过表达死亡蛋白相关激酶2对TGF-β_(1)诱导的人肺成纤维细胞自噬和胶原蛋白分泌的影响

Effect of overexpression of death-associated protein kinase 2 on TGF-β_(1)-induced autophagy and collagen secretion in human lung fibroblasts

目的结合生物信息学分析结果研究死亡蛋白相关激酶2(DAPK2)在肺纤维化中的作用及分子机制。方法从GEO数据库下载特发性肺纤维化(IPF)数据集GSE150910进行自噬相关基因的差异表达分析, 利用极端梯度提升法筛选出关键基因。用t分布随机邻域嵌入法在单细胞测序数据集GSE135893中验证关键基因在细胞中的表达, 采用Kaplan-Meier法在GSE28221和GSE93606数据集中绘制DAPK2高、低表达组患者的生存曲线。将12只6~8周龄的C57BL/6小鼠随机分为对照组和实验组, 每组6只, 实验组小鼠气管内滴注博来霉素构建肺纤维化模型, 取小鼠肺组织进行Masson染色、免疫组织化学染色和实时荧光定量聚合酶链式反应(RT-qPCR)检测DAPK2的蛋白和mRNA表达水平。将人肺成纤维细胞分为空载体慢病毒组、转化生长因子β_(1)(TGF-β_(1))组、空载体慢病毒+TGF-β_(1)组、过表达DAPK2慢病毒+TGF-β_(1)组, 应用蛋白质印迹法检测各组自噬相关蛋白以及Ⅰ型胶原蛋白(Collage Ⅰ)、Ⅲ型胶原蛋白(Collage Ⅲ)的表达, 并应用MDC染色检测各组细胞的自噬小体所占比例。结果 GSE150910数据集中筛选出32个差异表达的衰老相关基因, 其中8个上调, 24个下调。极端梯度提升法筛选出关键自噬相关基因DAPK2。单细胞测序分析提示DAPK2主要表达于肺成纤维细胞中, 且在IPF患者肺组织内成纤维细胞的表达量低于正常肺组织(P<0.05);高表达DAPK2的IPF患者总体生存时间较低表达DAPK2的IPF患者更长(P<0.05)。Masson染色提示小鼠肺纤维化模型造模成功, RT-qPCR及免疫组织化学染色结果提示DAPK2在博来霉素诱导的小鼠肺纤维化组织中为低表达(P值均<0.05)。与TGF-β_(1)组和空载体慢病毒+TGF-β_(1)组比较, 过表达DAPK2慢病毒+TGF-β_(1)组细胞中的LC3Ⅱ和Beclin 1蛋白表达水平升高, 而哺乳动物雷帕霉素靶蛋白、Collage Ⅰ、Collage Ⅲ降低(P值均<0.05);自噬相关蛋白及Collage Ⅰ、Collage Ⅲ在TGF-β_(1)组和空载体慢病毒+TGF-β_(1)组间差异均无统计学意义(P值均>0.05)。MDC染色结果提示过表达DAPK慢病毒+TGF-β_(1)组细胞内绿色点状荧光亮度明显增强, 荧光斑点的数目也增多。结论 DAPK2在博来霉素诱导的小鼠肺纤维化组织内呈低表达, 过表达DAPK2可以促进细胞自噬减轻TGF-β_(1)诱导的肺成纤维细胞分泌胶原蛋白, 从而抑制肺纤维化的进展。

Objective To investigate the role and molecular mechanism of death-associated protein kinase 2(DAPK2)in pulmonary fibrosis by bioinformatics analysis.Methods IPF dataset GSE150910 was downloaded from GEO database for differential expression analysis of autophagy-related genes,and the extreme Gradient Boosting algorithm was used to screen out the key genes.T-distributed stochastic neighbor embedding(T-SNE)was used to verify the expression of key genes in cells in the single-cell sequencing dataset GSE135893.Kaplan-Meier method was used to plot the survival curves of patients with high and low DAPK2 expression groups in GSE28221 and GSE93606 datasets.Twelve C57BL/6 mice aged 6-8 weeks were randomly divided into the control group and the experimental group,with 6 mice in each group.The experimental group was injected with Bleomycin(BLM)intratracheally to establish the pulmonary fibrosis model.The expression levels of DAPK2 protein and mRNA were detected by Masson staining,immunohistochemistry,and real-time quantitative polymerase chain reaction(RT-qPCR).Human lung fibroblasts(HLFs)were divided into empty vector lentivirus group,transforming growth factor-β_(1)(TGF-β_(1))group,empty vector lentivirus+TGF-β_(1) group,and overexpressing DAPK2 lentivirus+TGF-β_(1) group.Western blotting was used to detect the expressions of autophagy-related proteins,CollageⅠ,and CollageⅢin each group.MDC staining was used to detect the proportion of autophagosomes in each group.Results A total of 32 differentially expressed aging-related genes were screened from the GSE150910 dataset,among which 8 genes were up-regulated and 24 genes were down-regulated.The key autophagy-related gene DAPK2 was screened by extreme gradient boosting algorithm.Single cell sequencing analysis suggested that DAPK2 was mainly expressed in lung fibroblasts,and the expression of DAPK2 in lung fibroblasts of IPF patients was lower than that in normal lung tissues(P<0.05).The overall survival time of IPF patients with high DAPK2 expression was longer than that of IPF patients with low DAPK2 expression(P<0.05).Masson staining indicated that the mouse pulmonary fibrosis model was successfully established.RT-qPCR and immunohistochemical staining showed that DAPK2 was low expressed in bleomycin-induced pulmonary fibrosis tissues of mice(all P<0.05).Compared with TGF-β_(1) group and empty vector lentivirus+TGF-β_(1) group,LC3Ⅱand Beclin 1 protein expression levels were significantly higher in overexpression DAPK2 lentivirus+TGF-β_(1) group,while mTOR,CollageⅠ,and CollageⅢwere significantly lower(all P<0.05).There was no significant difference in autophagy-related proteins,CollageⅠ,and CollageⅢbetween TGF-β_(1) group and empty vector lentivirus+TGF-β_(1) group(all P>0.05).MDC staining results showed that the green dot fluorescence brightness and the number of fluorescent spots were significantly increased in the overexpression DAPK lentivirus+TGF-β_(1) group.Conclusions DAPK2 is low-expressed in BLM fibrosis tissues of mice.Over-expression of DAPK2 can promote cellular autophagy and reduce TGF-β_(1)-induced collagen secretion by lung fibroblasts,thereby inhibiting the progression of pulmonary fibrosis.