摘要
目的:探讨宣肺止嗽配方对慢性阻塞性肺疾病(COPD)模型大鼠白细胞介素-17(IL-17)信号通路的影响.方法:将60只Wistar大鼠随机分为空白组10只和造模组50只,采用气管滴注脂多糖联合被动烟熏建立COPD模型大鼠.造模成功后采用随机数字表法分为模型组、地塞米松组、宣肺止嗽方高、中、低剂量组(3.6、1.8、0.9g·kg-1·d-1).空白组和模型组给予10mL·kg-1·d-1生理盐水灌胃,宣肺止嗽方各干预组灌胃给药,地塞米松组给予2.57×10-4g·kg-1·d-1地塞米松灌胃,持续治疗28 d.肺功能检测仪进行肺功能指标水平测定;酶联免疫吸附测定法(ELISA)检测大鼠血清白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、IL-17、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量;免疫组化(IHC)检测肺组织IL-17A、白细胞介素-17受体A(IL-17RA)、核转录因子-κB激活剂1(Act1)、肿瘤坏死因子相关因子6(TRAF6)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)、核转录因子-κB p65(NF-KB p65)及磷酸化阳性表达;蛋白免疫印迹法(Western blot)检测肺组织IL-17A、IL-17RA、Act1、TRAF6蛋白表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)测定肺组织IL-17A、IL-17RA、Act1、TRAF6 mRNA的情况.结果:与空白组比较,模型组血清中IL-6、IL-8、IL-17、IL-1β、TNF-α含量明显升高(P＜0.05),肺功能流速指标和容量指标明显下降(P＜0.05),时间指标和其他指标均明显升高(P＜0.05),肺组织IL-17A、IL-17RA、Act1、TRAF6 mRNA和蛋白水平及下游NF-κB p65、p-NF-κB p65、p-p38 MAPK阳性表达均明显升高(P＜0.05);与模型组比较,各治疗组血清中IL-6、IL-8、IL-17、IL-1β、TNF-α含量均有不同程度的降低(P＜0.05),肺功能指标均有不同程度的改善(P＜0.05),宣肺止嗽方高、中剂量组 IL-17A、IL-17RA、Act1、TRAF6 mRNA 和蛋白水平及下游 NF-κB p65、p-NF-κB p65、p-p38 MAPK 阳性表达明显减弱(P＜0.05).结论:宣肺止嗽方能有效对抗COPD炎症反应,其机制可能与下调IL-17A、IL-17RA、Act1、TRAF6蛋白表达,进而抑制下游NF-κB、p38 MAPK信号通路,减少IL-6、IL-8、TNF-α、IL-17、IL-1β的释放,进而减轻气道炎症反应有关.
Abstract
Objective:To investigate the effect of Xuanfei Zhisou prescription on the interleukin-17(IL-17)signaling pathway in model rats with chronic obstructive pulmonary disease(COPD).Method:A total of 60 Wistar rats were randomly divided into a blank group(10 rats)and a model group(50 rats),and COPD model rats were established by tracheal infusion of lipopolysaccharide combined with passive fumigation.After modeling,the rats were divided into the model group,dexamethasone group,and high,medium,and low-dose Xuanfei Zhisou prescription groups(3.6,1.8,0.9 g·kg-1·d-1)according to the random number table.Rats in the blank group and model group were given normal saline of 10 mL·kg-1·d-1 by gavage administration,and the intervention groups of Xuanfei Zhisou prescription were given corresponding drugs.Rats in the dexamethasone group were given dexamethasone of 2.57×10-4 g·kg-1·d-1 for 28 days.The level of pulmonary function indexes in rats was measured by a pulmonary function detector.The contents of interleukin-6(IL-6),interleukin-8(IL-8),IL-17,interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).The positive expressions of IL-17A,IL-17RA,nuclear factor-κB activator 1(Act1),tumor necrosis factor-associated factor 6(TRAF6),p-p38 mitogen-activated protein kinase(p-p38 MAPK),nuclear factor-κB p65(NF-κB p65),and phosphorylation were detected by immunohistochemistry(IHC).The protein expression levels of IL-17A,IL-17RA,Act1,and TRAF6 in the lung tissue were detected by Western blot.The mRNA expressions of IL-17A,IL-17RA,Actl,and TRAF6 in the lung tissue were detected by Real-time polymerase chain reaction(Real-time PCR).Result:Compared with the blank group,the serum contents of IL-6,IL-8,IL-17,IL-1β,and TNF-α in the model group were significantly increased(P＜0.05),and the flow rate and volume indexes of pulmonary function in the model group were significantly decreased(P＜0.05),while the time indexes and other indexes were significantly increased(P＜0.05).The mRNA and protein expression levels of IL-17A,IL-17RA,Act1,and TRAF6 in pulmonary tissue and the positive expressions of downstream NF-κB p65,p-NF-KB p65,and p-p38 MAPK were increased(P＜0.05).Compared with the model group,the levels of IL-6,IL-8,IL-17,IL-1β,and TNF-α in the serum of all treatment groups were decreased to varying degrees(P＜0.05),and the indexes of pulmonary function were improved to different degrees(P＜0.05).The mRNA and protein levels of IL-17A,IL-17RA,Actl,and TRAF6 and the positive expression of downstream NF-κB p65,p-NF-κB p65,and p-p38 MAPK in high and medium-dose Xuanfei Zhisou prescription groups were significantly decreased(P＜0.05).Conclusion:Xuanfei Zhisou prescription can effectively resist inflammation of COPD rats.The mechanism may be related to down-regulating the protein expression of IL-17A,IL-17RA,Act1,and TRAF6,inhibiting downstream NF-κB and p38 MAPK signaling pathways,and reducing the release of IL-6,IL-8,TNF-α,IL-17,and IL-1β,thus reducing the airway inflammation response.
基金项目
国家重点研发计划重点专项(2017YFC0907202)
中央引导地方科技发展资金项目(22ZY1QA003)
甘肃省高等学校产业支撑计划(2022CYZC-53)
国家科技期刊平台
NSTL
维普
万方数据