摘要
1目的
本研究观察大鼠呼吸系统症状体征及肺功能，检测COPD肺气虚证大鼠辅助T细胞（Th）、调节性T细胞（Treg）、树突状细胞（DC）、热休克蛋白（HSP70）、基质金属蛋白酶系统（MMPs/TIMPs）、细胞信号转导转录激活因子（STAT）、JAKs/STATs信号通路、T-bet、RORγt、GATA变化，同时观察六味补气胶囊对上述指标的干预，探讨六味补气胶囊防治COPD肺气虚证大鼠的作用机制。
2方法
2.1动物分组
将大鼠随机分为正常组、模型组、模型空白组（以下简称模空组）、模型安慰剂组（以下简称模安组）、六味补气胶囊预防组（以下简称六预组）、六味补气胶囊治疗组（以下简称六味补气组）、金水宝组、脾氨肽组，每组10只。
2.2模型复制
除正常组大鼠外，其余大鼠于实验第1天、14天采用10%水合氯醛麻醉，手术暴露气管，注入1mg.mL-1脂多糖200μL至气管内。脂多糖滴入的次日，将大鼠置于动物气雾吸入仪中，用50g锯末加7.11g香烟烟丝混和点燃烟熏，每日烟熏1次，每次30min。造模周期28d。COPD模型成功标准依据肺组织影像学、肺功能及肺组织病理形态学变化。于第22天改烟熏次数为3次/天。二次复制肺气虚证模型成功依据肺功能及大鼠生物学症状、体征变化，大鼠生物学症状、体征变化包括反映肺气虚有无的一般情况、活动量、毛发、体质量、饮食量、咳嗽、呼吸频率等，其中呼吸频率为大鼠平静呼吸时每分钟呼吸次数。
2.3药物干预
于模型复制第1天开始，模空组、模安组、六预组大鼠预防给药剂量如下：模空组大鼠未予任何药物或安慰剂；模安组大鼠予生理盐水，按照10mL·kg-1·d-1剂量灌服，一天一次，连续灌服28d；六预组大鼠予六味补气胶囊混悬液，按照0.4g·kg-1·d-1（相当于临床成人常规用量的10倍）剂量灌服，一天一次，连续灌服28d。模型复制周期28d后治疗组给药，给药剂量按照体表面积计算得出。正常组、模型组灌服生理盐水10mL·kg-1·d-1，六味补气组灌服六味补气胶囊混悬液0.4g·kg-1·d-1，金水宝组灌服金水宝胶囊混悬液0.495g·kg-1·d-1，脾氨肽组灌服脾氨肽水溶液0.33mg·kg-1·d-1，各药物组剂量均相当于成人常规临床用量的10倍，1次/天，共给药30d。
2.4指标检测
分别于预防给药28d及治疗给药30d后观察和检测以下指标：呼吸系统症状体征、肺功能、肺组织病理形态学变化，血清细胞因子、Th1、Th2、Th17细胞变化，外周血Treg表达、肺组织DC、HSP70表达，肺组织MMPs/TIMPs、JAKs/STATs信号通路、T-bet、RORγt、GATA变化。
3结果
3.1COPD肺气虚证大鼠指标表达及相关性分析
3.1.1COPD肺气虚证大鼠呼吸系统改变
COPD肺气虚证大鼠出现蜷伏少动，拱背蜷卧，撮毛，食量减少，毛失光泽、发黄、易脱落，体质量较正常组减轻；模型组呼吸急促，呼吸频率明显高于正常组，呼吸道有分泌物从口鼻流出，偶可闻及咳嗽及气道痰鸣音，随着造模时间的延长。与正常组比较，模型组大鼠肺功能参数FEV0.3、FVC、FEV0.3/FVC降低；肺系数、肺泡炎积分及病理学总积分升高。3.1.2COPD肺气虚证大鼠细胞因子变化及相关性分析
COPD肺气虚证大鼠血清中IL-1β、IL-6、IL-12升高，IL-10、IL-13、IL-35表达降低。肺功能参数FEV0.3与IL-10呈正相关，FEV0.3/FVC与IL-13呈正相关，PEF与IL-35呈正相关，肺泡炎积分与IL-6呈正相关，肺病理学积分与IL-1β、IL-12呈正相关。肺功能参数FVC与IL-6呈负相关，FEV0.3/FVC与IL-1β呈负相关，呼吸频率与IL-13呈负相关。3.1.3COPD肺气虚证大鼠Th细胞变化及相关性分析
