粒状头样2在肺纤维化上皮间质转化中的作用研究

摘要

Objective:To explore the function of grainyheas-like2 (GRHL2) on the epithelial-mesenchymal transition (EMT) in pulmonary fibrosis rats.Methods:45 rats were randomly divided into normal control group,model group and sodium valproate group,15 rats in each group.Rats in the model group and sodium valproate group were injected with bleomycin along air tube to construct pulmonary fibrosis model,and rats in the normal control group was given the same amount of normal saline by the same method.The rats were injected with 200 mg ·kg-1·d-1 sodium valproate by intraperitoneally in sodium valproate group,the rats were injected with the same amount of normal saline in the normal control group and the model group for 28 d continuously.The pathological changes of lung tissue was detected by HE staining.The fibrosis of lung tissue was detected by Masson staining.The content of hydroxyproline (HYP) was detected by alkaline hydrolysis.The expression of GRHL2 was detected by immunohistochemistry and Western blot.The expression of EMT related protein E-cadherin,alpha smooth muscle cell actin (α-SMA),Vimentin,Wnt/β-catenin signaling pathway related proteins was detected by Western blot.Results:Compared with the normal control group,the alveolar structure of lung tissue was destroyed,a large number of collagen fibers were deposited,the collagen staining area ratio,Ashcroft score and HYP content were increased (P ＜ 0.01),the positive rate and expression of GRHL2 was decreased (P ＜ 0.01),the expression of E-cadherin was down-regulated (P ＜ 0.01),the expression of α-SMA,Vimentin,β-catenin,lymphoid enhancer-binding (LEF1) and matrix metalloproteinase 7 (MMP7) up-regulated in model group (P ＜ 0.01).Compared with the model group,the structure of alveolar tissue was more complete,the degree of collagen fiber deposition was lighter,the collagen staining area ratio,Ashcroft score and HYP content decreased (P ＜ 0.01),the positive rate and expression of GRHL2 was increased (P ＜ 0.01),the expression of E-cadherin was up-regulated (P ＜0.01),the expression of α-SMA,Vimentin,β-catenin,LEF1 and MMP7 down-regulated (P ＜ 0.01).Conclusion:In rats with pulmonary fibrosis,the expression of GRHL2 is down regulated,which activates the Wnt/beta-catenin signaling pathway,promotes the formation of EMT and aggravates the pulmonary fibrosis in rats,and valproate can reverse the process.%目的:探讨粒状头样2(grainyheas-like2,GRHL2)在肺纤维化上皮间质转化(epithelial-mesenchymal transition,EMT)中的作用研究.方法:45只大鼠随机分为正常对照组,模型组及丙戊酸钠组,每组15只,模型组及丙戊酸钠组沿着气管注射博来霉素构建大鼠肺纤维化模型,正常对照组采用同样方法给予等量生理盐水.丙戊酸钠组大鼠腹腔注射200 mg·kg-1·d-丙戊酸钠,正常对照组及模型组腹腔注射等量生理盐水,连续给药28 d.HE染色检测肺组织病理情况,Masson染色检测肺组织纤维化情况,碱水解法检测羟脯氨酸(HYP)含量,免疫组化及Western Blot检测GRHL2表达,Western Blot检测EMT相关蛋白E-cadherin,α-平滑肌细胞肌动蛋白(α-SMA),Vimentin,Wnt/β-catenin信号通路相关蛋白表达.结果:与正常对照组比较,模型组大鼠肺组织肺泡结构被破坏,大量胶原纤维沉积,胶原染色面积比、Ashcroft评分及HYP含量升高(P＜0.01),GRHL2阳性率及蛋白表达量降低(P＜0.01),E-cadherin表达量下调(P＜0.01),α-SMA,Vimentin,β-catenin,淋巴样增强结合因子(LEF)及基质金属蛋白酶7(MMP7)表达量上调(P＜0.01).与模型组比较,丙戊酸钠组大鼠肺泡组织结构较为完整,胶原纤维沉积程度较轻,胶原染色面积比、Ashcroft评分及HYP含量降低(P＜0.01),GRHL2阳性率及蛋白表达量提高(P＜0.01),E-cadherin表达量上调(P＜0.01),α-SMA,Vimentin,β-catenin,LEF1及MMP7表达量下调(P＜0.01).结论:大鼠肺纤维化过程GRHL2表达量下调,进而激活Wnt/β-catenin信号通路,促进EMT生成,加重大鼠肺纤维化程度,丙戊酸钠能扭转此过程.