mg/L of lipopolysaccharide(LPS,LPS group);75 mol/L adenovirus vector carrying mitofusion 2(Adv-Mfn2)was transfected into HELF(Adv-Mfn2+LPS group);at the same time,blank control group(complete medium culture)and Adv-vector+LPS group were set as controls.The cell proliferation was observed by sulforhodamine B(SRB)method at 0,12,24,36 and 48 hours.After Hoechst 33342 staining,the morphological changes were observed under confocal microscope.Western blotting was used to detect the protein expressions of Bcl-2 and caspase-3.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the gene expressions of Bcl-2 and caspase-3.Results After LPS stimulation for 12-48 hours,the cell proliferation rates in the LPS group increased gradually,which were significantly higher than those in the blank control group[12 hours:(10.75±1.51)%vs.(0.73±1.22)%,24 hours:(20.09±1.71)%vs.(1.15±1.12)%,36 hours:(20.58±1.55)%vs.(1.20±1.12)%,48 hours:(21.30±1.51)%vs.(1.23±1.10)%,all P<0.01].There was no statistically significant difference in the cell proliferation rate between the LPS group and the Adv-vector+LPS group.After overexpression of Mfn2,the cell proliferation rates at 12,24,36,48 hours in the Adv-Mfn2+LPS group were(8.93±1.14)%,(10.52±1.24)%,(10.72±1.30)%,and(10.91±1.20)%,which were significantly lower than those in the LPS group(all P<0.05).Confocal microscopy showed that some cells in the blank control group had nuclei of different sizes,and some nuclei fragmented or shrank to form apoptotic bodies.The nuclei of the cells in the LPS and Adv-vector+LPS groups were round or oval in size,and only a few apoptotic cells appeared.When Mfn2 was overexpressed,there were more apoptotic cells in the visual field in the Adv-Mfn2+LPS group than LPS group.Western blotting and RT-qPCR results showed that Bcl-2 expressions increased significantly after LPS stimulation in the LPS group as compared with the blank control group[Bcl-2 protein(Bcl-2/GAPDH):0.68±0.01 vs.0.29±0.01,Bcl-2 mRNA(2-ΔΔCT):2.23±0.34 vs.1.00±0.00,both P<0.01],and caspase-3 expressions decreased significantly[caspase-3 protein(caspase-3/GAPDH):0.37±0.02 vs.0.66±0.02,caspase-3 mRNA(2-ΔΔCT):0.31±0.05 vs.1.00±0.00,both P<0.01].Compared with LPS group,the expressions of Bcl-2 after overexpression of Mfn2 in the Adv-Mfn2+LPS group were down-regulated[Bcl-2 protein(Bcl-2/GAPDH):0.46±0.01 vs.0.68±0.01,Bcl-2 mRNA(2-ΔΔCT):1.45±0.14 vs.2.23±0.34,both P<0.01],and the expressions of caspase-3 were up-regulated[caspase-3 protein(caspase-3/GAPDH):0.54±0.02 vs.0.37±0.02,caspase-3 mRNA(2-ΔΔCT):0.88±0.10 vs.0.31±0.05,both P<0.01].Conclusion Mfn2 protein is involved in ARDS pulmonary fibrosis,which may be related to mitochondrial mediated inhibition of cell proliferation.