COPD肺气虚证大鼠血清IFN-γ、IL-17、Th1/Th2升高，血清IL-4和肺组织IL-4蛋白降低，肺组织IFN-γ、IL-17蛋白表达升高。肺功能参数FEV0.3/FVC与血清IL-4呈正相关，肺泡炎积分与Th1/Th2呈正相关。肺功能参数FEV0.3与血清IFN-γ、肺组织IFN-γmRNA呈负相关，FVC与IFN-γ蛋白呈负相关，FEV0.3/FVC与Th1/Th2呈负相关，PEF与IL-17蛋白呈负相关；肺泡炎积分与IL-4蛋白呈负相关，肺病理学积分与IL-17、IFN-γ蛋白呈正相关。
3.1.4COPD肺气虚证大鼠Treg变化及相关性分析
COPD肺气虚证大鼠外周血CD4+CD25+Treg、CD4+CD25+FoxP3+Treg降低，肺组织FoxP3蛋白表达降低。肺功能参数FEV0.3与FoxP3蛋白呈正相关，FVC与CD4+CD25+Treg呈正相关，FEV0.3/FVC与CD4+CD25+FoxP3+Treg呈正相关。肺泡炎积分与CD4+CD25+FoxP3+Treg呈负相关，肺病理学积分与FoxP3蛋白呈负相关。
3.1.5COPD肺气虚证大鼠DC、HSP70变化及相关性分析
COPD肺气虚证大鼠肺组织DC、HSP70阳性表达面积增加、平均光密度增强。肺泡炎积分、肺病理学积分分别与DC、HSP70光密度值呈正相关。肺功能参数FEV0.3与DC光密度呈负相关，PEF、FEV0.3/FVC与HSP70光密度呈负相关。
3.1.6COPD肺气虚证大鼠MMPs/TIMPs变化及相关性分析
COPD肺气虚证大鼠肺组织MMP-9、MMP-12呈强阳性表达，表达强度明显增强。且肺组织MMP-9基因和蛋白表达升高，TIMP1基因和蛋白表达降低。肺功能参数FEV0.3/FVC与TIMP1蛋白呈正相关，肺泡炎积分与MMP-9光密度呈正相关，肺病理学积分与MMP-12光密度呈正相关。FEV0.3与MMP-9蛋白呈负相关，FVC与MMP-9光密度呈负相关，FEV0.3/FVC与MMP-12光密度呈负相关；呼吸频率与TIMP1mRNA呈负相关。说明基质金属蛋白酶系统的表达紊乱，导致细胞外基质生成增加，进而影响COPD肺气虚证大鼠肺组织重塑，影响肺的通气功能，导致肺功能降低和肺组织损伤。
3.1.7COPD肺气虚证大鼠JAKs/STATs变化及相关性分析
COPD肺气虚证大鼠肺组织JAK1、STAT3mRNA表达，STAT4阳性面积率、平均光密度升高，JAK1、p-JAK1、STAT3、p-STAT3蛋白表达升高；STAT6阳性面积率、平均光密度较模型组降低。肺功能参数FEV0.3/FVC与STAT6呈正相关，呼吸频率与p-STAT3蛋白呈正相关，肺泡炎积分、肺病理学积分与STAT4呈正相关。FEV0.3与p-JAK1呈负相关，FVC与STAT3mRNA呈负相关，FEV0.3/FVC与STAT3呈负相关，肺泡炎积分与STAT6、p-JAK1呈负相关。
3.1.8COPD肺气虚证大鼠T-bet、RORγt、GATA变化及相关性分析
COPD肺气虚证大鼠肺组织GATA-3mRNA、蛋白表达降低，T-bet、RORγtmRNA和蛋白表达升高。肺组织T-bet、RORγt蛋白和T-bet/GATA-3比值较正常组升高，GATA-3蛋白降低。肺功能参数FEV0.3与肺组织GATA-3mRNA呈正相关，呼吸频率与RORγt蛋白呈正相关，肺泡炎积分与T-bet蛋白呈正相关，肺病理学积分与T-bet mRNA呈正相关(P<0.05，P <0.01)。FEV0.3与RORγt蛋白呈负相关，FEV0.3/FVC与T-bet mRNA呈负相关，PEF与RORγtmRNA呈负相关，肺病理学积分与GATA-3蛋白呈负相关(P<0.05)。
3.2六味补气胶囊对COPD大鼠的防治作用
3.2.1六味补气胶囊对COPD肺气虚证大鼠呼吸系统的防治
