TNF-α通过调控肺成纤维细胞旁分泌光蛋白聚糖诱导小气道上皮细胞向间充质细胞转化

TNF-αinduces the mesenchymal transition of small airway epithelial cells by regulating the paracrine secretion of lumican by lung fibroblasts

目的探索炎症介质肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)诱导肺成纤维细胞旁分泌光蛋白聚糖(lumican)对小气道上皮细胞间充质转化的影响与调控机制。方法体外培养人原代肺成纤维细胞(human primary lung fibroblasts;HLF)与人原代小气道上皮细胞(human primary small airway epithelial cells;HSAE),通过转录组测序与RT-qPCR检测lumican在两种细胞内的表达情况。利用lumican siRNA靶向沉默HLF lumican表达后,将HLF与HSAE进行共培养,检测TNF-α刺激下HSAE中上皮-间充质转化(epithelial-mesenchymal transi-tion;EMT)相关指标的变化。采用ERK磷酸化特异性抑制剂预处理HSAE后,检测lumican刺激下ERK/slug通路的活性变化。结果RNA测序与RT-qPCR结果显示lumican在HSAE中几乎不表达,而在HLF中表达量较高。TNF-α可以显著增加HLF中lumican的合成和分泌。TNF-α作用于HLF与HSAE共培养体系后可诱导HSAE中α-平滑肌肌动蛋白(alpha smooth muscle actin;α-SMA)和Ⅲ型胶原蛋白α1链(collagen typeⅢ,alpha 1 chain;COL3A1)的表达,下调E-钙黏蛋白(E-cadherin)的表达。Lumican siRNA预处理HLF后可以抑制上述改变。利用重组蛋白lumican处理HSAE后,发现HSAE中ERK磷酸化和Slug蛋白水平增加,α-SMA表达上调的同时E-cadherin表达下调,而ERK磷酸化抑制剂预处理可以显著抑制该变化。结论炎症介质TNF-α可能通过促进肺成纤维细胞旁分泌lumican,作用于小气道上皮细胞,激活ERK/Slug信号通路,引起小气道上皮细胞向间充质细胞转化,进而促进炎性损伤后的纤维化进程。

Objective To explore the effect and regulatory mechanism of the inflammatory mediator tumor necrosis factor-α(TNF-α)on the paracrine secretion of lumican by lung fibroblasts and its impact on the epithelial-mesenchymal transition(EMT)of small airway epithelial cells.Methods Primary human lung fibroblasts(HLF)and human small airway epithelial cells(HSAE)were cultured in vitro.The expression of the proteoglycan lumican in HLF and HSAE was detected using transcriptome sequencing and RT-qPCR.After silencing lumican expression in HLF using lumican siRNA,HLF and HSAE were co-cultured,and the changes in EMT-related markers in HSAE under TNF-αstimulation were detected.Following the pretreatment of HSAE with a specific ERK phosphory-lation inhibitor,the changes in ERK/Slug pathway activity under lumican stimulation were measured.Results RNA sequencing and RT-qPCR results showed that lumican was barely detectable in HSAE but highly expressed in HLF.TNF-αsignificantly induced the syn-thesis and secretion of lumican in HLF.In a co-culture system of HLF and HSAE,TNF-αtreatment increased the expression ofα-SMA(alpha smooth muscle actin)and COL3A1(collagen typeⅢ,alpha 1 chain)while decreasing the expression of E-cadherin in HSAE.These changes were reversed when HLF was treated with lumican siRNA.Furthermore,treating HSAE with recombinant lumi-can led to an increase in ERK phosphorylation and Slug protein levels,upregulation ofα-SMA,and downregulation of E-cadherin.These effects were significantly reduced when an inhibitor of ERK phosphorylation was used.Conclusion The inflammatory factor TNF-αmay promote fibrosis following inflammatory injury by stimulating lung fibroblasts to secrete lumican,which then acts on small airway epithelial cells to activate the ERK/Slug signaling pathway,thereby inducing EMT in these cells.