An Ning, Zhou Xian, Zhang Xiaoxia, Yang Tao, Xu Meixia
Institue of Anesthesia and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei, China.
Department of Intensive Care Unit, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan Fourth Hospital), Wuhan 430034, Hubei, China. Corresponding author: Xu Meixia, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 Jul;33(7):861-865. doi: 10.3760/cma.j.cn121430-20201222-00768.
To study the inhibitory effect of overexpression of mitofusion 2 (Mfn2) protein on acute respiratory distress syndrome (ARDS) pulmonary fibrosis and its mechanism.
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.
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): 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): 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): 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): 0.88±0.10 vs. 0.31±0.05, both P < 0.01].
Mfn2 protein is involved in ARDS pulmonary fibrosis, which may be related to mitochondrial mediated inhibition of cell proliferation.
研究线粒体融合蛋白2（Mfn2）过表达对急性呼吸窘迫综合征（ARDS）肺纤维化的抑制作用及其机制。
体外培养人胚肺成纤维细胞（HELF），当HELF贴壁率达80%时进行消化传代，选取状态良好的细胞进行实验。用5 mg/L脂多糖（LPS）复制ARDS细胞模型（LPS组）；将携带线粒体融合蛋白2的75 μmol/L腺病毒载体转染入HELF（Adv-Mfn2+LPS组）；同时设空白对照组（完全培养基培养）和Adv-载体+LPS组作为对照。采用磺酰罗丹明B（SRB）法于0、12、24、36和48小时观察细胞增殖情况。经Hoechst 33342染色后，在共聚焦显微镜下观察形态学变化。采用蛋白质免疫印迹法检测Bcl-2和半胱天冬酶-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和半胱天冬酶-3的基因表达。
LPS刺激12～48小时后，LPS组细胞增殖率逐渐升高，显著高于空白对照组[12小时：（10.75±1.51）%比（0.73±1.22）%，24小时：（20.09±1.71）%比（1.15±1.12）%，36小时：（20.58±1.55）%比（1.20±1.12）%，48小时：（21.30±1.51）%比（1.23±1.10）%，均P<0.01]。LPS组与Adv-载体+LPS组细胞增殖率差异无统计学意义。Mfn2过表达后，Adv-Mfn2+LPS组12、24、36、48小时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）%，显著低于LPS组（均P<0.05）。共聚焦显微镜观察显示，空白对照组部分细胞有大小不一的细胞核，部分细胞核碎片化或皱缩形成凋亡小体。LPS组和Adv-载体+LPS组细胞的细胞核呈圆形或椭圆形，仅出现少数凋亡细胞。Mfn2过表达时，Adv-Mfn2+LPS组视野中凋亡细胞比LPS组多。蛋白质免疫印迹法和RT-qPCR结果显示，与空白对照组比较，LPS刺激后LPS组Bcl-2表达显著增加[Bcl-2蛋白（Bcl-2/GAPDH）：0.68±0.01比0.29±0.01，Bcl-2 mRNA（2）：2.23±0.34比1.00±0.00，均P<0.01]，半胱天冬酶-3表达显著降低[半胱天冬酶-3蛋白（半胱天冬酶-3/GAPDH）：0.37±0.02比0.66±0.02，半胱天冬酶-3 mRNA（2）：0.31±0.05比1.00±0.00，均P<0.01]。与LPS组比较，Adv-Mfn2+LPS组Mfn2过表达后Bcl-2表达下调[Bcl-2蛋白（Bcl-2/GAPDH）：0.46±0.01比0.68±0.01，Bcl-2 mRNA（2）：1.45±0.14比2.23±0.34，均P<0.01]，半胱天冬酶-3表达上调[半胱天冬酶-3蛋白（半胱天冬酶-3/GAPDH）：0.54±0.02比0.37±0.02，半胱天冬酶-3 mRNA（2）：0.88±0.10比0.31±0.05，均P<0.01]。
Mfn2蛋白参与ARDS肺纤维化，可能与线粒体介导的细胞增殖抑制有关。