与模型组比较，六味补气预防和治疗组大鼠蜷伏少动，拱背蜷卧，撮毛，食量减少，毛失光泽、发黄、易脱落症状、体征改善，体质量升高，呼吸频率降低。六味补气预防和治疗组大鼠肺功能参数FEV0.3、FVC、FEV0.3/FVC升高。六味补气预防和治疗组大鼠气管、支气管有炎性细胞浸润，但数量较模型组减少；上皮坏死、脱落；肺泡间隔破坏较少，部分肺泡扩张成大小不等的囊泡；纤毛排列较规整。六味补气预防和治疗组肺系数、肺泡炎积分及病理学总积分降低。与六味补气组比较，脾氨肽组FEV0.3、FEV0.3/FVC、PEF降低；病理学总积分比较，六味补气组较脾氨肽组降低。
3.2.2六味补气胶囊对COPD肺气虚证大鼠细胞因子的防治
与模型组比较，六味补气预防和治疗组IL-1β、IL-6、IL-12降低；IL-10、IL-13、IL-35表达升高。与六味补气组比较，脾氨肽组IL-1β、IL-6表达升高，IL-10、IL-35表达降低；金水宝组IL-12表达升高。
3.2.3六味补气胶囊对COPD肺气虚证大鼠Th细胞的防治
与模型组比较，六味补气预防和治疗组血清IFN-γ、IL-17、Th1/Th2表达降低，IL-4升高；肺组织IL-4mRNA、IL-4蛋白升高、IFN-γmRNA、IL-17蛋白降低。与六味补气组比较，金水宝组血清IL-4降低、血清IL-17、Th1/Th2升高，脾氨肽组IFN-γ、IL-17升高。与六味补气组比较，金水宝组肺组织IL-17升高；脾氨肽组肺组织IL-4蛋白降低，IFN-γ升高。
3.2.4六味补气胶囊对COPD肺气虚证大鼠Treg的防治
与模型组比较，六味补气预防和治疗组外周血CD4+CD25+Treg、CD4+CD25+FoxP3+Treg升高，肺组织FoxP3蛋白表达升高。与六味补气组比较，脾氨肽组、金水宝组CD4+CD25+Treg、CD4+CD25+FoxP3+Treg表达降低；脾氨肽组FoxP3蛋白升高。
3.2.5六味补气胶囊对COPD肺气虚证大鼠DC、HSP70的防治
与模型组比较，六味补气预防和治疗组肺组织DC、HSP70光密度下降、阳性面积率减小。各治疗组之间统计分析显示，未见统计学差异(P>0.05)。
3.2.6六味补气胶囊对COPD肺气虚证大鼠MMPs/TIMPs的防治
与模型组比较，六味补气预防和治疗组肺组织MMP-9、MMP-12表达强度明显降低；TIMP1阳性表达增强。肺组织MMP-9基因和蛋白表达降低，TIMP1基因和蛋白表达升高。与六味补气组比较，脾氨肽组MMP-9光密度值升高，TIMP1降低；金水宝组、脾氨肽组MMP-9蛋白升高，TIMP1蛋白表达降低。
3.2.7六味补气胶囊对COPD肺气虚证大鼠JAKs/STATs的防治
与模型组比较，六味补气预防和治疗组肺组织STAT4阳性面积率、平均光密度降低，STAT6阳性面积率、平均光密度较模型组增大。JAK1mRNA表达及JAK1、p-JAK1、p-STAT3、STAT4蛋白蛋白表达下降；STAT6蛋白升高。与六味补气组比较，金水宝组JAK1、p-JAK1蛋白升高；脾氨肽组JAK1mRNA及JAK1、p-STAT3蛋白升高；脾氨肽组STAT6蛋白升高降低。
3.2.8六味补气胶囊对COPD肺气虚证大鼠T-bet、RORγt、GATA的防治
与模型组比较，六味补气预防和治疗组GATA-3mRNA、蛋白表达升高，T-bet、RORγt mRNA和蛋白表达降低。与六味补气组比较，金水宝组T-betmRNA和蛋白、T-bet/GATA-3、RORγt基因升高，GATA3蛋白降低；脾氨肽组T-bet蛋白、RORγt基因表达升高。
4结论
六味补气胶囊防治COPD肺气虚证大鼠的作用机制如下：
4.1改善COPD肺气虚证大鼠呼吸系统症状体征
中药六味补气胶囊通过改善COPD肺气虚证大鼠呼吸系统症状体征，升高肺功能参数。降低肺系数、肺泡炎积分、病理学总积分。降低肺组织炎性细胞浸润，改善COPD肺气虚证大鼠呼吸系统的症状体征，进一步提高肺功能水平和改善肺组织损伤。
4.2降低COPD肺气虚证大鼠炎症反应
