| ObjectiveAirway mucus hypersecretion is one of the important pathological features of chronic obstructive pulmonary disease(COPD).70 patients with AECOPD who met the criteria of phlegm-heat obstructing lung congestion were included,and their ACT,CCQ and mMRC scores were evaluated to observe the clinical treatment effect of Sang Ju Qing Jie decoction,and the molecular levels of serum NE,serum and sputum Muc5ac were detected to investigate the mechanism of its effect on inhibiting airway mucus secretion in AECOPD patients.We established a phlegm-heat obstructing lung COPD model in rats,and set up control group,model group,high,medium and low dose groups of Sang Ju Qing Jie decoction and dexamethasone group to detect the changes of lung function and the expression levels of serum and BALF inflammatory factors in rats of different groups.To investigate the mechanism of its effect on airway mucus secretion in rats.Research methods1.Clinical Research70 inpatients with phlegm-heat obstructing lung AECOPD were included as study subjects according to the corresponding inclusion criteria,and the control group and the treatment group were randomly divided according to the order of consultation time.The former group was treated with conventional interventions in the acute phase,including antiinfection,asthma,phlegm and cough relief,and the treatment group was treated with Sang Ju Qing Jie decoction orally on this basis.(1)After 7 days of treatment,the patients in the two groups were compared before and after treatment with the Chronic Obstructive Pulmonary Disease Assessment Test(CAT),the Modified Medical Research Council Respiratory Distress Rating Questionnaire(mMRC),and the Clinical COPD Questionnaire(CCQ)to analyze the degree of improvement of the quality of life of patients with COPD caused by phlegm and heat.(2)At the end of the 7 days intervention,the Chinese medicine symptom scores of the two groups of patients before and after the intervention were counted and compared to analyze the clinical treatment effect of Sang Ju Qing Jie decoction on patients with phlegmheat obstructing lung AECOPD.(3)After 7 days of treatment,the changes of NE and Muc5ac in serum and Muc5ac in sputum were measured by ELISA before and after treatment in both groups.2.Experimental research(1)Animal models of sputum-heat congestion COPD were established by fumigation combined with LPS airway drip method,and divided into control group,model group,Sang Ju Qing Jie Decoction high-dose group,medium-dose group,low-dose group and dexamethasone group according to the drug administration mode.The weight change curves of the model rats in each group and the lung function change after treatment were counted to analyze the effect of Sang Ju Qing Jie decoction on lung function of the model rats.(2)ELISA experiment was performed to detect the concentrations of IL-6,IL-8,MMP-2,MMP-3,slgA and TNF-α in the serum of model rats in each group;ELISA experiment was performed to detect the concentrations of IL-6,IL-8,MMP-2,MMP-3,slgA,TNF-αand Muc5ac in the alveolar lavage fluid of model rats;ELISA experiment was performed to detect the concentrations of sIgA,TMP-3 and Muc5ac in the lung tissue of model rats.The concentrations of slgA,TGF-β and VEGF in lung tissues of model rats were detected by ELISA,and the mechanism of the effect of Sang Ju Qing Jie decoction on antiinflammation and inhibition of mucin secretion in model rats was analyzed.(3)To observe the distribution of inflammatory cells in lung tissues of model rats by electron microscopy and lung lesions of model rats by HE staining,and to analyze the effects of Sang Ju Qing Jie decoction on the level of inflammatory cells and pathological changes in rats with chronic obstructive pulmonary disease.(4)Western blot assay was performed to detect the expression of NE,P38MARK and Muc5ac in the lung tissues of the model rats to further prove the mechanism of the effect of Sang Ju Qing Jie Decoction on the expression of Muc5ac.