扶正化纤方水煎液对博来霉素诱导小鼠肺纤维化及肺组织CFTR表达的影响

Effects of aqueous decoction of Fuzheng Huaxian Recipe on bleomycin-induced pulmonary fibrosis and CFTR expression in mouse lung tissue

目的 探讨囊性纤维化跨膜转导调节因子(CFTR)在小鼠特发性肺纤维化中的表达及扶正化纤方水煎液的干预作用。方法 84只C57BL/6小鼠分为空白组(12只)和造模组(72只),采用博来霉素构建特发性肺纤维化模型,造模成功后随机分为模型组,吡非尼酮组,扶正化纤方低、中、高剂量组,扶正化纤方+H-89组,每组12只,给予相应药物干预。称定小鼠体质量和肺质量,计算肺指数,HE、Masson染色观察肺组织炎症细胞浸润和胶原沉积情况,采用试剂盒检测肺组织羟脯氨酸(HYP)水平,酶联免疫吸附法(ELISA)检测肺组织环磷酸腺苷(cAMP)水平,RT-qPCR法检测肺组织CFTR mRNA表达,免疫组化法检测肺组织CFTR、白介素17B(IL-17B)蛋白表达,Western blot法检测肺组织CFTR、IL-17B、蛋白激酶A(PKA)、环磷腺苷效应元件结合蛋白(CREB)表达。结果 与空白组比较,模型组小鼠肺指数、HYP水平、炎性细胞浸润和胶原纤维沉积、血清IL-17B表达均升高(P<0.05),肺组织CFTR mRNA和蛋白、cAMP水平、p-PKA/PKA、p-CREB/CREB表达均降低(P<0.05);与模型组比较,扶正化纤方中、高剂量组小鼠肺指数、HYP水平、炎性细胞浸润和胶原纤维沉积、IL-17B表达降低(P<0.05),肺组织CFTR mRNA和蛋白、cAMP水平、p-PKA/PKA、p-CREB/CREB表达升高(P<0.05);H-89可减弱扶正化纤方的抗肺纤维化作用。结论 扶正化纤方可能通过激活cAMP/PKA通路,上调CFTR表达,进而发挥抗肺纤维化作用。

AIM To investigate the expression of cystic fibrosis transmembrane conductance regulator(CFTR) in mouse idiopathic pulmonary fibrosis and the intervention effects of aqueous decoction of Fuzheng Huaxian Recipe.METHODS Eighty-four C57 BL/6 mice were divided into the blank group(12 mice) and the group(72 mice) of idiopathic pulmonary fibrosis models established by bleomycin for further assignment of the model group, pirfenidone group, Fuzheng Huaxian Recipe groups(low dose, medium dose, and high dose use), and the group for combination use of Fuzheng Huaxian Recipe and H-89, with 12 mice in each group, for corresponding drug intervention. Subsequently, the mice had their body weight and lung mass weighed, and the lung index level calculated;their pulmonary inflammatory cell infiltration and collagen deposition observed by HE and Masson staining;their pulmonary hydroxyproline(HYP) level detected by kit;their pulmonary level of cyclic adenosine monophosphate(cAMP) detected by ELISA;their pulmonary CFTR mRNA expression detected by RT-qPCR;their expressions of CFTR and interleukin 17 B(IL-17 B) protein detected by immunohistochemistry;and their expression of CFTR, IL-17 B, protein kinase A(PKA) and cyclic adenosine monophosphate response element binding protein(CREB) detected by Western blot.RESULTS Compared with the blank group, the model group displayed increased levels of lung index, HYP, inflammatory cell infiltration and collagen fiber deposition, and IL-17 B expression(P<0.05), and reduced lung CFTR mRNA and protein, cAMP level, p-PKA/PKA, p-CREB/CREB expression(P<0.05). Compared with the model group, the groups intervened with medium dose and high dose Fuzheng Huaxian Recipe were observed with reduced levels of lung index, HYP, inflammatory cell infiltration and collagen fiber deposition, and IL-17 B expression(P<0.05), and increased expressions of lung CFTR mRNA and protein, cAMP level, p-PKA/PKA, p-CREB/CREB(P<0.05). H-89 weakened the anti-pulmonary fibrosis effect of Fuzheng Huaxian Recipe.CONCLUSION Fuzheng Huaxian Recipe may achieve an anti-pulmonary fibrosis effect via activating cAMP/PKA pathway in the up-regulation of CFTRexpression.