中药六味补气胶囊能够上调COPD肺气虚证大鼠血清IL-10、IL-13、IL-35表达，下调IL-1β、IL-6、IL-12表达水平，降低机体炎性反应，纠正血清细胞因子表达失衡状态，减少炎症介质渗出及对肺组织器官的刺激，改善COPD肺气虚证大鼠呼吸系统症状体征。
4.3调节COPD肺气虚证大鼠Th细胞平衡
中药六味补气胶囊通过上调IL-4表达，下调IFN-γ、IL-17，降低Th17细胞分泌，纠正Th1/Th2细胞平衡，降低炎性细胞分泌，调整肺组织细胞及体液免疫，减轻胞膜通透性，从而改善COPD肺气虚证大鼠呼吸系统症状体征。
4.4上调COPD肺气虚证大鼠Treg表达
中药六味补气胶囊通过上调CD4+Treg、CD4+CD25+Treg、FoxP3mRNA及FoxP3蛋白表达，抑制CD4+CD25+Treg向CD4+CD25-T细胞进一步分化，减少炎症介质的释放，减少炎症反应对组织的损伤，从而改善COPD肺气虚证大鼠呼吸系统症状体征。
4.5降低COPD肺气虚证大鼠DC、HSP70表达
中药六味补气胶囊通过下调DC、HSP70表达，降低炎性细胞对肺组织刺激，从而改善COPD肺气虚证大鼠呼吸系统症状体征。
4.6调节COPD肺气虚证大鼠MMPs/TIMPs系统
中药六味补气胶囊通过上调TIMP1表达，下调MMP-9、MMP-12，纠正MMP-9/TIMP1平衡状态，抑制机体炎症反应，从而改善COPD肺气虚证大鼠呼吸系统症状体征。
4.7调节COPD肺气虚证大鼠JAKs/STATs通路表达
中药六味补气胶囊可能是通过上调STAT6表达，下调JAK1、p-JAK1、p-STAT3表达，调控JAK/STAT信号转导，抑制AKs/STATs信号通路过激活状态，改善气道炎症和气流受限症状，从而改善COPD肺气虚证大鼠呼吸系统症状体征。
4.8调节COPD肺气虚证大鼠T-bet、RORγt、GATA表达
中药六味补气胶囊可能是通过下调T-bet、RORγt表达，上调GATA-3表达，从而改善COPD肺气虚证大鼠呼吸系统症状体征。
1Objective
To observe respiratory symptoms,signs and pulmonary function in COPDrats, and to detect changes of helper T cells (Th), regulatory T cells (Treg), dendritic cells (DC), heat shock protein (HSP70), matrix metalloproteinase system(MMPs/TIMPs),intracellular signal transduction activator of transcription(STAT),JAKs/STATs signaling pathway,T-bet,RORγt,GATA.Observe these indicators intervention,and explore the mechanism of Liuwei Buqi capsules in COPDrats with lung deficiency.2Methods
2.1Animal grouping
The rats were randomly divided into normal group,model group,the controlgroup (hereinafter referred to as blank group),the model in the placebo group(hereinafter referred to as analog security group),Liuwei Buqi capsules prevention group,Liuwei Buqi capsule treatment group,Jinshuibao group,spleen aminopeptidase groups.
