摘要
目的:研究苓桂术甘汤对香烟烟雾暴露联合脂多糖(LPS)气道滴注诱导的慢性支气管炎(CB)大鼠的疗效作用机制。方法:将60只SPF级SD大鼠按体质量分层法随机分为正常组、模型组、地塞米松组(1 mg·kg^(-1))、苓桂术甘汤高、中、低剂量组(30.06、15.03、7.515 g·kg^(-1))6组,每组10只。各组造模第1天、第14天分别予LPS(1 g·L^(-1))200μL气道滴注,正常组予等体积的生理盐水。除正常组外,其余各组第2~13天、15~30天予香烟烟雾暴露(10支/次/30 min,2次/d)构建大鼠CB模型。给药组分别在造模第2天连续灌胃30 d,观测大鼠精神行为状态与体质量。造模后30 d取大鼠肺组织,测定左肺湿/干质量比(W/D);苏木素-伊红(HE)染色观察肺组织及支气管病理形态学变化;阿利新蓝-过碘酸雪夫(AB-PAS)染色观察支气管黏液分泌情况与杯状细胞增生程度;酶联免疫吸附测定法(ELISA)检测血清中黏糖蛋白5AC(MUC5AC)、白细胞介素-13(IL-13)、白细胞介素-6(IL-6)与肿瘤坏死因子-α(TNF-α)的含量;免疫组化与全自动蛋白免疫印迹法(Western blot)检测肺组织中磷脂酶A2(PLA2)、热敏通道瞬时感受器电位香草素受体1(TRPV1)与瞬时感受器电位锚蛋白1(TRPA1)的表达变化。结果:与正常组比较,模型组大鼠精神憔悴,弓背蜷腹,口鼻有血性分泌物,死亡率为40%,体质量增长趋势显著下降,肺支气管结构损害严重,管壁大量炎性介质与炎症细胞浸润,充血水肿与纤维增生,杯状细胞大量增生,黏液过量分泌堆积,管腔明显狭窄变形,且肺水肿显著加重(P<0.01);血清MUC5AC、IL-13、IL-6、TNF-α及肺PLA2的组织表达与蛋白表达水平显著上调(P<0.01);与模型组比较,苓桂术甘汤高、中、低3组反应灵敏,无异常行为与分泌物,死亡率低,体质量持续增长稳定,肺支气管损伤减轻,杯状细胞增生与黏液分泌减少,肺水肿显著减轻(P<0.01);血清MUC5AC、IL-13、IL-6、TNF-α及肺PLA2、TRPA1、TRPV1的组织表达与蛋白表达水平显著下调(P<0.01)。结论:苓桂术甘汤可以有效减轻慢性支气管炎大鼠的肺部炎症与气道黏液高分泌状态,具有一定的抗炎化痰功效,其机制可能与PLA2-TRPV1/TRPA1通路有关。
Objective:To study the effect and mechanism of Linggui Zhugantang in treating chronic bronchitis(CB)induced by exposure to cigarette smoke combined with tracheal instillation of lipopolysaccharide(LPS).Method:Sixty SPF-grade SD rats were randomly divided into normal,model,dexamethasone(1 mg·kg^(-1)),and high-,medium-,and low-dose(30.06,15.03,7.515 g·kg^(-1),respectively)Linggui Zhugantang groups by the body weight stratification method,with 10 rats in each group.Each group was administrated with 200μL LPS(1 g·L^(-1))by tracheal instillation on days 1 and 14,respectively,while the normal group was administrated with an equal volume of normal saline.Except the normal group,the other groups were exposed to cigarette smoke on days 2-13 and 15-30(10 cigarettes/time/30 min,twice/day)for the modeling of CB.The rats were administrated with corresponding drugs by gavage for 30 consecutive days from day 2 of modeling,and the mental status,behavior,and body weights of the rats were observed and measured.The wet/dry mass ratio(W/D)of the left lung was measured 30 days after modeling.Hematoxylin-eosin staining was employed to observe the pathological changes in the lung and bronchial tissues.The bronchial mucus secretion and goblet cell proliferation were observed by Alcian blue-periodic acid Schiff(AB-PAS)staining.The levels of mucin 5AC(MUC5AC),interleukin(IL)-13,IL-6,and tumor necrosis factor(TNF)-αin the serum were determined by enzyme-linked immunosorbent assay.The expression of phospholipase A2(PLA2),transient receptor potential vanilloid receptor 1(TRPV1),and transient receptor potential ankyrin 1(TRPA1)in the lung tissue was quantitatively analyzed by immunohistochemistry and Western blot.Result:Compared with the normal group,the model group showcased abnormal mental status and behaviors,bloody secretion in the nose and mouth,the mortality rate of 40%,decreased body weight,severe lung bronchial structure damage,a large number of inflammatory mediators and inflammatory cell infiltration in the tube wall,hyperemia,edema,and fibroplasia,massive proliferation of goblet cells,excessive secretion and accumulation of mucus,stenosis and deformation of the lumen,and aggravation of pulmonary edema(P<0.01).In addition,the model group had higher levels of MUC5AC,IL-13,IL-6,and TNF-αin the serum and higher expression of PLA2 in the lung tissue than the normal group(P<0.01).Compared with the model group,the medication groups showed normal mental status and behaviors,reduced mortality rate,stable weight gain,reduced lung and bronchial injuries,decreased goblet cell proliferation and mucus secretion,and alleviated pulmonary edema(P<0.01).Furthermore,Linggui Zhugantang lowered the levels of MUC5AC,IL-13,IL-6,and TNF-αin the serum and down-regulated the protein levels of PLA2,TRPV1,and TRPA1 in the lung tissue(P<0.01).Conclusion:Linggui Zhugantang can reduce the pulmonary inflammation and airway mucus hypersecretion in the rat model of chronic bronchitis.It may exert the effects of reducing inflammation and resolving phlegm by regulating the PLA2-TRPV1/TRPA1 pathway.
作者
丁薇
汪文来
刘珍洪
陈香云
何湛湛
褚策
袁雨露
许咏琪
张雨欣
赵培彰
杨桢
赵红霞
DING Wei;WANG Wenlai;LIU Zhenhong;CHEN Xiangyun;HE Zhanzhan;CHU Ce;YUAN Yulu;XU Yongqi;ZHANG Yuxin;ZHAO Peizhang;YANG Zhen;ZHAO Hongxia(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102488,China;Institute of Basic Theory for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;Institute for Brain Disorders,Beijing University of Chinese Medicine,Beijing 100700,China;West China School of Medicine,Sichuan University,Chengdu 610041,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第14期1-9,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
中国中医科学院科技创新工程黑地黄丸重大攻关项目(CI2021A00606)
国家自然科学基金项目(82104822)
中国中医科学院自主选题项目(YZX202237,YZX202241)。