(5)The experimental data were statistically analyzed by SPSS26.0 and GraphPad Prism8 software,and the means of multiple groups were analyzed by ANOVA,and the comparison between two groups was performed by LSD t-test with P<0.05 difference was statistically significant,and P<0.01 was considered statistically significant.The mean and standard deviation were used to describe the indicators of normal distribution,for the comparison of components,the scheme of one-way variance or t-test was selected,and paired t-test was used for intra-group comparison,and repeated measurement data were analyzed by repeated measurement ANOVA with P<0.05 was considered a statistically significant difference,and P<0.01 was considered statistically significant.Results1.Clinical Research(1)There was no difference in the comparison of age and mean disease duration between patients in the treatment group as well as the control group(P>0.05)(2)There was no significant difference in CAT scores between the treatment group and the control group(P>0.05).After 7 days of treatment,two dropouts were dropped out in the control group and the treatment group,and the CAT score in the treatment group was significantly smaller than that in the control group,and the difference was statistically significant(P<0.05).(3)There was no significant difference in the grade of mMRC before treatment between the treatment group and the control group.After 7 days of treatment,the mMRC grade obtained by the treatment group was significantly lower and the difference was statistically significant(P<0.05).(4)There was no significant difference between the CCQ scores of patients in the two groups before treatment.After 7 days of treatment,the CCQ scores of patients in the control and treatment groups were significantly lower,and the CCQ scores of patients in the treatment group were significantly lower than those in the control group(P<0.01)(5)After 7 days of treatment,the scores of TCM symptoms in the treatment group were significantly lower than before treatment,and the data differences were statistically significant(P<0.01).The score of the treatment group was significantly lower and the difference was statistically significant(P<0.05).(6)The ELISA results showed that there was no significant difference between the serum NE,Muc5ac and sputum Muc5ac concentrations in the treatment groups before treatment(P>0.05).After 7 days of treatment,the mean values of NE,Muc5ac in serum and Muc5ac in sputum were significantly lower in both control and treatment groups than in the control group(P<0.05),but the differences were not statistically different.2.Experimental research(1)After 28 days of modeling,the weight of the control group was significantly higher than that of the model group,high-dose group,medium-dose group,low-dose group and dexamethasone group(P<0.05),and there was no significant difference between the other groups(P>0.05).(2)Using a special volume bin for animal lung function,the values of-PIF(peak inspiratory flow rate),PEF and MV of the model group were significantly lower than those of the control group(P<0.01).The values of PIF(peak inspiratory velocity),PEF(peak expiratory velocity)and MV(minute ventilation)in the model group were significantly lower than those in the control group(P<0.01),consistent with the characteristics of airflow limitation in COPD.After treatment,there was no statistically significant difference in the dexamethasone group compared with the model group PIF;the low,medium and high dose Sang Ju Qing Jie Decoctiongroups all P<0.01,a highly significant difference,indicating that the effect of Sang Ju Qing Jie decoction was obvious and concentration-dependent.In terms of PEF,compared with the model group,the dexamethasone group showed significant improvement;there was a significant difference between the middle and high doses of Sang Ju Qing Jie Decoction group and the dexamethasone group.Compared with the dexamethasone group,the low-dose Sang Ju Qing Jie Decoctiongroup P>0.05,the difference was not statistically significant.For MV,compared with the model group,the dexamethasone group P<0.01,with significant difference;compared with the dexamethasone group,P>0.05 in the low-dose Sang Ju Qing Jie Decoctiongroup,with no statistically significant difference.05,the difference was not statistically significant;P<0.01 in both the middle and high dose Sang Ju Qing Jie decoction groups.There is a highly significant difference between the middle and high doses of Sang Ju Qing Jie Decoction and dexamethasone.