2.2model copied
In addition to the normal group,the remaining rats were smoked plus lipopolysaccharide (LPS) tracheal instillation method to esta blish COPD lung deficiency rat model.28days after the model copied, success criteria of COPDmodel based on radiological, pathological changes in lung function and lung tissue.Smoking for3times/day in the first22days.Copy lung deficiencysecondary lung function and symptoms based on the successful model of ratbiology,signs, symptoms of rat biology,signs of change,including whether thelung qi reflect the general situation,activity,hair,body weight,food intake,cough,respiratory frequency,wherein the frequency of the respiratory frequency andrespiratory quiet breathing rats per minute.
2.3Drug Intervention
The rats were randomly divided into normal group,model group,LiuweiBuqi group,Jinshuibao group,spleen aminopeptidase groups of10.In additionto the normal group,the remaining rats were used smoke plus lipopolysaccharide tracheal instillation method to establish the rat model of COPD accompanied lung deficiency.At twenty-eight days after the model successfully copied,administration,dose of the groups:normal group, model group were given normalsaline(0.09g·kg-1),Liuwei Buqi group:Liuwei Buqi capsule(0.4g·kg-1),Jin shuibao group:Jinshuibao capsules (0.495g·kg-1),Spleen group:spleen aminopeptidase(0.33mg·kg-1).Once a day for consecutive30days.
2.4index detection
Observation and testing of the following indicators:Respiratory symptomsand signs,lung function,pathological changes in the lung tissue,serum cytokines,Th1,Th2,Th17cells in peripheral blood Treg expression in lung tissue,DC and HSP70expression in lung tissue,MMPs/TIMPs,JAKs/STATs signaling pathway,T-bet,RORγt,GATA change.
3results
3.1Expression of index and correlation analysis in COPD rat with lungdeficiency
3.1.1Change of respiratory system in COPD rat with lung deficiency
There were appears crouched less dynamic,Arch lying curled summarizedhair,decreased appetite,hair loss of luster,yellow, easy to fall off in COPD rat with lung deficiency.Compared with the control group,body weight was reduced in model group,and respiratory frequency was significantly higher thannormal group, respiratory secretions from the nose and mouth out,be heardand even airway cough and sputum,with the extension of modeling time.Compared with the normal group,the lung function parameters such as FEV0.3,FVC,FEV0.3/FVC was lower;lung coefficient,integral and pathological alveolitis total score were higer in the model group.
3.1.2Changes of factor lung deficiency and correlation analysis in COPD ratExpression of IL-1β, IL-6, IL-12increased,IL-10,IL-13,IL-35of serum wasreduced in COPD lung deficiency rat.lung Function parameters FEV0.3waspositively correlated with IL-10,FEV0.3/FVC was positively correlated withIL-13,PEF was positively correlated with IL-35,alveolitis integral waspositively correlated with IL-6,IL-1β, IL-12were positively correlated withlung pathology integral.Lung function parameters FVC and IL-6werenegatively correlated,FEV0.3/FVC was negatively correlated withIL-1β.Respiratory rate was negatively correlated with IL-13.
