线粒体融合蛋白2对内毒素致急性呼吸窘迫综合征肺纤维化的影响

Effects of Mnf2 on pulmonary fibrosis in LPS-induced ARDS

目的从细胞水平观察线粒体融合蛋白2(Mfn2)对内毒素(LPS)致急性呼吸窘迫综合征(ARDS)肺纤维化形成的影响。方法LPS以浓度梯度及时间梯度作用于人胚肺成纤维细胞(HELF),检测细胞内胶原蛋白表达情况,建立ARDS细胞模型;以复制缺陷型腺病毒作为载体,将Mfn2蛋白转移到HELF中,通过Western blot检测HELF中Mfn2蛋白水平,并运用MTT观察HELF增殖情况,考马斯亮蓝法测定细胞内总蛋白、Sircol胶原测定试剂盒测定胶原蛋白含量。结果LPS浓度为0.5、1、5、10μg/mL刺激HELF 24 h,细胞内胶原蛋白水平分别为(70.97±1.03)、(71.31±0.82)、(76.50±1.31)、(76.84±1.44)μg/mg,与对照组(69.08±2.48)μg/mg相比较差异有统计学意义(P值分别为0.028、0.011、0.000、0.000),且在LPS 5μg/mL时作用显著;LPS(5μg/mL)处理HELF 6、12、24、48 h,细胞内胶原蛋白水平分别为(70.67±0.96)、(73.39±0.80)、(76.01±0.59)、(76.91±0.79)μg/mg,与对照组(69.66±0.99)μg/mg比较差异有统计学意义(P值分别为0.031、0.000、0.000、0.000),并且在24 h时作用明显;LPS以浓度及时间依赖性诱导HELF胶原蛋白的表达,建立ARDS细胞模型适宜的条件为5μg/mL LPS刺激HELF 24 h;同时发现LPS组较对照组Mfn2蛋白表达明显下调(P=0.000),转染Adv-Mfn2后,LPS+Adv-Mfn2组较LPS组Mfn2蛋白表达明显上调(P=0.000);与LPS组相比较,LPS+Adv-Mfn2组HELF 12、24、48 h细胞增殖率(0.93±0.06、1.48±0.03、1.76±0.01)明显降低(P<0.05),且胶原蛋白水平(72.12±0.30)、(73.21±0.26)、(73.46±0.25)μg/mg明显下降(P<0.05)。结论过表达Mfn2能减轻内毒素诱导的肺成纤维细胞增殖并减少胶原产生。

Objective To observe the effect of Mfn2 protein on the formation of pulmonary fibrosis in ARDS induced by lipopolysaccharide(LPS)at the cellular level.Methods LPS acted on human embryonic lung fibroblasts(HELF)with the concentration gradient and time gradient,the expression level of intracellular collagen was detected and the ARDS cell model was established.With replication-of deficient adenovirus as the carrier,the Mfn2 protein was tranfered to HELF,the level of Mfn2 protein in HELF was detected by Western blot,and the proliferation situation of HELF was observed by MTT,the Coomassie brilliant blue method was used to determine the total intracellular protein level and the Sircol collagen assay kit was used to determine the collagen content.Results The LPS concentrations of 0.5,1,5,10μg/mL were used to stimulate HELF for 24 h,and the intracellular collagen levels were(70.97±1.03),(71.31±0.82),(76.50±1.31),(76.84±1.44)μg/mg respectively,compared with(69.08±2.48)μg/mg in the control group,and the differences were statistically significant(P=0.028,0.011,0.000,0.000),moreover the effect was significant when the LPS concentration was 5μg/mL.When HELF was treated with 5μg/mL LPS for 6,12,24,48 h,the intracellular collagen levels were(70.67±0.96),(73.39±0.80),(76.01±0.59),(76.91±0.79)μg/mg respectively,compared with(69.66±0.99)μg/mg in the control group,and the differences were statistically significant(P=0.031,0.000,0.000,0.000),moreover the effect was obvious at 24h.LPS induced the expression of HELF collagen in a concentration and time-dependent manner,the appropriate condition for establishing the ARDS cell model was 5μg/mL LPS to stimulate HELF for 24 h.Meanwhile it was also found that the expression of Mfn2 protein in the LPS group was significantly down-regulated compared with the control group(P=0.000);after transfection with Adv-Mfn2,the expression of Mfn2 protein in the LPS+Adv-Mfn2 group was significantly up-regulated compared with the LPS group(P=0.000);compared with the LPS group,the cell proliferation rates(0.93±0.06,1.48±0.03,1.76±0.01)at 12,24,48 h in the LPS+Adv-Mfn2 group were significantly decreased(P<0.05),moreover the collagen levels were significantly reduced[(72.12±0.30),(73.21±0.26),(73.46±0.25)μg/mg,P<0.05].Conclusion The overexpression of Mfn2 can reduce the LPS-induced lung fibroblast proliferation and reduce the collagen production.