| Background:Chronic Obstructive Pulmonary Disease(COPD)is a chronic disease characterized by persistent respiratory symptoms and airflow limitation,usually due to airway and/or alveolar abnormalities caused by exposure to toxic particles or gases.Currently,COPD treatment in Western medicine is mainly symptomatic,but the effect is not satisfactory and need lifelong medication.Following the principles of holism and treatment according to pattern differentiation,methods of traditional Chinese medicine(TCM)are used to achieve the goals of preventing disease before it arises and controlling the development of existing,so as to prevent and control the occurrence and development of COPD.Guben Zhike Decoction(GBZKD)is derived from the experience of treating COPD by Professor Enxiang Chao,a top master of Chinese medicine.Professor Chao Enxiang attached great importance to the position and role of healthy qi in the onset and progression of diseases,and put forward the theory of’reinforcing the healthy qi and consolidating the defensive qi’.Therefore,based on the classic prescription ’Yupingfeng decoction’,Professor Chao developed GBZKD.Preliminary clinical studies have shown that GBZKD has a good effect in improving patients’ lung function,and exert a broad range of effects of human immune function by regulating T cell subsets.The results of a large number of animal experiments have shown that GBZKD is capable of improving lung function,reversing the pathological changes of respiratory tract,reducing overexpression of inflammation cytokines,decreasing the ratio of αβT cells/γδT cells and increasing the protein and gene expression of KGF and KGFR in lung tissue of COPD mice.Therefore,it is of certain significance to further study the efficacy and mechanism of GBZKD in treating COPD model mice.Objective:Injury repair is an orderly process under the co-action of various cell growth factors,which can be divided into four stages:inflammatory response,cell proliferation,matrix deposition and tissue remodeling.Injury repair in respiratory tract is one of the main pathogenesis of COPD,which runs through the whole course of COPD.The study aim to study the regulation effect of GBZKD on injury repair in the respiratory tract of COPD in three aspects:injury repair of respiratory mucosal inflammation,injury repair of respiratory structure,aberrant repair of injured respiratory tract;so as to better fulfilling the theory of ’reinforcing the healthy qi and consolidating the defensive qi’ and ’lung governing skin and hair’.Methods:In this study,100 healthy ICR mice were randomly divided into blank control group(n=30),COPD group(n=35)and GBZKD group(n=35).In this study,COPD group and GBZKD group were modeled by cigarette smoke exposure and nasal administration of lipopolysaccharide(LPS)for 90 days.From the 61st day,mice in GBZKD group were given intragastric traditional Chinese medicine liquid while mice in blank control group and model control group were given intragastric distilled water for 28 days.In part one:Body weight monitoring and lung function detection were used to evaluate the preparation of COPD model mice and the efficacy of GBZKD.In terms of regulation effect of GBZKD on the injury repair of respiratory mucosal inflammation in COPD model mice,forty inflammatory cytokines of lung tissue in mice were systematically detected by antibody array.Immunohistochemical method was used to verify the inflammatory factors that statistically significant between blank control group and COPD control group or/and between COPD group and GBZKD group.In terms of regulation effect of GBZKD on the injury repair of respiratory structure and aberrant repair of injured respiratory tract,structural damage and aberrant injury repair of respiratory tract structure,alveolar structure,extracellular matrix and type II alveolar epithelial cells were determined by pathological evaluation,immunohistochemistry and transmission electron microscopy.In part two:The composition of GBZKD was analyzed based on mass spectrometry technology to discuss the mechanism of its regulatory role of injury repair in respiratory tract of COPD mice.Besides,label-free proteomics was performed to clarify the mechanism of overall regulation by GBZKD in the injury repair of respiratory tract in COPD mice.In part three,western-blot and RT-qPCR were used to verify the JAK-STAT signaling pathway that appeared in the enrichment of the results in part one and part two of this study.The protein expression levels of JAK1,p-JAK1,STAT3,p-STAT3,SOCS3 as well as gene expression levels of JAK1 mRNA,JAK2 mRNA,JAK3 mRNA,STAT1 mRNA,STAT3 mRNA,SOCS3 mRNA were determined in this study.Results:1)In terms of weight monitoring:GBZKD can prevent the low weight gain situation of COPD model mice.2)In terms of lung function detection:Compared with blank control group,FEV0.05,FEV0.1,FEV0.2,PEF,Crs and Cst were significantly decreased in COPD group(P<0.01),while Rrs,Ers and Rn were significantly increased in COPD group(P<0.01).There were no significant differences in G and H between