Ac-SDKP在博莱霉素诱导的肺纤维化小鼠中作用机制的探讨

目的 探讨小鼠肺间质纤维化模型中,Ac SDKP通过抑制内质网应激进而干预上皮-间质转化过程而延缓肺纤维化发展的机制.方法 将24只9周龄健康雄性C57BL/6小鼠体质量(25±2)g]随机分为3组:对照组(N组)、肺纤维化模型组(M组)、肺纤维化+Ac-SDKP组(P组),每组8只.选取其中16只小鼠,采用气管内注入博莱霉素(5 mg/kg)法建立小鼠肺纤维化模型,21d后随机选取8只纤维化小鼠,腹腔内埋入Ac-SDKP微量注射泵,持续干预21 d.于各组造模21 d后处死小鼠,取肺组织,观察肺组织病理学变化;测定羟脯氨酸含量,判断造模是否成功并评估肺组织纤维化严重程度;Western blot、免疫组化法检测内质网应激相关蛋白GRP78、CHOP,间质组织标志物α-SMA、Vementin,上皮组织标志物E-cadherin蛋白表达水平.结果 ①肉眼观察:N组双肺表面及切面光滑,未见结节形成;M组双肺表面及切面均可见散在结节性病变;P组肺组织表面也可见小结节,但较M组稀少.②HE及Masson染色:M组出现明显的纤维化改变;P组与M组相比,肺泡炎及肺纤维化的程度均有所减轻(P＜0.05).③羟脯氨酸含量测定:M组较N组含量升高;P组较M组含量降低,P组较N组含量仍有所升高(P值均＜0.05).④免疫组化结果:N组肺组织中αSMA、Vimentin、CHOP及GRP78少量表达,M组表达较N组明显增加,P组较M组表达减少,但仍多于N组(P值均＜0.05);N组肺组织中E-cadherin表达较多,M组表达较N组减少,P组较M组表达增加,但少于N组(P值均＜0.05).⑤Western blot结果:与N组比较,M组CHOP、GRP78、Vimentin蛋白相对表达量均升高,E-cadherin蛋白相对表达量降低;而P组各蛋白相对表达量均介于N组和M组之间(P值均＜0.05).结论 在肺纤维化发病过程中,Ae-SDKP可能通过抑制内质网应激减缓上皮-间质转化,从而发挥其抗肺间质纤维化的作用.

Effects of Ac-SDKP on bleomycin induced pulmonary fibrosis mice

Objective To investigate the effect of Ac-SDKP on reducing lung epithelial mesenchymal transition by inhibiting the endoplasmic reticulum stress in pulmonary fibrosis mice.Methods 24 male C57BL/6 mice of nine weeks old were randomly divided into the following groups:① control group (group N,n =8).② fibrosis model group (group M,n =8).Mice were subjected to bleomycin instillation intratracheally in a dose of 5 mg/kg.③fibrosis model group+ Ac-SDKP group (group P,n =8).Same as the group M but Ac-SDKP was administered (800 μg.kg-1.d-1) via a miniosmotic pump implanted into the abdominal cavity after bleomycin until 21 days.After the formation of the pulmonary fibrosis model,lung tissues were collected for HE staining,masson staining and measuring the content of hydroxyproline (HYP) to evaluate the histological changes of lungs and observe whether the model of pulmonary fibrosis in mice was successful.The expressions of proteins related to endoplasmic reticulum stress GRP78,CHOP,epithelial cell marker E cadherin and mesenchymal markers Vementin,α-SMA were assessed by immunohistochemistry and Western blot.Results ①Morphological observation:in group N,surface and cut surface of lung tissues were smooth,there was no nodule formation.In group M,there were many visible scattered gray nodules in surface and cut surface of lung tissues.Group P was the same as group M,but the numbers were lesser.② Histopathological examination:on the 21 day,the histopathological findings of fibrosis were induced in group M,which showed that bleomycin induced pulmonary fibrosis in mice model was successful.Compared with group M,fibrosis changes were lesser in group P.③The level of HYP in group M was significantly upregulated,the content of HYP in group P was lower than that in group M (P ＜0.05).④ Immunohistochemistry:compared with group N,the expressions of α-SMA,Vimentin,CHOP and GRP78 were higher in group M,and the expressions of them in group P were between group N and group M (P ＜0.05).The expression of E-cadherin was lower in group M,and the expression of it in group P was between group N and group M (P ＜0.05).⑤Western blot:protein expressions of GRP78,CHOP,Vementin in group M were significantly higher than those in group N,and E-cadherin in group M was lower than that in group N,which in group P were between group M and group N (P ＜0.05).Conclusions In the process of pulmonary fibrosis,Ac-SDKP plays an important role in alleviating pulmonary fibrosis by reducing endoplasmic reticulum stress and epithelialmesenchymal transition.