温肺化纤颗粒通过调控TGF-β1/Smad信号通路改善肺纤维化

Wenfei Huaxian Granules ameliorated pulmonary fibrosis through modulating the signaling pathway of TGF-β1/Smad

目的:探究温肺化纤颗粒抗博来霉素诱导小鼠肺纤维化的作用及其机制。方法:将C57BL/6J雄性小鼠随机分为Control组、Model组、WFHX-low-dose(12.5 g·kg^(–1))组、WFHX-high-dose(50 g·kg^(–1))组以及吡非尼酮(Pirfenidone,180 mg·kg^(–1))组,采用气管插管法灌注博来霉素(0.75 mg·kg^(–1))建立肺纤维化模型。自造模第2天起灌胃给药干预,持续给药28 d。HE和Masson染色法检测肺组织肺泡炎及纤维化程度;RT-qPCR法检测肺组织白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)、胶原蛋白Ⅰ(collagenⅠ)以及上皮间质转化(epithelial-mesenchymal transition,EMT)相关标志物的mRNA表达;Western blot法检测肺组织IL-6、TNF-α、CollagenⅠ、EMT相关标志物以及转化生长因子β1(transforming growth factorβ1,TGF-β1)/Smad信号通路相关蛋白表达;免疫组织化学染色法(IHC)检测p-Smad3、Smad7表达。结果:与Control组比较,Model组的组织纤维化评分显著升高,IL-6、TNF-α、collagenⅠ以及间质标志物纤维连接蛋白(fibronectin,Fn)、波形蛋白(vimentin)、α-平滑肌肌动蛋白(α-SMA)的mRNA及蛋白表达水平显著上调(P<0.01),上皮标志物钙黏蛋白E(E-cadherin)显著下调(P<0.05)。同时,TGF-β1与p-Smad2、p-Smad3蛋白表达增加,Smad7表达减少;与Model组比较,给药干预后显著降低了肺组织纤维化程度,并且WFHX给药组均在不同程度上改善上述因子的表达,显著抑制炎症因子IL-6、TNF-α释放,减轻肺组织炎症反应,减少collagenⅠ的分泌,改善EMT,减少TGF-β1生成并抑制Smad2、Smad3磷酸化,上调Smad7的表达。结论:温肺化纤颗粒可通过减轻炎症反应,抑制EMT,抑制TGF-β1/Smad信号通路发挥抗纤维化作用。

OBJECTIVE To explore the effect and mechanism of Wenfei Huaxian Granules for BLM-induced pulmonary fibrosis in mice.METHODS The C57BL/6J male mice were randomized into five groups of Control,Model,WFHX-low-dose(12.5 g·kg^(–1)),WFHX-high-dose(50 g·kg^(–1))and Pirfenidone(180 mg·kg^(–1)).The model of pulmonary fibrosis was established by an endotracheal intubation infusion of bleomycin(0.75 mg·kg^(–1)).The gavage intervention began from the second day of modeling and continued for 28 days.Degrees of alveolitis and fibrosis were detected after hematoxylin-eosin and Masson staining.The mRNA expressions of interleukin-6(IL-6),tumor necrosis factorα(TNF-α),collagenⅠand epithelial-mesenchymal transition(EMT)related markers in lung tissues were detected by real-time quantitative polymerase chain reaction(RT-qPCR).And the expressions of IL-6,TNF-α,collagenⅠ,EMT-related markers and transforming growth factorβ1(TGF-β1)/Smad signaling pathway-related proteins in lung tissues were detected by Western blot.The expressions of p-Smad3 and Smad7 were detected by immunohistochemical(IHC)stain.RESULTS As compared with Control group,tissue fibrosis score was significantly higher in Model group and the mRNA and protein expression levels of IL-6,TNF-α,collagenⅠ,mesenchymal markers fibronectin(Fn),vimentin andα-smooth muscle actin(α-SMA)were significantly up-regulated(P<0.01)while epithelial marker calmodulin E(E-cadherin)was markedly down-regulated(P<0.05).Meanwhile,the protein expressions of TGF-β1,p-Smad2 and p-Smad3 spiked while Smad7 expression declined.As compared with Model group,intervention could lessen the degree of fibrosis in lung tissues.In all WFHX-dosed groups,the expressions of the above factors improved somewhat.The releases of IL-6 and TNF-αwere blunted and inflammatory responses of lung tissues attenuated.Secretion of collagenⅠand production of TGF-β1 became suppressed.EMT improved.Phosphorylation of Smad2/Smad3 was silenced and the expression of Smad7 became up-regulated.CONCLUSION Wenfei Huaxian Granules may exert anti-fibrotic effects through blunting inflammatory responses,silencing EMT and suppressing the signaling pathway of TGF-β1/Smad.