复方葶苈子汤通过调控HMGB1介导的细胞焦亡及免疫失衡治疗COPD相关性肺动脉高压

Compound Tinglizi Decoction intervenes COPD-associated pulmonary hypertension through regulation of HMGB1-mediated pyroptosis and immune imbalance

研究高迁移率族蛋白B1(high mobility group box 1,HMGB1)介导的肺动脉平滑肌细胞焦亡及免疫失衡对慢性阻塞性肺疾病相关性肺动脉高压(chronic obstructive pulmonary disease-associated pulmonary hypertension,COPD-PH)大鼠的影响,及复方葶苈子汤的干预机制。将90只大鼠随机分为正常组,模型组,复方葶苈子汤低、中、高剂量组,辛伐他汀组。使用香烟烟雾联合脂多糖(lipopolysaccharide,LPS)气道内滴入的方法建立COPD-PH大鼠模型,模型制备时间为60 d。复方葶苈子汤低、中、高剂量组分别给予复方葶苈子汤4.93、9.87、19.74 g·kg^(-1)大鼠灌胃,以辛伐他汀片1.50 mg·kg^(-1)予辛伐他汀组大鼠灌胃,连续干预14 d后,进行大鼠肺功能、平均肺动脉压力检测和动脉血气分析,取肺组织进行苏木素-伊红染色(hematoxylin-eosin,HE),观察大鼠肺组织病理形态的改变,采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative PCR,qRT-PCR)检测大鼠肺组织中相关mRNA表达,蛋白免疫印迹法(Western blot,WB)检测大鼠肺组织中相关蛋白表达,酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测肺组织中炎症因子水平,透射电子显微镜观察肺动脉平滑肌细胞的超微结构。复方葶苈子汤可以使COPD-PH大鼠用力肺活量(forced vital capacity,FVC)、0.3 s用力呼气量(forced expiratory volume in 0.3 second,FEV0.3)、FEV0.3/FVC、用力呼气峰流量(peek expiratory flow,PEF)、肺动态顺应性(respiratory dynamic compliance,Cdyn)、动脉氧分压(arterial partial pressure of oxygen,PaO_(2))和血氧饱和度(arterial oxygen saturation,SaO_(2))升高,使呼气阻力(resistance of expiration,Re)、平均肺动脉压力(mean pulmonary arterial pressure,mPAP)、右心肥厚指数(right ventricular hypertrophy index,RVHI)和动脉二氧化碳分压(arterial partial pressure of carbon dioxide,PaCO_(2))下降;可以抑制COPD-PH大鼠肺组织中HMGB1、晚期糖基化终末产物受体(receptor for advanced glycation end products,RAGE)、pro caspase-8、cleaved caspase-8和gasdermin D(GSDMD)蛋白表达,HMGB1、RAGE、caspase-8和GSDMD mRNA表达,抑制肺动脉平滑肌细胞焦亡;可以抑制COPD-PH大鼠肺组织中干扰素-γ(IFN-γ)和白细胞介素-17(IL-17)分泌,促进白细胞介素-4(IL-4)和白细胞介素-10(IL-10)分泌;还可以减轻COPD-PH大鼠气管、肺泡及肺动脉的病变程度;并且复方葶苈子汤的作用效果呈剂量依赖性。复方葶苈子汤可以改善COPD-PH大鼠肺功能、肺动脉压力、动脉血气情况、炎症反应以及气管、肺泡及肺动脉的病变情况,其作用机制与HMGB1介导的肺动脉平滑肌细胞焦亡及辅助性T细胞1(helper T cell 1,Th1)/辅助性T细胞2(Th2)、辅助性T细胞17(Th17)/调节性T细胞(regulatory T cell,Treg)失衡有关。

This paper aimed to investigate the effects of high mobility group box 1(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis and immune imbalance on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPDPH)in rats and the intervening mechanism of Compound Tinglizi Decoction.Ninety rats were randomly divided into a normal group,a model group,low-dose,medium-dose,and high-dose Compound Tinglizi Decoction groups,and a simvastatin group.The rat model of COPD-PH was established by fumigation combined with lipopolysaccharide(LPS)intravascular infusion,which lasted 60 days.Rats in the low,medium,and high-dose Compound Tinglizi Decoction groups were given 4.93,9.87,and 19.74 g·kg^(-1)Compound Tinglizi Decoction by gavage,respectively.Rats in the simvastatin group were given 1.50 mg·kg^(-1)simvastatin by gavage.After 14 days,the lung function,mean pulmonary artery pressure,and arterial blood gas of rats were analyzed.Lung tissues of rats were collected for hematoxylin-eosin(HE)staining to observe the pathological changes.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to determine the expression of related mRNA in lung tissues,Western blot(WB)was used to determine the expression of related proteins in lung tissues,and enzyme linked immunosorbent assay(ELISA)was used to determine the levels of inflammatory factors in the lung tissues of rats.The ultrastructure of lung cells was observed by transmission electron microscope.The forced vital capacity(FVC),forced expiratory volume in 0.3 second(FEV0.3),FEV0.3/FVC,peek expiratory flow(PEF),respiratory dynamic compliance(Cdyn),arterial partial pressure of oxygen(PaO_(2)),and arterial oxygen saturation(SaO_(2))were increased,and resistance of expiration(Re),mean pulmonary arterial pressure(mPAP),right ventricular hypertrophy index(RVHI),and arterial partial pressure of carbon dioxide(PaCO_(2))were decreased by Compound Tinglizi Decoction in rats with COPDPH.Compound Tinglizi Decoction inhibited the protein expression of HMGB1,receptor for advanced glycation end products(RAGE),pro caspase-8,cleaved caspase-8,and gasdermin D(GSDMD)in lung tissues of rats with COPD-PH,as well as the mRNA expression of HMGB1,RAGE,and caspase-8.Pulmonary artery smooth muscle cell pyroptosis was inhibited by Compound Tinglizi Decoction.Interferon-γ(IFN-γ)and interleukin-17(IL-17)were reduced,and interleukin-4(IL-4)and interleukin-10(IL-10)were incresead by Compound Tinglizi Decoction in lung tissues of rats with COPD-PH.In addition,the lesion degree of trachea,alveoli,and pulmonary artery in lung tissues of rats with COPD-PH was improved by Compound Tinglizi Decoction.Compound Tinglizi Decoction had dose-dependent effects.The lung function,pulmonary artery pressure,arterial blood gas,inflammation,trachea,alveoli,and pulmonary artery disease have been improved by Compound Tinglizi Decoction,and its mechanism is related to HMGB1-mediated pulmonary artery smooth muscle cell pyroptosis and helper T cell 1(Th1)/helper T cell 2(Th2),helper T cell 17(Th17)/regulatory T cell(Treg)imbalance.