Optimization Of Bufei Yishen Recipe Based On The Curative Effect Of COPD And The Mechanism Of Inhibiting Airway Mucus Hypersecretion

Objective1 To evaluate the effect of Bufei Yishen Zufen formula on chronic obstructive pulmonary disease(COPD)rat model induced by cigarette smoke exposure combined with bacterial infection,and to determine the safe and effective dose of Bufei Yishen Zufen formula.2 To evaluate the effects of Bufei Yishen Zufen Jingjian formulas based on the pharimacodynamic function,and to determine the optimal Bufei Yishen Zufen Jingjian formula.3 To observe the effect of Bufei Yishen Zufen Jingjian formula on improving mucus hypersecretion on COPD rats,and to preliminarily explore its mechanism via the EGFR/PI3K/mTOR pathway.MethodExperiment 1:84 SD rats were randomly divided into the normal group(Normal),model group(Model),Bufei Yishen formula group(BYF),high dose of Bufei Yishen Zufen group(BYZF-H),medial dose of Bufei Yishen Zufen group(BYZF-M),low dose of Bufei Yishen Zufen group(BYZF-L)and aminophylline group(APL).The COPD model was prepared by cigarette smoke exposure combined with Klebsiella pneumoniae infection during weeks 1-12,and the corresponding drugs were administered during weeks 13-20.Pulmonary function was detected by a whole body plethysmography and a PFT measurement system.Pathological technique was used to observe the pathological changes of the lung tissues.The levels of interleukin(IL)-1β in the bronchoalveolar lavage fluid(BALF),IL-6 and matrix metalloprotein(MMP)-9 in the serum were detected by enzyme-linked immunosorbent assays(ELISAs).A total antioxidant capacity(T-AOC)kit and a lipid peroxidation(LPO)kit were used to determine the changes T-AOC and LPO.R-value comprehensive evaluation method was used to evaluate the effects of Bufei Yishen formula,different doses of Bufei Yishen Zufen formula and aminophylline on COPD rats.Experiment 2:144 SD rats were randomly divided into the normal group(Normal),model group(Model),Bufei Yishen formula group(BYF),Bufei Yishen Zufen formula group(BYZF),Bufei Yishen Zufen Jingjian 1 group(BYZF-J1),Bufei Yishen Zufen Jingjian 2 group(BYZF-J2),Bufei Yishen Zufen Jingjian 3 group(BYZF-J3),Bufei Yishen Zufen Jingjian 4 group(BYZF-J4),Bufei Yishen Zufen Jingjian 5 group(BYZF-J5),Bufei Yishen Zufen Jingjian 6 group(BYZF-J6),aminophylline group(APL)and acetylcysteine group(NAC).The COPD model was prepared during weeks 1-8,and the corresponding drugs were administered to the rats during weeks 9-16.Pulmonary function and histopathology of lung tissues were detected.The levels of tumor necrosis factor(TNF)-α IL-1β and MUC5AC in the BALF;IL-6,MMP-9 and tissue inhibitor of metalloproteinase 1(TIMP-1)in the serum were detected by ELISAs.The changes of T-AOC and LPO in the serum were determined by a T-AOC kit and a LPO kit.The expression of Collagen Ⅰ and Collagen Ⅲ in the lung tissues were detected by immunohistochemistry(IHC).R-value comprehensive evaluation method was used to evaluate the effects of Bufei Yishen formula,Bufei Yishen Zufen formula,Bufei Yishen Zufen Jingjian 1-6,aminophylline and acetylcysteine on COPD rats.Experiment 3:Pathological techniques were used to observe pathological changes of the airway in the normal group(Normal),model group(Model),Bufei Yishen Zufen Jingjian group(BYZF-J),and acetylcysteine group(NAC).The expression of MUC5AC,MUC5B,EGFR,Akt and p-mTOR were observed by IHC.Real-time quantitative polymerase chain reaction(qPCR)was used to detect