| Objective:The impact of chronic obstructive pulmonary disease(Chronic Obstructive Pulmonary Disease,COPD)on human health is enormous and profound.The Acute Exacerbation of Chronic Obstructive Pulmonary Disease(AECOPD)is the main reason to accelerate the progress of the disease.At present,the drugs used clinically to treat AECOPD are mainly anti-inflammatory and bronchodilators drugs,but they still have relative limitations.There is a need for more alternative drugs for AECOPD in clinical practice.Method:(1)Firstly,by means of network pharmacology,the active compounds and corresponding targets in Qingyuan Huatan granule were searched through database,the biological processes and signal transduction pathways related to Qingyuan Huatan granule and AECOPD were identified,and the mechanism hypothesis of Qingyuan Huatan granule on AECOPD was put forward.(2)Secondly,SPF male C57BL/6 mice were selected to establish the model of AECOPD disease by smoking and intratracheal instillation of LPS.After 8 weeks,the general conditions of mice,the pathological changes of HE in lung tissue and the average lining interval were observed.The number of inflammatory cells in bronchoalveolar lavage fluid and the ratio of neutrophils to lymphocytes were calculated.The apoptosis of neutrophils in lung tissue was detected by immunofluorescence,and the levels of HMGB1-RAGE and apoptotic protein C-casp3 were detected by Western blot and PCR.(3)Thirdly,16S rDNA and targeted metabonomics were used to analyze the community species information and the level of short-chain fatty acids in mouse feces,and the species composition information and inter-group composition differences were obtained by OTU cluster analysis,inter-sample ? diversity index analysis and multi-group comparison,and the short-chain fatty acids in the samples were identified by Masshunter quantitative software to determine the content of short-chain fatty acids.Finally,the correlation between metabolites and sample species was analyzed to determine whether there was a correlation between differential metabolites and flora,and to mine the key bacteria that caused metabolic differences.(4)Finally,the double intervention model of mice was established by bacterial depletion combined with AECOPD.At the eighth week,the general condition of mice,the pathological changes of HE in lung tissue and the count of inflammatory cells in bronchoalveolar lavage fluid were observed.The expression of HMGB1-RAGE and apoptotic protein C-casp3 in related inflammatory pathway was detected by Western blot and PCR.Results:(1)The results of network pharmacology showed that the mechanism of Qingyuan Huatan granule was mainly manifested in the regulation of inflammatory reaction.Phenotypic analysis showed that CRP,IL-6 and IL-1 ? in the target of Qingyuan Huatan granule were related to frequent acute aggravation phenotype,MPO was related to neutrophil inflammation,and MMP-1 and MMP-9 were related to the degree of emphysema.In the pathway,it mainly involves RAGE and its ligands signal pathway,"PI3K-Akt" signal pathway,and can regulate the outcome of Apoptosis.Combined with the literature research,we finally put forward the scientific hypothesis that Qingyuan Huatan granule may interfere with neutrophil apoptosis by mediating "HMGB1-RAGE" signal pathway,and then affect the disease process of AECOPD.(2)Qingyuan Huatan granule could significantly improve the inflammation of lung tissue in AECOPD mice.Compared with the model group,the body weight of mice in western medicine group,middle dose group,high dose group and emodin group increased steadily,and the lung pathology showed that the infiltration of inflammatory cells in lung tissue in the traditional Chinese medicine high dose group and western medicine group were alleviated.Mild inflammatory cell infiltration can be seen in the traditional Chinese medicine middle dose group and emodin group,and the average lung lining interval was significantly lower than that in the model group(P<0.05);The number of neutrophils and the ratio of neutrophils to lymphocytes in bronchoalveolar lavage fluid of each drug group were lower than those of the model group,the changes in lymphocytes were not obvious,especially in the middle dose group(P<0.05).The results of immunofluorescence showed that there was no obvious morphological display of C-casp3 and Ly6G fluorescent cells in the blank control group.Obvious neutrophils could be seen in the model group,and there was no C-casp3 in and around the cells.Different degrees of Ly6G and C-casp3 could be seen in each treatment group,indicating that there were different degrees of neutrophil apoptosis in the treatment group.The results of Western blot showed that C-casp3 and C-casp3/Casp3 in the lung tissue of the AECOPD model group were significantly lower than those of the blank control group(P<0.05),while Casp3,HMGB1 and RAGE were significantly higher than those of the blank control group(P<0.05,P<0.01).After treatment with different drugs,C-casp3 and C-casp3/Casp3 showed an upward trend,and there were significant differences compared with the model group(P<0.01).Among them,the effects of the middle dose group and emodin group were better than those of the high dose group of traditional Chinese medicine and the western medicine group(P<0.05,P<0.01).In terms of Casp3 expression,after drug intervention,the level of Casp3 in each group decreased significantly,compared with the model