3.1.3Changes of cytokines and correlation analysis in COPD ratsIFN-γ,IL-17,Th1/Th2of serum were elevated,IL-4and IL-4protein in lungtissue were decreased,expression of IFN-γ,IL-17protein lung tissue were increased.Lung function parameters such as FEV0.3/FVC were positively correlatedwith IL-4,Th1/Th2were positively correlatedalveolitis integral.FEV0.3andIFN-γ,IFN-γmRNA of lung tissue were negatively correlated,FVC and IFN-γprotein was negatively cor related,FEV0.3./FVC was negatively correlated withTh1/Th2,PEF and IL-17protein was negatively correlated;alveolitis integraland IL-4protein was negatively correlated.IFN-γ,IL-17protein werepositively correlated with lung pathology integral.
3.1.4Changes of Treg and correlation analysis in COPD rats
CD4+CD25+Treg,CD4+CD25+FoxP3+Treg were reduced, and expression of FoxP3protein in lung tissue was reduced.Lung function parameters such asFEV0.3was positively correlated with FoxP3protein,FVC was positively correlated with CD4+CD25+Treg,FEV0.3/FVC and CD4+CD25+FoxP3+Treg was positively correlated.Alveolitis integral and CD4+CD25+FoxP3+Treg was negatively correlated.Pulmonary pathology integral and FoxP3protein was negatively correlated.
3.1.5Changes of DC,HSP70and correlation analysis in COPD rat
Expression of DC,HSP70were increased,the average optical density was increased.Alveolitis integral was positive correlation with lung pathology,DC,HSP70optical density values,respectively.Lung function parameters suah asFEV0.3was negatively correlated with DC optical density.PEF,FEV0.3/FVC were negatively correlated with HSP70optical density.
3.1.6Changes of MMPs/TIMPs and correlation analysis
Expression of MMP-9,MMP-12in lung tissue were significantly enhanced,and MMP-9gene and protein expression in lung tissue were increased,TIMP1mRNA and protein expression were decreased.Lung function parameters FEV0.3/FVC and TIMP1protein was positively correlated,alveolitis integral waspositively correlated with optical density of MMP-9,lung pathology integraloptical density and MMP-12was positively correlated.FEV0.3and MMP-9protein was negatively correlated,FVC and the optical density of MMP-9wasnegatively correlated,FEV0.3/FVC and MMP-12optical density was negativelycorrelated.Respiratory rate was negatively correlated with TIMP1mRNA.
3.1.7Changes of JAKs/STATs and correlation analysis
JAK1,STAT3mRNA expression,and STAT4positive area ratio were increases,JAK1,p-JAK1,STAT3,p-STAT3protein expression were increased.FEV0.3/FVC was positively correlated with STAT6, respiratory rate and p-STAT3protein was positively correlated, integral alveolitis,pulmonary pathology integraland STAT4were positively correlated.FEV0.3and p-JAK1negative corre lation,FVC was negatively correlated with STAT3mRNA,FEV0.3/FVC was negatively correlated with STAT3,alveolitis points and STAT6,p-JAK1werenegatively correlated.
3.1.8Changes of T-bet,RORγt,GATA and correlation analysis
GATA-3mRNA,protein expression were decreased,and T-bet,mRNA andprotein expression of RORγt were increased in COPD rat.Compared with thenormal group,T-bet,RORγt protein and T-bet/GATA-3ratio were increased,andGATA-3protein was reduced.FEV0.3and lung tissue GATA-3mRNA waspositive correlation,respiratory rate and RORγt protein was positively correlated,alveolitis integral and T-bet protein was positively correlated,and lungpathology score and T-bet mRNA was positively correlated (P<0.05orP<0.01).FEV0.3and RORγt protein was negatively correlated,FEV0.3/FVC andT-bet mRNA was negatively correlated,PEF and RORγt mRNA was negatively correlated,pulmonary pathology integral and GATA-3protein wascorrelated negatively (P<0.05).
