Mechanism Research Of Prevention And Treatment Of Liuwei Buqi Capsule On Chronic Obstructive Pulmonary Disease Rat Model With Fei-qi Deficiency Syndrome

1ObjectiveTo 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.2Methods2.1Animal groupingThe 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 copiedIn 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 InterventionThe 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 detectionObservation 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.3results3.1Expression of index and correlation analysis in COPD rat with lungdeficiency3.1.1Change of respiratory system in COPD rat with lung deficiencyThere 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 ratsCD4+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 ratExpression 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 analysisExpression 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 analysisJAK1,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 analysisGATA-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 rats3.2.1Liuwei Buqi Capsules on prevention of respiratory in ratCompared 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 lungCompared 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 ratsCompared 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,HSP70preventionCompared 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 preventionCompared 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 preventionCompared 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 preventionCompared 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.4ConclusionMechanisms 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 ratsLiuwei 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 ratsLiuwei 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 ratsLiuwei 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 ratsLiuwei 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 ratsLiuwei 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 ratsLiuwei 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.