Clinical Observation Of Shenqi Huatan Decoction In The Treatment Of AECOPD And The Effect Of Serum Containing Shenqi Huatan Decoction On RhoA/ROCK Signaling Pathway Of Airway Smooth Muscle Cells

1 BackgroundChronic obstructive pulmonary disease（COPD）is one of the main causes of chronic diseases and death in the world.In recent years,the research on COPD has been a hot spot in the respiratory field.According to the global initiative on chronic obstructive pulmonary disease（COPD）,issued in 2021,COPD is the third leading cause of death in the world,and it is predicted that its incidence rate and mortality rate will rise further in the next few decades.At present,the pathogenesis of COPD is not completely clear,its etiology is due to the complex interaction between environmental factors and genetic factors.Exposure to inhaled pollutants or cigarette smoke can activate alveolar epithelium and inflammatory cells to cause airway inflammation,which is the main pathogenesis of COPD.This study is divided into three parts:theoretical study,clinical study and in vitro study.Using the traditional medical theory and modern molecular medical technology,we carried out the correlation research on COPD,a major disease.Based on the theoretical research,the relevant literature on the etiology and pathogenesis of chronic obstructive pulmonary disease（COPD）was extracted by classification,and the pathogenesis and treatment principles and methods of COPD were sorted out.Through clinical observation and in vitro research,the internal mechanism of the treatment of COPD with Yiqi Huoxue Huatan method was clarified,so as to provide an objective basis for the further application of Yiqi Huoxue Huatan method in the treatment of COPD.2 ObjectiveUnder the guidance of traditional Chinese medicine thought of"the unity of holistic concept and syndrome differentiation",this paper discusses the internal pathogenesis of COPD in TCM occurrence and development.Through theoretical research,it reveals the pathological essence of COPD with"deficiency of origin and excess of origin"and the therapeutic significance of Supplementing Qi,activating blood circulation and resolving phlegm.Objective to observe the efficacy and safety of Yiqi Huoxue Huatan method in the treatment of acute exacerbation of chronic obstructive pulmonary disease（COPD）with Qi deficiency,blood stasis and phlegm syndrome,and analyze the correlation between TGF-β1 and Rock1 and other indicators.The effects of Shenqi Huatan Decoction containing serum on the proliferation,migration,apoptosis,F-actin formation and cytoskeleton reorganization of airway smooth muscle cells stimulated by TGF-β1 were observed by using Rho A/rock and MAPK/ERK signaling pathways in vitro.The expression changes of molecules related to the above pathways were detected after drug intervention,and the effects of Shenqi Huatan Decoction on Qi metabolism were observed Objective to investigate the role of airway smooth muscle cells in the treatment of COPD,and to explore the molecular mechanism of airway remodeling,so as to provide scientific basis for the adjuvant treatment of COPD with traditional Chinese medicine.3 MethodsTheoretical research part:through the collection and screening of literature related to the etiology and pathogenesis of COPD,extract the pathogenesis of COPD occurrence and development of traditional Chinese medicine,reveal the essence and treatment of COPD,and elaborate the structural function of Rho A/rock signal pathway and TGF-β1 and its correlation with COPD through literature research,so as to provide reference for further clinical research and systemic research of qihuatan formula in the treatment of COPD In vitro experiments provide theoretical basis.Clinical research part:according to the principle of randomized controlled design,60 patients with AECOPD were divided into control group and treatment group,30 cases in each group.The control group was given basic treatment such as controlled oxygen therapy,antibiotics,antispasmodic and antiasthmatic drugs and expectorant therapy,while the treatment group was given Shenqi Huatan Decoction on the basis of which,both groups were treated for 10 days.Before and after