基于TRPV4/P2X3信号通路探讨祛风宣肺方干预咳嗽敏感性增高豚鼠的疗效机制  

Therapeutic mechanism study of Qufeng Xuanfei recipe on guinea pigs with increased cough sensitivity based on TRPV4/P2X3 signal pathway

作　　者：李先莉 马建岭[2] 史利卿[2] 温绍惠[2] 王丽云[2] 李扭扭[2] 季坤[2] 王亚杰 罗景舒 Li Xianli;Ma Jianling;Shi Liqing;Wen Shaohui;Wang Liyun;Li Niuniu;Ji Kun;Wang Yajie;Luo Jingshu(Beijing University of Chinese Medicine,Beijing 100029,China;Department of Fever and Respiratory Diseases,Dongfang Hospital,Beijing University of Chinese Medicine,Beijing 100078,China)

机构地区：[1]北京中医药大学,北京100029 [2]北京中医药大学东方医院呼吸热病科,北京100078

出　　处：《国际呼吸杂志》2023年第8期897-903,共7页International Journal of Respiration

基　　金：国家自然科学基金(81373588);北京市自然科学基金(7192115、7184224);东方医院"1166"人才培养项目。

摘　　要：目的:观察祛风宣肺方对咳嗽敏感性增高豚鼠咳嗽次数、气道反应性及瞬时受体电位香草素4(TRPV4)/P2X3信号通路相关指标的影响,初步探讨其疗效机制。方法:本研究为实验性研究。采用随机数字表法,将25只SPF级雄性健康豚鼠随机分为空白组、模型组、中药组、TRPV4抑制剂组、P2X3抑制剂组,每组5只。除空白组外,其余各组建立咳嗽敏感性增高豚鼠模型。造模成功后,空白组及模型组给予生理盐水,中药组给予祛风宣肺方,TRPV4抑制剂组及P2X3抑制剂组分别给予GSK2193874、AF-353,干预7 d。用TRPV4激动剂GSK1016790A测定各组豚鼠的咳嗽次数,肺功能仪检测豚鼠气道阻力,酶联免疫吸附试验法检测血清白细胞介素6(IL-6)、IL-8、IL-1β水平及支气管肺泡灌洗液(BALF)中TRPV4、P2X3、P物质(SP)蛋白表达水平,蛋白质印迹法检测肺组织中TRPV4、P2X3、SP蛋白表达。结果:与空白组比较,模型组咳嗽次数增多[(4.00±0.82)次比(14.75±2.22)次,P<0.01],气道阻力[氯化乙酰胆碱浓度为400 mg/L:(2.32±1.07)cmH_(2)O·s/L比(6.50±2.03)cmH_(2)O·s/L,800 mg/L:(2.88±0.86)cmH_(2)O·s/L比(9.47±3.52)cmH_(2)O·s/L]、血清炎性因子[IL-6、IL-8、IL-1β分别为(16.32±0.17)ng/L比(24.49±0.19)ng/L、(33.48±1.36)ng/L比(59.33±2.15)ng/L、(10.71±1.22)ng/L比(38.79±2.22)ng/L]、BALF及肺组织相关指标[BALF中TRPV4、P2X3、SP分别为(46.14±0.35)μg/L比(91.62±0.25)μg/L、(78.37±0.35)μg/L比(156.62±0.25)μg/L、(38.20±1.75)μg/L比(79.84±2.45)μg/L,肺组织中TRPV4、P2X3、SP分别为(0.34±0.01)比(1.09±0.12)、(0.28±0.09)比(0.77±0.18)、(0.26±0.04)比(0.87±0.09)]升高(均P<0.01)。与模型组相比,中药组咳嗽次数减少[(2.00±0.82)次],气道阻力[氯化乙酰胆碱浓度为400 mg/L:(4.01±0.43)cmH_(2)O·s/L,800 mg/L:(3.77±0.73)cmH_(2)O·s/L]、血清炎性因子[IL-6、IL-8、IL-1β分别为(18.94±0.43)ng/L、(36.42±1.60)ng/L、(25.20±3.22)ng/L]、BALF及肺组织相关指标[BALF中TRPObjective To observe the effects of Qufeng Xuanfei recipe on cough frequency,airway responsiveness,and related indexes of transient receptor potential vanilloid 4(TRPV4)/P2X3 signal pathway in guinea pigs with increased cough sensitivity,and to explore its therapeutic mechanism.Methods This was an experimental study.Using the random number table method,25 SPF male healthy guinea pigs were randomly divided into blank group,model group,traditional Chinese medicine group,TRPV4 inhibitor group,and P2X3 inhibitor group,with five animals in each group.Except for the blank group,a guinea pig model with increased cough sensitivity was established in other four groups.After successful modeling,the blank and model groups were given normal saline,the traditional Chinese medicine group was given Qufeng Xuanfei Recipe,and the TRPV4 inhibitor group and P2X3 inhibitor group were given GSK2193874 and AF-353,respectively,for 7 days of intervention.The TRPV4 agonist GSK1016790A was used to measure the number of coughs of guinea pigs in each group.Pulmonary function test was used to detect airway resistance in guinea pigs.The expression levels of serum IL-6,IL-8,IL-1β,and TRPV4,P2X3 and SP in bronchoalveolar lavage fluid(BALF)were detected by ELISA,and the protein expressions of TRPV4,P2X3,and SP in lung tissue were detected by Western Blot.Results Compared with the blank group,the number of coughs([4.00±0.82]times vs[14.75±2.22]times,P<0.01),airway resistance(acetylcholine chloride:concentration of 400 mg/L[2.32±1.07]cmH2O·s/L vs[6.50±2.03]cmH2O·s/L,800 mg/L[2.88±0.86]cmH2O·s/L vs[9.47±3.52]cmH2O·s/L),serum inflammatory factors(IL-6,IL-8,IL-1β:[16.32±0.17]ng/L vs[24.49±0.19]ng/L,[33.48±1.36]ng/L vs[59.33±2.15]ng/L,[10.71±1.22]ng/L vs[38.79±2.22]ng/L respectively),and the expressions of TRPV4,P2X3,and SP in BALF([46.14±0.35]μg/L vs[91.62±0.25]μg/L,[78.37±0.35]μg/L vs[156.62±0.25]μg/L,[38.20±1.75]μg/L vs[79.84±2.45]μg/L respectively)and lung tissue([0.34±0.01]vs[1.09±0.12],[0.28±0.09]vs[0.77±0.18],[

关 键 词：慢性咳嗽 咳嗽敏感性增高 气道神经源性炎症 祛风宣肺方 瞬时受体电位香草素4 P2X3 P物质 

分 类 号：R285.5[医药卫生—中药学]