阳和平喘颗粒对哮喘大鼠HMGB1相关信号通路蛋白及炎症因子的影响

Effect of Yanghe Pingchuan Granule on HMGB1-Related Signalling Pathway Proteins and Inflammatory Factors in Asthmatic Rats

目的探讨阳和平喘颗粒对哮喘大鼠相关炎症因子及高迁移率组蛋白(HMGB1)、Toll样受体4(TLR4)、核因子-κB(NF-κB)信号通路调控的影响。方法60只SD雄性大鼠按随机数字表法分为正常组,模型组,地塞米松组(0.25 g/kg),阳和平喘颗粒(简称阳和)低(3.87 g/kg)、中(7.74 g/kg)、高(15.48 g/kg)组,每组10只。联合应用氢氧化铝和卵蛋白致敏诱发大鼠,建立哮喘的动物模型,药物干预4周。采用HE染色观察肺组织病理炎症评分和形态学变化;吉姆萨染色计肺泡灌洗液中嗜酸性粒细胞(Eos)、中性粒细胞(Neu)、淋巴细胞(Lym)数量;动物肺功能仪检测各组大鼠肺功能;免疫组化法检测肺组织HMGB1、TLR4、NF-κB蛋白表达量;酶联免疫吸附测定法检测大鼠血清中IL-5、IL-4、IL-13含量;Western Blot法检测各组大鼠肺组织HMGB1、TLR4、磷酸化核因子-κB(p-NF-κB)、磷酸化核因子-κB抑制蛋白(p-IκB)蛋白表达情况;实时荧光定量聚合酶链式反应(qPCR)检测各组大鼠肺组织HMGB1、TLR4、NF-κB、核因子-κB抑制蛋白(IκB)mRNA转录水平。结果与正常组比较,模型组HE染色见大鼠肺组织大量炎性浸润及渗出,肺泡炎症增加,上皮细胞明显脱落,管壁平滑肌厚度增加,用力肺活量(FVC)、0.3秒用力呼气容积(FEV0.3)、FEV0.3/FVC、呼气峰流量(PEF)、最大每分钟通气量(MVV)及IκB表达下降,肺组织HMGB1、TLR4、NF-κB表达升高,大鼠血清中IL-5、IL-4、IL-13水平升高(P<0.05,P<0.01)。与模型组比较,阳和各剂量组及地塞米松组气道炎症渗出物减少,肺泡炎症减少,平滑肌厚度降低,FVC、FEV0.3、PEF、MVV升高,IL-5、IL-4、IL-13水平降低,肺组织HMGB1、TLR4、NF-κB表达减少,IκB表达水平上升(P<0.05,P<0.01)。与阳和低剂量组比较,阳和中、高剂量组和地塞米松组肺泡炎症评分及气管平滑肌厚底下降,IL-5、IL-4、IL-13水平降低,MVV、PEF、FVC、FEV0.3明显升高,肺组织中HMGB1、TLR4、NF-κB表达减少,IκB表达增加(P<0.05,P<0.01)。与阳和中剂量组比较,阳和高剂量组及地塞米松组肺泡炎症评分及平滑肌厚度降低,PEF、MVV、FEV0.3、FEV0.3/FVC水平上升,IL-4、IL-5表达水平降低,肺组织HMGB1、TLR4、NF-κB表达减少,IκB表达增加(P<0.05,P<0.01)。阳和高剂量组与地塞米松组各指标比较,差异无统计学意义(P>0.05)。结论阳和平喘颗粒可通过调节HMGB1/TLR4/NF-κB信号通路,降低Th2相关细胞因子水平,抑制Th2炎症的进展,有效改善哮喘大鼠气道炎症。

Objective To investigate the effect of Yanghe Pingchuan Granule(YHPCG)on the regulation of inflammatory factors and high-mobility histone(HMGB1),toll-like receptor 4(TLR4)and nuclear factor kappa B(NF-κB)signalling pathways in asthmatic rats.Methods Totally 60 SD male rats were divided into normal group,model group,dexamethasone group(0.25 g·kg^(-1)),low-dose YHPCG group(3.87 g·kg^(-1)),medium-dose YHPCG group(7.74 g·kg^(-1)),and high-dose YHPCG group(15.48 g·kg^(-1))according to random number method,10 rats in each group.The animal model of asthma was established.After 4 weeks of drug intervention,the pathological inflammatory score and morphological changes of lung tissue were observed by HE staining.The number of eosinophil(Eos),neutrophil(Neu)and lymphocyte(Lym)in alveolar lavage fluid were measured by Giamsa staining.The lung function of rats in each groups was detected by animal lung function instrument.The expression levels of HMGB1,TLR4,and NF-κB protein in lung tissue were detected by immunohistochemistry.The contents of IL-5,IL-4 and IL-13 in the serum of rats were determined by enzyme-linked immunosorbent assay.Western Blot was used to detect the protein expression of HMGB1,TLR4,phosphorylated nuclear factor kappa B(p-NFκB),and phosphorylated nuclear factor kappa B inhibitor protein(p-IκB)and in lung tissues of rats.The transcription levels of HMGB1,TLR4,NF-κB,and nuclear factor kappa B inhibitor protein(IκB)mRNA in lung tissues of rats were detected by real-time fluorescence quantitative polymerase chain reaction(q PCR).Results Compared with the normal group,HE staining in the model group showed substantial inflammatory infiltration and exudation,alveolar inflammation increased,obvious exudation of epithelial cells,increased thickness of smooth muscle of tube wall,the expression of HMGB1,TLR4,and NF-κB in lung tissue and the levels of IL-5,IL-4,and IL-13 in serum increased,while forced vital capacity(FVC),forced expiratory volume in 0.3 second(FEV0.3),average flow rate in 0.3 seconds(FEV0.3/FVC),peak expiratory flow(PEF),minute ventilation volume(MVV)and the expression of IκB decreased(P<0.05,P<0.01).Compared with the model group,the airway inflammatory exudate and alveolar inflammation decreased,smooth muscle thickness,IL-5,IL-4,and IL-13 levels decreased,HMGB1,TLR4 and NF-κB expression decreased,FVC,FEV0.3,PEF,MVV,and IκB increased in YHPCG groups and dexamethasone group(P<0.05,P<0.01).Compared with the low-dose group,alveolar inflammation,smooth muscle thickness,and the levels of IL-5,IL-4 and IL-13 in the medium and high-dose groups along with dexamethasone group decreased,while the expression of HMGB1,TLR4 and NF-κB in lung tissues decreased,and FVC,FEV0.3,PEF,MVV,and IκB expression increased(P<0.05,P<0.01).Compared with the medium-dose group,alveolar inflammation,smooth muscle thickness and the levels of IκB expression increased IL-4,IL-5 decreased,while the expression of HMGB1,TLR4,NF-κB in lung tissues decreased,and PEF,MVV,FEV0.3,FEV0.3/FVC and IκB expression increased in the high-dose group and dexamethasone group(P<0.05,P<0.01).Finally,there was no significant difference between the high-dose group and dexamethasone group(P>0.05).Conclusion YHPCG could alleviate airway inflammation in asthmatic rats by regulating the HMGB1/TLR4/NF-κB signalling pathway,reducing the level of Th2 related cytokines,and inhibiting the progression of Th2 inflammation.