treatment,the scores of symptoms and signs of the two groups were recorded to evaluate the curative effect of TCM syndromes,the dyspnea index（MMRC）and lung function（FEV1%）were monitored before and after treatment PRED,FEV1/FVC),blood gas analysis（Pa CO₂,Pa O₂）,infection index（WBC,neut%,hs CRP）,coagulation index（FBG,DD level）;in addition,the TGF-β1 and Rock1 of patients before and after treatment were detected,and the correlation between TGF-β1 and other related indexes of patients in the treatment group before treatment was analyzed,so as to lay the foundation for the mechanism research of follow-up cell experiment.In vitro research:this project is supported by the National Natural Science Foundation of China（No.81473675）.The rat serum containing Shenqi Huatan formula was used to test airway smooth muscle cells in vitro.In Experiment 1,airway smooth muscle cells were successfully isolated from the lungs of neonatal SD rats,and the optimal concentration of Shenqi Huatan formula（SQHT）on airway smooth muscle cells stimulated by TGF-β1 was screened from different levels,so as to provide the basis for the follow-up study of signal pathway mechanism.In Experiment 2,the following experiments were carried out according to the optimal concentration of serum containing SQHT selected in Experiment 1.CCK8 method was used to detect the proliferation ability of cells;Transwell method was used to detect the migration ability of cells;flow cytometry was used to detect the apoptosis ability of cells,and Western blot was used to detect the apoptosis ability of cells The levels of Bax and Bcl-2 were detected by RT-q PCR,and the changes of F-actin were detected by immunofluorescence.In Experiment 3,Western blot and RT q PCR were used to detect the key proteins in Rho A/rock and MAPK/ERK signaling pathways,including the changes of Rho A,Rock1,Rock2,p-ERK and ERK;in Experiment 4,in order to further prove that SQHT plays an important role in airway smooth muscle cell remodeling and inflammation,Western blot was used At the same time,the changes of MMP9 and TIMP-1 protein in the cell supernatant were verified by ELISA method,so as to provide scientific basis for the clinical application of Yiqi Huoxue Huatan method in the treatment of COPD.4 Results4.1 Theoretical researchThe main characteristic of COPD is the deficiency of origin and excess,the deficiency of origin is the deficiency of right,and the deficiency of lung,spleen and kidney qi is the main.It is characterized by the combination of phlegm and blood stasis.Qi deficiency,blood stasis and phlegm obstruction are the key to the pathogenesis of lung distension.The fundamental treatment of lung distension is to replenish qi,activate blood circulation and remove phlegm.Rho A/ROCK is one of the most common dysfunctional signaling pathways in COPD,and MAPK/ERK signaling pathway plays a role downstream.TGF-β1 stimulation is closely related to airway remodeling and airway smooth muscle in COPD.Shenqi Huatan formula has the effect of Supplementing Qi,activating blood circulation and resolving phlegm,so it may mediate the above two signaling pathways to intervene the stimulation of TGF-β1 to treat COPD.4.2 Clinical research4.2.1 Comparison between groups before treatmentBefore treatment,there was no significant difference in baseline data（gender,age,course of disease and severity）,symptom and sign score,MMRC,FEV1%PRED,FEV1/FVC,Pa CO₂,Pa O₂,WBC,neut%,hs CRP,FBG,DD,TGF-β1 and Rock1levels between the two groups（P>0.05）,which proved that the clinical data were comparable.4.2.2 Comparison of curative effect after treatment4.2.2.1 Effect of Yiqi Huoxue Huatan method on TCM SyndromeIntra group comparison after treatment:the scores of cough,wheezing,sputum volume,sputum state,diet,cyanosis of lips,chest tightness,fatigue and pulmonary rales in the two groups were statistically significant（P<0.01）.Comparison between groups after treatment:compared with the control group,the single score of cough,sputum volume,sputum state,diet,cyanosis of lips,fatigue and pulmonary rales in the treatment group had statistical significance（P<0.05）,while the single score of chest tightness and wheezing had no statistical significance（P>0.05）.In