group,the middle dose group,high dose group and emodin group were significantly lower than those in the model group(P<0.01).At the same time,the protein expression of HMGB1 and RAGE in the treatment group was significantly lower than that in the model group,especially the middle dose group and emodin group(P<0.01,P<0.05).The results of PCR showed that the expression of HMGB1mRNA and RAGE mRNA in the lung tissue of the model group was significantly higher than that of the normal group,and the expression levels of HMGB1mRNA and RAGEmRNA were down-regulated after drug treatment in each group(P<0.01).Compared with the model group,the differences were statistically significant(P<0.01).Compared with the western medicine group,the expression level of HMGB1mRNA in the middle dose group and emodin group was lower,and the difference was statistically significant(P<0.01,P<0.05).There was no significant difference in the expression of RAGE mRNA among the treatment groups(P>0.05).(3)In terms of the effect on intestinal flora and metabolites,the contents of acetic acid,propanoic acid and butanoic acid in the model group were significantly lower than those in the blank control group,especially butanoic acid(P<0.01).Compared with the model group,the contents of acetic acid and propanoic acid increased slightly in the western medicine group,while the contents of acetic acid decreased,propanoic acid and butanoic acid increased significantly in the middle dose group,high dose group and emodin group,and the contents of propanoic acid and butanoic acid were the highest in the middle dose group.After comprehensive analysis,butanoic acid was used as the main differential metabolite,and the correlation between butyric acid and the main differential bacteria between groups was analyzed.The results showed that the levels of Akkermansiaceae,Rikenellaceae,Ruminococcaceae and Sutterellaceae were positively correlated with butanoic acid,Enterococcaceae was negatively correlated with the level of butanoic acid.The change trend of Akkermansiaceae and butanoic acid between groups was basically the same.(4)Fecal microflora transplantation could significantly reverse the inflammation of lung tissue in AECOPD mice,the body weight of mice in the transplanting normal group,transplanting traditional Chinese medicine group and transplanting emodin group were increased steadily after microflora transplantation.The pathological examination of lung tissue showed that the destruction of alveolar structure and the infiltration of inflammatory cells in the lung tissue were significantly less than those in the model group,and the mean linear intercept of lung were lower than that in the model group(P<0.05).The neutrophil count and the ratio of neutrophils to lymphocytes(NLR)in the bronchoalveolar lavage fluid were lower than those in the model group.The results of Western blot showed that C-casp3 and C-casp3/Casp3 in lung tissue of mice in transplanting normal group,transplanting traditional Chinese medicine group and transplanting emodin group were significantly higher than those in model group(P<0.01),and the effect of transplanting traditional Chinese medicine group was slightly better than that of transplanting normal group and transplanting emodin group,but the difference was not statistically significant.Casp3 in each transplantation group decreased significantly,compared with the model group,the difference was statistically significant(P<0.05).The HMGB1 and RAGE of mice in each transplantation group showed a downward trend.Compared with the model group,there was significant difference between the transplanting traditional Chinese medicine group and transplanting emodin group(P<0.05),but there was no significant difference between the transplantation groups(P>0.05).The results of PCR showed that compared with the blank control group,the expression of HMGB1mRNA and RAGE mRNA in the lung tissue of the model group increased significantly(P<0.01,P<0.05),and compared with the model group,the expression levels of HMGB1mRNA and RAGE mRNA in each transplantation group were down-regulated(P<0.01,P<0.05),especially in the transplanting traditional Chinese medicine group,but there was no significant difference between the transplantation groups(P>0.05).Conclusion:Qingyuan Huatan granule can affect HMGB1-RAGE pathway,down-regulate the expression of pro-inflammatory factor HMGB1 and its receptor RAGE protein and mRNA,up-regulate the level of apoptosis executive protein C-casp3,and induce apoptosis of inflammatory cells dominated by neutrophils,so as to reduce the neutrophil-mediated lung inflammation and have the effect of "treating lung".AECOPD model mice have disorder of short chain fatty acids and intestinal flora.Qingyuan Huatan granule can increase the level of butyric acid in model mice,regulate the structure and abundance of related intestinal microflora,corrected the disorder of intestinal flora and metabolites,indicating that Qingyuan Huatan granule has the characteristics of "regulating intestine".After the intervention of Qingyuan Huatan granule,the transplantation of fecal flora can significantly reduce the inflammatory reaction in lung tissue of AECOPD mice,regulate the expression of HMGB1-RAGE pathway and apoptotic protein C-casp3,and reflect the characteristics of "treating lung from intestine" and"lung-intestine co-management" of Qingyuan Huatan granule. |
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