(3)As shown by ELISA,compared with the control group,the concentrations of IL6,TNF-α,IL-8,MMP-3 and MMP-2 in the serum of the model animals were significantly increased(P<0.01).The concentrations of IL-6,IL-8,MMP-2,the indexes of MMP-3 and TNF-α in the low-dose group were significantly lower than those in the model group(P<0.01),while the concentrations of IL-6,IL-8,MMP-2,the indexes of MMP-3 and TNFa in the high-dose group were also significantly lower than those in the model group(P<0.01).The mean serum concentrations of IL-6,MMP-2,MMP-3 and TNF-α in the rats of the high-dose group were significantly lower than those in the low-dose group and the dexamethasone group(P<0.01),the index concentrations in the high-dose group were significantly lower than those in the low-dose and dexamethasone groups.The mean values of serum TNF-α,IL-8 and MMP-3 were not significantly different between the low-dose group and the dexamethasone group(P>0.05).Compared with the control group,serum slgA was significantly lower in the model group rats(P<0.01).In the high-dose group,the serum levels of slgA were significantly higher than those in the low-dose group,the medium-dose group and the dexamethasone group in a concentration-dependent manner,but were still significantly lower than those in the control group(P<0.01).(4)The ELISA results showed that the mean concentrations of IL-6,IL-8,MMP-2,MMP-3 in the alveolar lavage fluid of rats,the indexes of TNF-α and Muc5ac in the model group were significantly higher than those in the control group(P<0.01).The concentrations of IL-6,IL-8,MMP-2,MMP-3,TNF-α and Muc5ac in the alveolar lavage fluid of rats in the low-dose group were significantly lower than those in the model group(P<0.05),and the mean values of IL-6,IL-8,MMP-2,MMP-3,TNF-α,and Muc5ac concentrations in the highdose group were significantly lower than the mean values of concentrations in the low-dose and dexamethasone groups(P<0.01),among which,there was no significant difference between the mean concentrations of IL-6 lavage solution in the high-dose group and the medium-dose group,the medium-dose group and the low-dose group,and the low-dose group and the dexamethasone group(P>0.05),no significant differences existed between the mean values of IL-8 and Muc5ac lavage solution concentrations in the low-dose group and the dexamethasone group(P>0.05),and there was no significant difference in the mean values of MPP-2 and MMP-3 lavage fluid concentrations between the high-dose group and the medium-dose group,and between the low-dose group and the dexamethasone group(P>0.05).Compared with the control group,slgA was significantly lower in the alveolar lavage fluid of rats in the model group(P<0.01),and the mean value of sIgA lavage fluid in the high-dose group was significantly higher than that in the low-dose and dexamethasone groups(P<0.01),and the mean value of sIgA lavage solution in the medium-dose group was significantly lower than that in the low-dose and dexamethasone groups(P<0.05),(P<0.01),and there was no significant difference between the high-dose group and the medium-dose group,and the low-dose group and the dexamethasone group(P>0.05).(5)The results of ELISA experiments showed that the mean values of TGF-βconcentration in the lung tissue of rats in the model group were significantly higher compared with the control group(P<0.01).The mean values of TGF-β concentrations in the lung tissues of rats in the high,the liver fibrosis in the medium and low mulberry chrysanthemum groups and dexamethasone groups was significantly less than that in the model group(P<0.01).The mean TNF-β pulmonary tissue values in the medium-dose group were significantly lower than those in the low-dose and dexamethasone groups(P<0.05),and no significant differences existed between the control and high-dose groups,the high-dose and medium-dose groups,and the low-dose and dexamethasone groups(P>0.05).Compared with the control group,the mean values of sIgA and VEGF concentrations in the lung tissue of the model group rats were significantly lower(P<0.01),but the mean values of VEGF concentrations in the model group were not significantly different from those in the dexamethasone group(P>0.05).The mean values of sIgA and VEGF concentrations in the lung tissues of rats in the low-dose and dexamethasone groups were significantly higher than those in the model group(P<0.05).SIgA and VEGF in the low-dose and dexamethasone groups were significantly higher than those in the model group(P<0.01),while the above indexes in the serum of rats in the highdose group were significantly higher than those in the low-dose and medium-dose groups(P<0.05),but still lower than the control group(P<0.01).