the mRNA of MUC5AC,MUC5B,EGFR,PI3K,Akt,and mTOR in the airway and western blotting(WB)technique was used to detect the protein expression of EGFR,PI3K,Akt,mTOR,p-EGFR,p-PI3K,p-Akt and p-mTOR in the airways.ResultExperiment 1:In terms of pulmonary function,compared with the normal group,the tidal volume(VT),minute volume(MV),peak expiratory flow rate(PEF),forced vital capacity(FVC),forced exhalation in 0.1 second volume(FEV0.1),and FEV0.1/FVC in the model group were decreased significantly(P<0.05,P<0.01),while the functional residual capacity(FRC)increased(P<0.01).The R-value comprehensive evaluation showed that each treatment group had significant improvement on pulmonary function in COPD rats(P<0.05,P<0.01),and the intensity was ranked as following:BYZF-M,BYF,APL,BYZF-H and BYZF-L.The lung pathology of model rats showed that the alveolar cavity was enlarged and the bronchiole basement membrane was thickened.There was large number of inflammatory cells in the bronchial and alveolar space.The average alveolar intercept(mean linear intercept,MLI)in the model rats was significantly increased than that in the normal(P<0.01),and the mean alveolar number(MAN)was significantly lower(P<0.01).Compared with the model group,the lung tissue pathology in the treatment groups was improved,and the MLIs in the BYF,BYZF-H,BYZF-M and APL groups were significantly decreased(P<0.01,P<0.05).The MAN of each treatment group was significantly increased(P<0.01,P<0.05).R-value comprehensive evaluation method was used for the 15 indicators including pulmonary function(VT,MV,PEF,FVC,FEV0.1,FEV0.1/FVC,FRC),lung histopathology(MLI,MAN),ratio of neutrophil in the BALF,inflammatory factors(IL-1β,IL-6),MMP-9,T-AOC and LPO to evaluate the effects of the treatments.Results showed that each treatment group had significant effect on COPD model rats(P<0.05,P<0.01).The effect was ranked as following:BYZF-H,BYZF-M,BYF,BYZF-L and APL.Experiment 2:In terms of pulmonary function,R-value comprehensive evaluation showed that pulmonary function in the BYF,BYZF,BYZF-J1,BYZF-J2,BYZF-J3,BYZF-J5,BYZF-J6,APL and NAC groups were significantly improved compared with that in the normal(P<0.05,P<0.01).The effect was ranked as following:APL,NAC,BYZF,BYF,BYZF-J2,BYZF-J5,BYZF-J6,BYZF-J3,BYZF-J1 and BYZF-J4.The pathological changes of lung tissue and MLI in all treatment groups were improved than those in the model(P<0.01);MANs in the BYF,BYZF,BYZF-J1,BYZF-J3,BYZF-J5,BYZF-J6 groups were significantly increased than that in the model(P<0.05,P<0.01).In terms of inflammatory factors,the levels of IL-1β,TNF-α in the BALF and IL-6 in the serum were significantly increased in COPD rats compared with those in the normal rats(P<0.05,P<0.01).Compared with the model group,levels of TNF-α in the BYZF,BYZF-J3,and BYZF-J6 groups were significantly decreased(P<0.05).The level of IL-1β was significantly decreased in the BYF,BYZF-J3,BYZF-J4,BYZF-J5,BYZF-J6,and NAC groups(P<0.05,P<0.01).The level of IL-6 was significantly decreased in the BYF,BYZF-J1--BYZF-J6,and NAC groups(P<0.05,P<0.01).R-value comprehensive evaluation was used to evaluat the effects of the drugs on inhibiting inflammation and oxidative stress,and the order of effect was:BYZF,BYZF-J6,BYZF-J3,BYF,BYZF-J4,BYZF-J5,BYZF-J1 BYZF-J2,NAC and APL.As for the improvement of collagen deposition and metalloproteinase imbalance in COPD rats,the comprehensive evaluation of R-value showed that the BYZF,BYZF-J1,BYZF-J5 and NAC groups were significantly improved than