blank control group and COPD group(P>0.05).Compared with COPD group,FEV0.05,FEV0.1,FEV0.2 and Crs were significantly increased in GBZKD group(P<0.05),while Rrs,Ers and Rn were significantly decreased in GBZKD group(P<0.05).There were no significant differences in PEF,G,H and Cst between GBZKD group and COPD group(P>0.05).3)In terms of pathological morphology:HE staining results showed that the pathological morphology of lung tissue in COPD group was consistent with the characteristics of COPD.GBZKD could improve the pathological features of airways(50-100μm in diameter),respiratory bronchioles and alveoli in COPD mice.Transmission electron microscopy results showed that vacuolation,mitochondria swelling,cristae rupture and disappearance of lamellar bodies have occurred in type Ⅱ alveolar epithelial cells in lung tissue of COPD mice.Mitochondria swelling and disappearance of lamellar bodies in type Ⅱ alveolar epithelial cells of GBZKD-treated mice were reduced compared to those in COPD group.4)In terms of antibody array:Compared with the blank control group,there were 8 significantly increased inflammatory factors in COPD group,which were IL-6,GM-CSF,TNF-α,IFN-γ,IL-β,IL-3,IL-17 and IL-12p70(P<0.05).Compared with COPD group,there were 7 significantly decreased inflammatory factors in COPD group,which were IL-6,GM-CSF,TNF-α,IFN-γ,IL-1β,IL-3 and TCA-3(P<0.05).5)In terms of immunochemistry assay:Compared with blank control group,IL-6,GM-CSF,TNF-α,IFN-γ,α-SMA,MMP-9,TIMP-1,MMP-9/TIMP-1 and HIF-1α were significantly increased in COPD group(P<0.05).There was no significant difference in Collagen 1 between blank control group and COPD group(P>0.05).Compared with COPD group,IL-6,GM-CSF,TNF-α,IFN-γ,α-SMA,MMP-9,TIMP-1,MMP-9/TIMP-1 and HIF-lα were significantly decreased in GBZKD group(P<0.05).There was no significant difference in Collagen 1 between COPD group and GBZKD group(P>0.05).6)In terms of component analysis of GBZKD:A total of 41 components of GBZKD were identified,and many of which were proved to be involved in the regulation of injury repair in respiratory tract through anti-inflammatory,anti-oxidation,and inhibition of airway remodeling.7)In terms of label-free proteomics:There were 287 differential proteins between the COPD group and blank control group,184 differential proteins between the GBZKD group and COPD group.Through GO enrichment analysis,KEGG enrichment analysis and REACTOME enrichment analysis of differential proteins,it was found that GBZKD could overall regulate injury repair in the respiratory tract of COPD mice through multiple pathways such as neutrophil degranulation,complement pathway,extracellular matrix,and JAK-STAT signaling pathway,as well as multiple biomarkers involved in COPD progress.8)In terms of RT-qPCR assay:Compared with blank control group,JAK1 mRNA,JAK2 mRNA,JAK3 mRNA,STAT3 mRNA and SOCS3 mRNA were significantly increased in COPD group(P<0.05).There was no significant difference in STAT1 mRNA between blank control group and COPD group(P>0.05).Compared with COPD group,JAK1 mRNA and STAT3 mRNA were significantly decreased in GBZKD group(P<0.05);SOCS3 mRNA was significantly increased in GBZKD group(P<0.05).There were no significant difference in JAK2 mRNA,JAK3 mRNA and STAT1 mRNA between COPD group and GBZKD group(P>0.05).In terms of western-blot assay:Compared with blank control group,p-JAK1,p-STAT3,p-JAK1/JAK1 and p-STAT3/STAT3 were significantly increased in COPD group(P<0.05).There were no significant differences in JAK1,STAT3 and SOCS3 between blank control group and COPD group(P>0.05).Compared with COPD group,p-JAK1,p-STAT3 and p-STAT3/STAT3 were significantly decreased in GBZKD group(P<0.05).There were no significant differences in JAK1,STAT3 and P-JAK1/JAK1 between COPD group and GBZKD group(P>0.05).Conclusion:1)By reducing the overexpression of inflammatory factors in the lung tissue of COPD model mice,GBZKD plays a role in regulating injury repair of respiratory mucosal inflammation.By relieving the damage of respiratory tract structure,alveolar type Ⅱ epithelial cell ultrastructure and extracellular matrix in COPD model mice,it plays a role in regulating injury repair of respiratory structure,aberrant repair of injured respiratory tract.These results fulfills the theory of ’reinforcing the healthy qi and consolidating the defensive qi’and ’lung governing skin and hair’ in traditional Chinese medicine.2)GBZKD has the characteristic of multi-component,multi-target and multi-pathway in overall regulation of injury repair in the respiratory tract of COPD mice,which embodies the theory of ’holistic concept’ in traditional Chinese medicine.3)GBZKD can inhibits JAK-STAT signaling by reducing the phosphorylation of JAK1 and STAT3,and up-regulating the expression of SOCS3,which reflects the mechanism of GBZKD’s overall regulation of respiratory tract injury repair through ’reinforcing the healthy qi and consolidating the defensive qi’ and ’lung governing skin and hair’ in traditional Chinese medicine. |
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