(6)The distribution of inflammatory cells in the lung tissues of rats in each group was observed by electron microscopy,and the lung vesicles in the lung tissues of rats in the control group were arranged in a regular manner and no inflammatory cell infiltration was observed.The lung vesicles in the lung tissue of the rats in the model group were arranged in a disorderly manner without any rules,and there were a large number of inflammatory cells infiltrating the lung tissue,with neutrophils and lymphocytes accounting for the majority.In the lung tissues of rats with COPD model,the disorderly arrangement of pulmonary vesicles was reduced and the number of infiltrating inflammatory cells,mainly neutrophils,was significantly reduced.The infiltrating inflammation in the high dose group was significantly less than that in the medium and low dose groups.The number of eosinophils infiltrated in the lung tissue of rats in the dexamethasone group was basically the same as that in the low-dose group of Sang Ju Qing Jie decoction.(7)HE staining was performed to observe the lung tissue lesions of the model rats.The lung tissue of the control rats was intact and no obvious inflammatory cell infiltration was observed.In the COPD model group,the lung structure was disorganized,and a large number of inflammatory cells infiltrated and epithelial cup-like cells proliferated,suggesting that the modeling was successful.In the high-dose group,a small number of inflammatory cells were seen in the lung tissue with enlarged alveolar cavity,while in the middle-dose group,the number of inflammatory cell aggregates was reduced and the alveolar septum was widened with inflammatory cell infiltration.In the low-dose group,there were moderate interstitial inflammatory changes in the lung tissues of rats,and the same alveolar septum widening and inflammatory cell infiltration were observed.In the dexamethasone group,inflammatory cell infiltration with macrophage infiltration was observed in the lung tissue of rats.(8)The results of Western blot showed that the expression levels of Muc5ac,NE and P38MARK molecules were significantly up-regulated in the lung tissues of rats in the model group compared with the control group.The mean values of P38MARK and NE in the highdose group were significantly lower than those in the medium-dose group,low-dose and dexamethasone groups(P<0.01).The mean values of Muc5ac in the high-dose group were significantly lower than those in the low-dose group and dexamethasone group(P<0.01).The mean values of P38MARK and NE in the medium-dose group were significantly lower than those in the dexamethasone group(P<0.01),and there were no significant differences between the mean values of P38MARK and NE in the medium-dose group and the low-dose group,and between the mean values of P38MARK and NE in the low-dose group and the dexamethasone group(P>0.05).There were no significant differences between the mean Muc5ac values in the high-dose group and the medium-dose group,the mean Muc5ac values in the medium-dose group and the dexamethasone group,and the mean Muc5ac values in the low-dose group and the dexamethasone group(P>0.05).Conclusion(1)On the basis of conventional treatment,the combined use of Sang Ju Qing Jie decoction can significantly improve the degree of pulmonary inflammatory response,improve patients’ quality of life,improve patients’ clinical symptoms,reduce airway mucus secretion and improve patients’ therapeutic effects in AECOPD patients with phlegm-heat obstructing lung。(2)Based on the results of animal behavior、lung function and HE staining,it can be assumed that the phlegm-heat obstructing lung COPD model in rats were successfully seted.(3)Sang Ju Qing Jie Decoction can reduce the pulmonary ventilation function of COPD rats and significantly improve the obstructive ventilation dysfunction of the rat airway in a concentration-dependent manner.(4)Sang Ju Qing Jie Decoction can reduce the infiltration of inflammatory cells in the lung tissue of model animals,reduce the degree of histopathological changes in the lung,inhibit the concentration of IL-6,IL-8,MMP-2,MMP-3 and TNF-α in the serum and alveolar lavage fluid of model rats,and have anti-inflammatory effects,and further reduce the secretion of Muc5ac in the alveolar lavage fluid.(5)Sang Ju Qing Jie Decoction can increase the concentration of sIgA in the serum and alveolar lavage fluid of model rats,reduce the expression of TGF-β in the lung tissue of model rats,increase the expression of sIgA and VEGF in the lung tissue of model rats,and have the ability to improve the immunity of respiratory tract(6)Sang Ju Qing Jie Decoction reduces the release of inflammatory factors such as inflammatory alveolar lavage fluid and lung tissue,which in turn reduces the expression of NE as well as P38MARK molecule protein in lung tissue and blocks the expression of Muc5ac protein in lung tissue,indicating that its effect is related to the inhibitory effect on the activation of P38MARK signaling pathway. |
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