3.2Liuwei Buqi Capsules on prevention and treatment in COPD rats
3.2.1Liuwei Buqi Capsules on prevention of respiratory in rat
Compared with the model group,Liuwei Buqi prevention and treatment ofrats crouched less dynamic,Archlying curled summarized hair,decreasedappetite,hair loss of luster,yellow, easy to fall off the symptoms and signs,bodyweight increased respiratory rate reduced.Liuwei Buqi prevention and treatment of lung function parameters in rats FEV0.3,FVC,FEV0.3/FVC increased.Liuwei Buqi prevention and treatment group rat tracheal,bronchial inflammatory cell infiltration, but reduce the number compared with the modelgroup;epithelial necrosis,loss;less destruction of the alveolar septa,some alveolar expansion into vesicles of different sizes;cilia arranged in a more regular.Liu Wei bu qi prevention and treatment of lung coefficient,integral and pathological alveolitis total score lower.Group compared with Liuwei Buqi,spleen aminopeptidase group FEV0.3,FEV0.3/FVC,PEF lower;pathology total score comparison,Liuwei Buqi group were lower than the spleen aminopeptidase.
3.2.2Liuwei Buqi Capsules on cytokines in ratCompared with the model group,IL-1β,IL-6,IL-12were reduced in LiuweiBuqi prevention and treatment group;IL-10,IL-13,IL-35expression were increased.Compared with Liuwei Buqi,IL-1β,IL-6expression were increased inspleen aminopeptidase group,IL-10, IL-35expression were decreased,expression of IL-12was increased in Jinshuibao group.
3.2.3Liuwei Buqi Capsules on Th cell lung
Compared with the model group,expression of IFN-γ,IL-17,Th1/Th2ofserum were decreased in Liuwei Buqi prevention and treatment,IL-4,IL-4mRNA and IL-4protein levels was increased;IFN-γ mRNA,IL-17protein inlung tissue were reduced.Compared with Liuwei Buqi group, IL-4in serumwas decreased IL-17,Th1/Th2were increased in spleen aminopeptidasegroup,and IFN-,IL-17were increased in Jinshuibao.Compared with LiuweiBuqi group,IL-17in lung tissue was increased in Jinshuibao;andIL-4,IFN-γprotein in lung tissue was reduced spleen aminopeptidase.
3.2.4Liuwei Buqi Capsules on Prevention of Treg in rats
Compared with the model group,CD4+CD25+Treg,CD4+CD25+FoxP3+Tregof peripheral blood were increased in Liuwei Buqi prevention and treatment,FoxP3protein expression in lung tissue was increased. Compared with LiuweiBuqi group,CD4+CD25+Treg,CD4+CD25+FoxP3+Treg,FoxP3protein expression were reduced in spleen aminopeptidase group and Jinshuibao group.
3.2.5Liuwei Buqi Capsules on DC,HSP70prevention
Compared with the model group,DC,HSP70optical density of lung tissuewere decreased,the positive area ratio was decreases in Liuwei Buqi prevention and treatment.Statistical analysis between treatment groups showed nosignificant difference(P>0.05).
3.2.6Liuwei Buqi Capsules on MMPs/TIMPs prevention
Compared with the model group,MMP-9,MMP-12expression level of lungtissue were significantly decreased in Liuwei Buqi prevention and treatment;TIMP1expression was enhanced.MMP-9gene and protein expression in lungtissue were reduced,TIMP1mRNA and protein expression were increased.Compared with Liuwei Buqi group, MMP-9optical density values was increased in spleen aminopeptidase group,TIMP1was lower in Jinshuibao group,MMP-9protein was increased,TIMP1protein expression was decreased inspleen aminopeptidase group.
