利拉鲁肽对博来霉素诱导大鼠肺纤维化的作用及其机制研究

Effects and potential mechanisms of liraglutide on bleomycin-induced pulmonary fibrosis in rat

目的:研究利拉鲁肽(liraglutide,Li)对博来霉素(bleomycin,BLM)诱导大鼠肺纤维化的影响及其机制。方法:将SD大鼠随机分为4组:对照组(Con)、BLM组、BLM+Li组、Li组。气管内一次性注入BLM(4 U/kg)后,BLM+Li组、Li组大鼠予以每日皮下注射Li(0.2 mg/kg)。28 d后处死大鼠,采用HE、Masson三色染色法评估肺组织病理情况;甲苯胺蓝染色法评估肥大细胞浸润情况;碱水解法测定肺组织羟脯胺酸水平;RTq PCR法检测肺组织I型胶原蛋白α1、Ⅱ型胶原蛋白α1、Ⅲ型胶原蛋白α1、肿瘤坏死因子(tumor necrosis factor,TNF)-α、白介素(interleukin,IL)-6、IL-1β、转化生长因子β1(transforming growth factorβ1,TGF-β1)mRNA水平;ELISA法检测肺组织TNF-α、IL-6、IL-1β含量;免疫荧光法检测上皮细胞钙连蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达;Western blot法检测E-cadherin、紧密连接蛋白1(zonaoccluden-1,ZO-1)、闭合蛋白(Occludin)、α-SMA、TGF-β1的表达。结果:与BLM组相比,BLM+Li组肺组织炎症细胞浸润、肺泡结构破坏及纤维化程度减轻,羟脯胺酸及胶原含量显著降低。Li能抑制BLM诱导的肺组织TNF-α、IL-6、IL-1β、TGF-β1表达上调,并抑制上皮细胞标志物E-cadherin、ZO-1、Occludin下调及间质细胞标志物α-SMA的上调。结论:Li可减轻BLM诱导的大鼠肺纤维化,这种保护作用与其减轻肺内炎症反应、降低TGF-β1的表达及抑制上皮细胞间质转化有关。Li有望成为未来治疗肺纤维化的候选药物之一。

Objective:This study aims to investigate the effects of liraglutide(Li)on bleomycin(BLM)-induced pulmonary fibrosis in rat and the potential mechanisms underlying.Methods:SD rats were randomly divided into four groups:control group(Con),BLM group,BLM+Li group and Li group.After BLM(4 U/kg)was intratracheally administered once to induce pulmonary fibrosis,Li(0.2 mg/kg)was subcutaneously injected daily in BLM+Li and Li group while the control group was given the same volume of normal saline.On the day 28,all rats were anesthetized and the lung tissues were rapidly excised.Pulmonary injury and pulmonary fibrosis was evaluated by the hematoxylin-eosin and masson’s trichrome staining.Mast cells were detected with toluidine blue staining.The hydroxyproline content in the lung was detected by alkali hydrolysis assay.Collagen type I alpha 1,collagen typeⅡalpha 1,collagen typeⅢalpha 1,tumor necrosis factor(TNF)-α,interleukin(IL)-6,IL-1βand transforming growth factorβ1(TGF-β1)m RNA were detected by real time quantitative PCR.ELISA was performed to analyze the level of TNF-α,IL-6 and IL-1βin the lung homogenates.E-cadherin andα-smooth muscle actin(α-SMA)expression were evaluated by immunofluorescence.The protein levels of E-cadherin,zona occluden 1(ZO-1),Occludin,α-SMA and TGF-β1 were detected by Western blot.Results:Compared with the BLM group,Li reduced inflammatory cell infiltration,interstitial thickness,collagen deposition,significantly reduced the expression of TNF-α,IL-6,IL-1βand TGF-β1;up-regulated the expression of E-cadherin,ZO-1,Occludin and down-regulated the expression ofα-SMA.Conclusion:Li attenuates BLM-induced pulmonary fibrosis in rat,which might be attributed to its effects on alleviating inflammation,suppressing the expression of TGF-β1 and inhibiting epithelial-mesenchymal transition.Li might be a potential therapeutic option for pulmonary fibrosis in the future.