脂多糖抑制小鼠肺成纤维细胞自噬的观察性研究

An observational study on autophagy in mouse lung fibroblasts inhibited by lipopolysaccharide

目的观察体外原代培养的小鼠肺成纤维细胞在脂多糖(lipopolysaccharide,LPS)作用下,自噬微管相关蛋白轻链3(microtubule-associated protein 1 light chain 3,MAP1LC3)(以下简称LC3)和Beclin-1的表达情况,以明确LPS对肺成纤维细胞自噬的调控作用.方法将培养至4~7代的小鼠肺成纤维细胞接种于6孔培养板,密度1×10^4个/ml.待细胞贴壁后,更换无血清培养基饥饿过夜,采用随机数字表法将其分为4组(每组3孔):PBS对照组(Con组)、250μg/L LPS组(LPS250组)、500μg/L LPS组(LPS500组)、1 000μg/L LPS组(LPS1 000组),除Con组外,其余组分别加入以上终浓度LPS,各组在加入PBS或LPS后30 h裂解细胞并提取总蛋白,通过Western blot法比较小鼠肺成纤维细胞经不同浓度LPS刺激后LC3和Beclin-1蛋白的表达情况;同时,采用随机数字表法将传代细胞分为2组(每组3孔):IF-Con组(加入PBS作为对照)和IF-LPS组(加入1 000μg/L的LPS),采用免疫荧光技术比较IF-Con组和IF-LPS组中小鼠肺成纤维细胞内自噬小体的表达情况.结果随着LPS刺激浓度的增高,LC3蛋白表达量下调,而LC3-Ⅱ/LC3-Ⅰ在Con组最高,随LPS刺激浓度的升高逐渐降低,LPS1 000组中,LC3-Ⅱ/LC3-Ⅰ较Con组降低(P<0.05);Beclin-1蛋白的表达量在Con组、LPS250组和LPS500组间差异无统计学意义(P>0.05),而在LPS1 000组中,Beclin-1蛋白表达较Con组明显下调(P<0.05).同时,免疫荧光实验结果显示IF-LPS组中小鼠肺成纤维细胞内自噬小体的表达量较IF-Con组明显降低.结论在体外培养的小鼠肺成纤维细胞中应用1 000μg/L的LPS可以下调LC3和Beclin-1的蛋白表达,并抑制自噬小体的形成,表明LPS可以抑制肺成纤维细胞的自噬.提示LPS抑制肺成纤维细胞的自噬可能是肺纤维化发生的机制之一.

ObjectiveTo evaluate the effects of lipopolysaccharide(LPS)on the expression of autophagy-associated proteins,Beclin-1 and microtubule-associated protein 1 light chain 3(LC3)in primary mouse lung fibroblasts in vitro.MethodsPrimary cultured mouse lung fibroblasts were seeded in 6-well plates at a density of 1×10^4/ml.After adherence,the cells were starved by free-serum culture media overnight and then divided into four groups according to random number table(n=3):a phosphate buffer saline(PBS)control group(group Con),a 250μg/L LPS group(group LPS250),a 500μg/L LPS group(group LPS500)and a 1 000μg/L LPS group(group LPS1 000).The cells were exposed to PBS and the corresponding concentrations of LPS for 30 h before total protein extraction.The expression of Beclin-1 and LC3 were determined by Western blot.Meanwhile,the cells were divided into two groups according to random number table(n=3):group IF-Con and group IF-LPS.The formation of autophagosomes was observed by immunoflourence.ResultsThe amount of LC3 protein was down-regulated as the increase of LPS concentrations.The ratio of LC3-Ⅱ/LC3-Ⅰwas apparently higher in group Con than that in other groups,which was gradually decreased as the concentrations of LPS increased.A lower ratio of LC3-Ⅱ/LC3-Ⅰwas found in group LPS1 000 than that in group Con(P<0.05).There was no obvious difference in Beclin-1 expression among groups Con,LPS250 and LPS500.However,a remarkably decreased amount of Beclin-1 was found in group LPS1 000 than that in group Con(P<0.05).Furthermore,the fibroblasts showed a marked weaker fluorescence expression of autophagosomes in group IF-LPS compared to those in group IF-Con.ConclusionsThe expression of LC3 and Beclin-1 can be down-regulated by 1 000μg/L LPS,and the formation of autophagosomes were inhibited by LPS at a concentration of 1 000μg/L.The findings indicates that LPS inhibits autophagy in mouse lung fibroblasts,which could be part of the internal mechanism of pulmonary fibrosis.