terms of effective rate,in the treatment group,16 cases were markedly effective,12 cases were effective and 2 cases were ineffective,the total effective rate was 93.33%;in the control group,9 cases were markedly effective,17 cases were effective and 4 cases were ineffective,the total effective rate was 86.67%.After treatment,the total effective rate of the two groups was similar（P>0.05）,but the difference between the two groups was statistically significant（P<0.01）.4.2.2.2 Effect of Yiqi Huoxue Huatan method on dyspnea indexIntra group comparison after treatment:the MMRC scores of the two groups were statistically significant（P<0.01）.After treatment,the MMRC score of the two groups was statistically significant（P<0.05）.4.2.2.3 Effect of Yiqi Huoxue Huatan method on lung function of patientsIntra group comparison after treatment:FEV1%PRED and FEV1/FVC of the two groups were statistically significant（P<0.01）.Comparison between groups after treatment:FEV1%PRED and FEV1/FVC in the treatment group were statistically significant（P<0.01）.4.2.2.4 Effect of Yiqi Huoxue Huatan method on WBC,net%,hs CRP,TGF-β1 and Rock1After treatment,there were significant differences in WBC,net%,hs CRP,TGF-β1 and Rock1 between the two groups（P<0.01）.Comparison between the two groups after treatment:WBC,net%,hs CRP,TGF-β1,Rock1 indexes of the two groups were statistically significant（P<0.01）.4.2.2.5 Effect of Yiqi Huoxue Huatan method on blood gas analysis indexesIntra group comparison after treatment:Pa O₂ and Pa CO₂ of the two groups were significantly higher than those before treatment（P<0.01）.Comparison between the two groups after treatment:the indexes of Pa O₂and Pa CO₂ in the two groups were statistically significant（P<0.01）.4.2.2.6 Effect of Yiqi Huoxue Huatan method on coagulation indexesIntra group comparison after treatment:FBG and DD of the two groups were significantly higher than those before treatment（P<0.01）.After treatment,the FBG and DD indexes of the two groups were statistically significant（P<0.01）.4.2.3 Safety evaluation of Yiqi Huoxue Huatan methodThere was no significant difference in liver and kidney function between the two groups before and after treatment,and there were no obvious adverse reactions such as rash,nausea and vomiting,allergy,bleeding tendency in the treatment group.4.2.4 Correlation analysis between TGF-β1 and various indexesBefore treatment,the serum TGF-β1 level was positively correlated with WBC（r=0.466,P=0.009）,neutral%（r=0.450,P=0.012）,hs-CRP（r=0.388,P=0.034）,Pa CO₂（r=0.432,P=0.017）and DD（r=0.394,P=0.031）,and negatively correlated with Pa O₂（r=-0.467,P=0.009）,FEV1%PRED（r=-0.379,P=0.039）and FEV1/FVC（r=-0.344,P=0.045）,There was no significant correlation with FBG（r=0.156,P=0.409）.Combined with the above results,it is suggested that TGF-β1 may be involved in the inflammation,coagulation and respiratory function changes of AECOPD.Further study also showed that the serum TGF-β1 level of AECOPD patients was positively correlated with the Rock1（r=0.309,P=0.043）in Rho A/rock,an important signaling pathway of COPD.4.3 In vitro study4.3.1 Experimental Study 1After DAPI immunochemical staining,according to the fluorescence color of the second fluorescent antibody,the nucleus of the cells was blue,while the positive expression ofα-smooth muscle actin was green,and the main expression area was cytoplasm.The cells were identified as ASMCs with good specificity and activity,which could be used for subsequent experiments.Different concentrations of SQHT containing serum were given to intervene ASMCs stimulated by TGF-β1.The results showed that compared with the blank group,the TGF-β1 model group had statistical significance in cell proliferation,migration and cytoskeleton reorganization（P<0.01）;compared with the model group,except that there was no significant difference in the proliferation ability of 5%SQHT（P>0.05）,5%,10%and 20%SQHT groups had statistical significance There was significant difference between5%and 10%SQHT（P<0.05,P<0.01）;there was no significant difference between10%and 20%SQHT（P>0.05）.Compared with the blank group,Rho A,Rock1,Rock2,Bax and Bcl-2 among the treatment groups（P<0.01）.The effects of 10%and20%treatment