Ac—SDKP在博莱霉素诱导的肺纤维化小鼠中作用机制的探讨

Effects of Ac-SDKP on bleomycin induced pulmonary fibrosis mice

目的 探讨小鼠肺间质纤维化（IPF）模型中，Ac-SDKP通过抑制内质网应激（ERS）进而干预上皮-间质转化（EMT）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（体重25±2g）随机分为3组：正常对照组（N组，n=8只）、肺纤维化模型组（M组，n=8只）、肺纤维化＋Ac-SDKP治疗组（P组,n=8只）。选取其中16只小鼠，采用气管内注入博来霉素（5mg/kg）法建立小鼠肺纤维化模型，21天后随机选取8 只纤维化小鼠，腹腔内埋入Ac-SDKP微量注射泵（内含Ac-SDKP混合溶液：Ac-SDKP 800ug/kg.d，冰乙酸0.1M，卡托普利100mg/kg.d），持续干预21天。于各组造模21天后处死小鼠，取肺组织，观察肺组织病理学变化；羟脯氨酸含量测定，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。2、HE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＜0.05）。4、免疫组化结果：（1）N组肺组织中，α-SMA 、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＜0.05）（2）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＜0.05）。5、Western blot 结果：与N组比较，M组CHOP、Grp78、Vimentin表达量均升高；Ecadherinn的表达量降低；而P组各指标表达量均介于N组和M组之间（P＜0.05）。 结论 在肺纤维化发病过程中，Ac-SDKP可能通过抑制ERS减缓EMT，从而发挥其抗肺间质纤维化的作用。

Objective To investigate the effect of Ac - SDKP on reducing the endoplasmic reticulum stress reduced by lung epithelial mesenchymal transition in the bleomycin model of pulmonary fibrosis of mice. Methods 24 male C57BL/6 mice of 9 weeks old were randomly allocated 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. （3） fibrosis model group＋ Ac-SDKP group （group P, n=8）.Same as the group M but Ac-SDKP was administered （800ug/kg.d） via a mini-osmotic pump implanted into the abdominal cavity after bleomycin until 21 day. After the formation of the IPF model, lung tissues were collected. HE staining, Masson staining and measuring the content of hydroxyproline （HYP） in the lung tissues were using to evaluate the histological changes in the lungs and observe whether the model of pulmonary fibrosis in mice was successful. The expression of proteins related Endoplasmic reticulum stress GRP78、CHOP,epithelial cells marker E-cadherin and mesenchymal marker Vementin、α-SMA were assessed by Immunohistochemistry、Western blot. Results 1. Morphological observation：In group N, surface and cut surface of lung tissues are smooth, there is no nodule formation; in Group M, there are many visible scattered gray nodules in surface and cut surface of lung tissues ; in group P are the same as group M, but the numbers are lesser. 2. Histopathological examination： on the 21 day, the histopathological findings of fibrosis were induced in group M. It is showing that tracheal injection of BLM induced pulmonary fibrosis in mice model was successful. Compared with group M, fibrosis changes were lesser in the group P. 3.The content of hydroxyproline（HYP）：The level of HYP in mice of group M was significantly up regulated;the content of HYP in group P was lower than that in group M （P 〈 0.05）; 4. Immunohistochemistry：（1） Compared with group N, the expression of α-SMA、 Vimentin、CHOP and Grp78 is higher in group M, and the expression in group P is between group N and group M（P〈0.05）. （2） The expression of E-cadherin is lower in group M, and the expression in group P is between group N and group M （P〈0.05）. 5. Western blot： Protein expression of GRP78、CHOP、Vementin in group M were significantly higher than those in group N, and E-cadherin was lower than that in group M, which in group P were between those in group M and group N（P 〈 0.05）. Conclusion In the process of pulmonary fibrosis, Ac- SDKP plays an important role of anti-fibrosis in alleviating pulmonary fibrosis by reducing ERS reduced EMT.