FSTL3在不同诱因所致的小鼠急性肺损伤中的表达

Expression of FSTL3 in acute lung injury in mice caused by different inducements

目的探讨人卵泡抑素样蛋白3(FSTL3)在不同诱因所致的小鼠急性肺损伤中的表达及其意义。方法采用腹腔注射脂多糖(LPS)构建脓毒血症肺损伤模型,采用左肺门夹闭后开放的方式构建缺血再灌注(IR)损伤模型。检测肺损伤不同时间点小鼠血清中FSTL3的蛋白表达水平,肺组织中FSTL3的蛋白及mRNA表达水平。使用FSTL3中和抗体(FSTL3-NA)或FSTL3基因敲除小鼠评估FSTL3抑制对两种诱因所致的小鼠肺损伤的影响。使用免疫印记法检测小鼠肺组织中肿瘤坏死因子-α(TNF-α),单核细胞趋化蛋白-1(MCP-1),超氧化物歧化酶(SOD)1和SOD2的蛋白表达水平。应用GraphPad Prism 9.2统计软件对数据进行分析。计量资料多组间比较采用单因素方差分析,两组间比较采用t检验。结果小鼠血清中FSTL3的蛋白表达水平于LPS腹腔注射后12 h显著升高,18 h达到峰值,后趋于稳定,均显著高于0 h(P<0.05);缺血2 h,再灌注2 h后小鼠血清中FSTL3蛋白表达水平显著升高,再灌注6 h达到峰值,8 h后蛋白表达水平仍显著高于0 h(P<0.05)。Western blotting和转录实时定量聚合酶链反应显示,LPS刺激18 h后小鼠肺组织中FSTL3的蛋白和mRNA表达水平达到峰值(P<0.05);缺血2 h,再灌注2 h后,小鼠肺组织中FSTL3的蛋白和mRNA表达水平显著升高(P<0.05),再灌注4 h后,FSTL3的蛋白和mRNA表达水平均达到峰值(P<0.05)。FSTL3-NA处理可明显减轻脓毒血症小鼠肺病理损伤,下调肺组织TNF-α[(1.23±0.11)和(2.45±0.27)]和MCP-1[(0.34±0.02)和(2.23±0.04)]的蛋白表达水平,上调SOD1[(3.05±0.16)和(1.87±0.32)]和SOD2[(2.45±0.19)和(1.61±0.24)]的蛋白表达水平(均P<0.05)。FSTL3基因敲除可明显减轻IR小鼠肺病理损伤,下调肺组织TNF-α[(1.22±0.19)和(4.13±0.21)]和MCP-1[(0.88±0.03)和(3.10±0.15)]的蛋白表达水平,上调SOD1[(2.27±0.16)和(0.41±0.06)]和SOD2[(2.04±0.13)和(0.19±0.05)]的蛋白表达水平(均P<0.05)。结论LPS或IR刺激后,小鼠血清及肺组织中的FSTL3明显升高,FSTL3抑制或FSTL3基因敲除可明显减轻小鼠肺组织炎症和氧化应激。

Objective To investigate the expression of human follistatin-like protein 3(FSTL3)in acute lung injury caused by different inducement in mice and its significance.Methods Mouse model of septic lung injury was established by intraperitoneal injection of lipopolysaccharide(LPS),and the model of ischemia-reperfusion(IR)injury was established by clamping the left pulmonary hilum followed by opening.The protein expression level of FSTL3 in serum and the protein and mRNA expression level of FSTL3 in lung tissue were detected at different time points of lung injury.FSTL3 neutralizing antibodies(FSTL3-NA)or FSTL3 gene knockout mice were employed to evaluate the effect of FSTL3 inhibition on lung injury in mice inflicted by 2 types of injuries.Western blotting was applied to detect the protein levels of tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein-1(MCP-1),superoxide dismutase(SOD)1 and SOD2 in the lung tissues.Statistical software GraphPad Prism 9.2 was used to analyze the data.One-way AVOVA was used to compare the measurement data between groups,and student′s t test was performed for intergroup comparison.Results The serum protein level of FSTL3 was increased significantly in the mice at 12 h after intraperitoneal injection of LPS,reached a peak at 18 h,and then stabilized,both of which were significantly higher than 0 h(P<0.05).After 2 h ischemia and 2 h reperfusion,the serum protein level of FSTL3 was elevated significantly,summited at 6 h of reperfusion,and the protein level after 8 h reperfusion was still notably higher than that at 0 h(P<0.05).Western blotting and RT-PCR showed that the protein and mRNA levels of FSTL3 in the lung tissues reached the peaks after 18 h of LPS stimulation(P<0.05);after 2 h of ischemia and 2 h of reperfusion,its protein and mRNA levels in lung tissue were increased significantly(P<0.05),and reached the peaks after 4 h reperfusion(P<0.05).FSTL3-NA treatment obviously alleviated the lung pathological injury in sepsis mice,and down-regulated the protein levels of TNF-α[(1.23±0.11)vs(2.45±0.27)]and MCP-1[(0.34±0.02)vs(2.23±0.04)]in the lung tissue,while up-regulated the protein levels of SOD1[(3.05±0.16)vs(1.87±0.32)]and SOD2[(2.45±0.19)vs(1.61±0.24)](all P<0.05).FSTL3 gene knockout also obviously alleviated the lung pathological injury in IR injured mice,and down-regulated the protein levels of TNF-α[(1.22±0.19)vs(4.13±0.21)]and MCP-1[(0.88±0.03)vs(3.10±0.15)]in the lung tissue and up-regulated those of SOD1[(2.27±0.16)vs(0.41±0.06)]and SOD2[(2.04±0.13)vs(0.19±0.05)](all P<0.05).Conclusion After LPS or IR stimulation,FSTL3 level is significantly increased in both mouse lung tissue and serum,while its inhibition or knockout could significantly reduce inflammation and oxidative stress in murine lung tissue.