基于“肺主行水”理论探究小青龙汤调节肺水转运蛋白的作用机制  

Mechanism of Xiao Qinglongtang Regulating Lung Water Transport-related Proteins Based on Theory of Lung Controlling Water Movement

作　　者：张迪 张冬梅[2] 陆瑞敏 吉静 许宗颖 王旭 陈萌 ZHANG Di;ZHANG Dong-mei;LU Rui-min;JI Jing;XU Zong-ying;WANG Xu;CHEN Meng(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China;Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China)

机构地区：[1]北京中医药大学中医学院,北京100029 [2]北京中医药大学东直门医院,北京100700

出　　处：《中国实验方剂学杂志》2022年第8期1-11,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：2020年度北京中医药大学基本科研业务费重点攻关项目(2020-JYB-ZDGG-001);2021年北京中医药大学校级科研纵向发展基金(2021-zxfzjj-012)。

摘　　要：目的:观察小青龙汤及其方元对于肺水转运蛋白的调节作用,初步阐释"肺主行水"的生物学内涵,并从该角度探索其作用机制。方法:根据经方的组方规律,将小青龙汤(11.22 g·kg^(-1))拆分为桂枝甘草(2.70 g·kg^(-1))、芍药甘草(2.70 g·kg^(-1))、姜辛味(3.90 g·kg^(-1))、半夏麻黄(3.27 g·kg^(-1))4个方元。通过"形寒+饮冷+冷水浴"法建立寒饮蕴肺证大鼠病理模型,给予小青龙汤及其方元进行干预。肺功能分析系统测定大鼠用力肺活量(FVC)、功能残气量(FRC)、平均呼气中期流量(MMEF)、吸气时间(tI)和呼气时间(tE)等参数;苏木素-伊红(HE)染色观察大鼠肺组织病理形态学改变;免疫组化法(IHC)检测大鼠肺组织中水通道蛋白(AQP)1、AQP5、上皮细胞钠通道α亚单位(α-ENaC)和钠钾泵(Na^(+)-K^(+)-ATPase)表达;酶联免疫吸附测定法(ELISA)检测肺组织中肿瘤坏死因子-α(TNF-α)含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测肺组织中环磷酸腺苷(cAMP)、蛋白激酶A(PKA)和cAMP反应元件结合蛋白(CREB)mRNA分子表达;蛋白免疫印迹法(Western blot)检测肺组织中cAMP、PKA、CREB和磷酸化(p)-CREB蛋白表达。结果:与正常组比较,模型组大鼠FVC、FRC和MMEF功能显著下降(P<0.01),tI与tE时间明显延长(P<0.05,P<0.01);肺组织中TNF-α含量显著升高(P<0.01);肺组织中cAMP、PKA、CREB mRNA及蛋白表达显著降低(P<0.01);肺组织中AQP5及α-ENaC表达明显减少;大鼠肺泡腔内充满水肿液,周围组织充血,炎性细胞浸润,支气管黏膜上皮黏连。与模型组比较,小青龙汤及其方元组可以明显增强模型大鼠FVC、FRC与MMEF功能(P<0.05,P<0.01),部分方元组可缩短tI与tE时间(P<0.05,P<0.01);小青龙汤组、桂枝甘草组及半夏麻黄组肺组织TNF-α的含量显著下调(P<0.01);小青龙汤组cAMP、PKA和CREB的mRNA的表达显著上调(P<0.01),桂枝甘草组、姜辛味组及半夏麻黄组显著上调cAMP和PKA的mRNA表达(P<0.01);�Objective:To observe the regulatory effect of Xiao Qinglongtang and its ingredients on lung water transport-related proteins,and to explain the biological connotation of lung governing water movement,based on which the regulatory mechanism of Xiao Qinglongtang will be explored.Method:According to the composition rules of classical formula,Xiao Qinglongtang(11.22 g·kg^(-1)),Guizhi Gancao(2.70 g·kg^(-1)),Shaoyao Gancao(2.70 g·kg^(-1)),Jiangxinwei(3.90 g·kg^(-1))and Banxia Muahuang(0.032 7 g·kg^(-1))were prepared.The pathological model of syndrome of cold fluid accumulated in lung of rats was established by the"coldness of body+drinking cold+cold bath"method,and Xiao Qinglongtang and its ingredients were administrated to intervene with the model rats.Lung function parameters of forced vital capacity(FVC),functional residual capacity(FRC),mean mid-expiratory flow(MMEF),inspiratory time(t I),and inspiratory time(t E)were determined by lung function analyzer.Hematoxylin and eosin(HE)staining was used to observe the changes in pathological morphology.The expression of aquaporin(AQP)1,AQP5,epithelial sodium channelαsubunit(α-ENa C)and Na^(+)-K^(+)-ATPase in lung tissues of rats,the content of tumor necrosis factor-α(TNF-α),the m RNA expression of cyclic adenosine monophosphate(c AMP),protein kinase A(PKA)and c AMP-response element binding protein(CREB),and the protein expression of c AMP,PKA,CREB,and phosphorylated-CREB (p-CREB) were detected by immunohistochemistry (IHC),enzyme-linked immunosorbent assay(ELISA),Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and Western blot,respectively.Result:Compared with normal group,functions of FVC,FRC and MMEF in model group were significantly decreased (P<0.01),and the time of t I and t E was significantly prolonged(P<0.05,P<0.01).The content of TNF-αin lung tissue was significantly increased(P<0.01).The m RNA and protein expressions of c AMP,PKA and CREB in lung tissue were significantly decreased(P<0.01).The expression of AQP5 andα-ENAC

关 键 词：小青龙汤 肺主行水 水通道蛋白 上皮钠通道 钠泵 

分 类 号：R2-0[医药卫生—中医学] R33