3.2.7Liuwei Buqi Capsules on COPD JAKs/STATs prevention
Compared with the model group,STAT4positive area ratio,the mean opticaldensity of lung tissue were decreased in Liuwei Buqi prevention and treatment.JAK1mRNA expression of JAK1,p-JAK1,p-STAT3STAT4protein expression were decreased.Compared with Liuwei Buqi group,JAK1,p-JAK1protein levels; spleen aminopeptidase group JAK1mRNA and JAK1,p-STAT3protein levels,STAT6protein concentration were decreased in Jinshuibaogroup and spleen aminopeptidase group.
3.2.8Liuwei Buqi Capsules on T-bet,RORγt,GATA prevention
Compared with the model group,GATA-3mRNA,protein expression wereelevated in Liuwei Buqi prevention and treatment group,T-bet,RORγt mRNAand protein expression were decreased.Compared with Liuwei Buqi group,T-bet mRNA and protein,T-bet/GATA-3,RORγt gene increases,GATA3proteinwere reduced in Jinshuibao group;RORγt gene,T-bet protein expression wereincreased in spleen aminopeptidase group.
4Conclusion
Mechanisms of Liuwei Buqi capsules on prevention in COPD rats with lungdeficiency as follows.
4.1Improve symptoms and signs of respiratory in COPD rats
Liuwei Buqi capsules can improve symptoms and signs of respire atory inrats,increased lung function parameters.Reduced lung coef ficient,alveolitispoints,pathology total points.Reduced lung tissue inflammatory cell infiltration,improve COPD lung deficiency rat respiratory symptoms and signs,tofurther enhance the level and improve lung function in lung tissue damage.
4.2Reduce lung inflammation in COPD rats
Liuwei Buqi capsules can increase IL-10,IL-13,IL-35expression, decreaseIL-1β, IL-6,IL-12expression levels in serum,reduce the body's inflammatoryres ponse,corrected serum cytokine expression imbalance,reduceinflammatory mediators and lung tissues and organs oozing stimulation toimprove COPD lung deficiency rat respiratory symptoms and signs.
4.3Regulate Th cell lung balance in COPD rats
Liuwei Buqi capsules can upregulate the expression of IL-4,down IFN-γ,IL-17,reduce Th17cells secrete to correct the Th1/Th2balance,reduce the secretion of inflammatory cells,regulating cell-mediated immunity and humoralimmune function,reduced lung tissue cell membrane permeability,thus improving the signs and symptoms of COPD lung respiratory deficiency in rats.
4.4Upregulate expression of Treg in COPD rats
Liuwei Buqi Capsules can upregulate CD4+Treg,CD4+CD25+Treg,FoxP3mRNA and FoxP3protein expression,inhibit of CD4+CD25+Treg to CD4+CD25-T cells,reduce the release of inflammatory mediators, reducing the inflammatory response to tissue damage,thereby improving the signs and symptoms of COPD lung respiratory deficiency in rats.
4.5Reduce DC,HSP70expression in COPD rats
Liuwei Buqi capsules can down DC,HSP70expression,reduce inflam matory cell lung tissue stimulation,thereby improving COPD lung deficiency ratrespiratory symptoms and signs.
4.6Adjust MMPs/TIMPs system in COPD rats
Liuwei Buqi capsules can upregulate the expression of TIMP1,down MMP-9,MMP-12,correct MMP-9/TIMP1balance,inhibit the body's inflammatoryresponse,thus improving COPD symptoms and signs of respir atory lung deficiency in rats.
4.7Adjust JAKs/STATs pathway expression in COPD rats
Liuwei Buqi capsules may be regulated by STAT6expression,down JAK1,p-JAK1,p-STAT3expression and regulation of JAK/STAT signal,inhibit JAKs/STATs signaling pathway active,improving airway inflamemation and airflowlimitation symptoms,thereby improving COPD symptoms and signs of respiretory lung deficiency in rats.
4.8Adjust T-bet,RORγt,GATA expression in COPD rats
Liuwei Buqi capsules may be reduced by T-bet,RORγt expression, increaseed expression of GATA-3,thereby improving COPD lung deficiency rat respiretory symptoms and signs.
引文
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