the model group(P<0.05,P<0.01).R-value comprehensive evaluation method was used for all 19 indicators including pulmonary function(VT,MV,PEF,EF50,FVC,FEV0.1,FEV0.1/FVC),lung histopathology(MLI,MAN),levels of IL-1β,TNF-α,IL-6,MMP-9,TIMP-1,T-AOC,LPO,MUC5AC,and expression of Collagen Ⅰ and Collagen Ⅲ to comprehensively evaluate the effect of each treatment on COPD rats.The treatments all had significant effects on COPD rats(P<0.05,P<0.01),and the effect was ranked as BYZF,BYZF-J5,NAC,BYF,BYZF-J3,BYZF-J6,BYZF-J2,BYZF-J1,APL and BYZF-J4.Experiment 3:Airway pathology showed that the basement membranes in the model group were thickened and the submucosal glands were enlarged.The arrangement of epithelial cells and cilia were disordered,and the bronchioles were narrow.The AB-PAS staining showed that in the model group,a large number of blue-stained goblet cells were observed in the airway epithelium.The pathological changes of airway in the NAC.and BYZF-J groups were alleviated,and the improvement was more obviously in the BYZF-J group.Compared with the normal group,the expression of MUC5AC,MUC5B in the lung tissues of the model rats were significantly increased(P<0.01).Compared with the model group,the protein and mRNA expressions of MUC5AC,MUC5B in the NAC and BYZF-J groups were significantly decreased(P<0.01).Compared with the normal group,the expression of EGFR,Akt,mTOR,and PI3K mRNA in the model rats airways were significantly increased(P<0.05,P<0.01).The expressions of mRNA of EGFR,Akt and mTOR in the NAC group and mRNA of EGFR,Akt,mTOR and PI3K in the BYZF-J group were significantly lower than those in the model groups(P<0.05,P<0.01).The expression of EGFR,Akt,PI3K,mTOR and phosphorylated proteins in the airway tissues of COPD rats were significantly higher than those in the normal rats(P<0.05,P<0.01).Compared with the model group,NAC could significantly reduce the expression of EGFR,PI3K,Akt,p-EGFR,p-PI3K,p-Akt and p-mTOR(P<0.05,P<0.01)and BYZF-J could significantly decreased the expression of EGFR,PI3K,Akt,mTOR,p-E GFR,p-PI3K,p-Akt and p-mTOR(P<0.05,P<0.01).Conclusion1 Bufei Yishen Zufen formula has curative effect on COPD rats and the high dose and medium doses are excellent,which can significantly improve the pulmonary function and lung pathology,reduce inflammation and oxidative stress on C.OPD rats.There is no difference between Bufei Yishen Zufen formula and Bufei Yishen formula.2 Each of the Bufei Yishen Zufen Jingjian formula can improve the pulmonary function and lung tissue pathology on COPD rats,as well as reduce inflammation,oxidative stress and collagen deposition in lung tissue,and improve protease/anti-protease imbalance.Among them,the BYZF-J1,BYZF-J2,BYZF-J3,BYZF-J5,BYZF-J6 showed equivalent curative effect with Bufei Yishen Zufen formula and Bufei Yishen formula.The optimized Jingjian formula is BYZF-J5 that named as the Bufei Yishen Zufen Jingjian formula.3 Bufei Yishen Zufen Jingjian formula effectively improved the airway remodeling,reduced the number of goblet cells and inhibited the hypertrophy of submucosal gland on COPD rats.It significantly reduced the expression of MUC5AC and MUC5B that inhibited the mucus hypersecretion effectively.The mechanism may be related to the regulation of the EGFR/PI3K/mTOR pathway.4 The efficacy of Bufei Yishen Zufen formula and Bufei Yishen Zufen Jingjian formula on COPD needs further clinical demonstration and the mechanism of inhibiting airway mucus hypersecretion needs further study.