groups on Rho A,Rock1,Rock2,Bax and Bcl-2 were significantly higher than those of 5%treatment group（P<0.01）,and there was no significant difference between 10%and 20%treatment groups on Rho A,Rock1,Rock2,Bax and Bcl-2（P>0.05）.In conclusion,considering the cytotoxic effect,we chose 10%SQHT serum as the optimal concentration for subsequent experiments.4.3.2 Experimental Study 2Drugs can inhibit the proliferation,migration and cytoskeleton reorganization of airway smooth cells.Compared with the blank group,the model group had statistical significance in proliferation,migration and skeleton reorganization（P<0.01）;compared with the model group,the treatment groups had statistical significance in proliferation,migration and skeleton reorganization（P<0.01）;compared with the treatment groups,there was no significant difference in inhibition of proliferation,migration and anti apoptosis between the 10%SQHT group and fasudil group（P>0.05）In terms of cytoskeleton reorganization,10%SQHT group was better than fasudil group（P<0.01）,and they had synergistic effect in inhibiting cell proliferation,migration,cytoskeleton reorganization and anti apoptosis expression,and 10%SQHT+fasudil group was the best（P<0.05,P<0.01）.Compared with the blank group,Bax,Bcl-2,Bax m RNA and bcl-2 m RNA in the model group were significantly different（P<0.01）;compared with the model group,except fasudil group,Bax,Bcl-2,Bax m RNA and bcl-2 m RNA in the three treatment groups were significantly different（P<0.01）;compared with the treatment group,Bax,Bcl-2,Bax m RNA and bcl-2 m RNA in 10%SQHT group were significantly different（P<0.01）The expression of Bax,Bcl-2,Bax m RNA and bcl-2 m RNA in the combined treatment group were better than those in the 10%SQHT and fasudil groups（P<0.01）.4.3.3 Experimental Study 3Drugs can regulate the abnormal expression of key proteins in Rho A/ROCK and MAPK/ERK signaling pathways.Compared with the blank group,Rho A,Rock1,Rock2,Rho A m RNA,Rock1 m RNA,Rock2 m RNA,p-ERK,ERK and p-ERK/ERK were significantly increased in the model group（P<0.01）.Compared with the model group,Rho A,Rock1,Rock2,Rho A m RNA,Rock1 m RNA,Rock2 m RNA,p-ERK,ERK and p-ERK/ERK in each treatment group were significantly decreased（P<0.01）.Compared with fasudil,the 10%SQHT treatment group was superior to fasudil in inhibiting Rho A,Rock1,Rock2,Rho A m RNA,Rock1 m RNA,Rock2 m RNA,p-ERK,ERK and p-ERK/ERK（P<0.05,P<0.01）;the combined treatment group was superior to the other two treatment groups in inhibiting Rho A,Rock2,Rho A m RNA,Rock1 m RNA,p-ERK and ERK（P<0.01）.There was no difference in the inhibition of Rock1,Rock2 m RNA and p-ERK/ERK between the combined treatment group and SQHT group（P>0.05）,and it was better than fasudil group（P<0.01）.4.3.4 Experimental study 4Drugs can regulate the abnormal expression of snail,slug,MMP9 and TIMP-1.Compared with the blank group,snail,snail m RNA,slug,slug m RNA,MMP-9 and TIMP-1 in the model group were significantly increased（P<0.01）.Compared with the model group,snail,snail m RNA,slug,slug m RNA,MMP-9 and TIMP-1 in each treatment group were significantly decreased（P<0.01）.Compared with fasudil,10%SQHT treatment group was superior to fasudil in inhibiting snail,slug m RNA,MMP-9 and TIMP-1（P<0.01）,and had no significant difference in inhibiting slug and snail m RNA（P>0.05）;combined treatment group was superior to the other two treatment groups in inhibiting slug,slug m RNA,MMP-9 and TIMP-1（P<0.01）.There was no difference in snail and snail m RNA between the combined treatment group and SQHT group（P>0.05）,and it was better than fasudil group（P<0.01）.5 ConclusionSQHT,which is based on the method of Invigorating Qi,activating blood circulation and phlegm,can improve the TCM syndrome effect,dyspnea index,lung function,infection index,blood gas analysis index and coagulation index in the acute exacerbation period of chronic obstructive pulmonary disease,and it is safe and has no side effects.The mechanism of its intrinsic action may be related to the signaling pathways such as TGF-β1 and Rho A/ROCK.By downregulating the expression of TGF-β1 and Rho A/ROCK related gene protein,the development of MAPK/ERK signaling pathway is negatively regulated.