| Objective 1 86 patients with cough variant asthma(CVA)were investigated in TCM syndromes to explore the distribution of CVA syndromes and provide further reference for the clinical treatment of CVA.2 Observe the effect of Manke prescription on IL-1? and TNF-? expression in the upstream of NF-?B signal pathway,and TGF-?1,MMP-9 expression in the downstream of NF-?B signal pathway and NF-?B specific inhibitor I?B expression in CVA rats,to explore the molecular mechanism of man ke prescription in the treatment of CVA.Methods 1 Clinical research On the basis of consulting a large number of relevant literature and combined with the clinical experience of tutors,a CVA clinical questionnaire was developed.For the data of 86 patients who met the inclusion criteria,the statistical software was used to analyze the relationship between gender,age,occupation,area,smoking and syndrome,and summed up the distribution characteristics of CVA syndrome in traditional Chinese Medicine.2 Experimental study 72 healthy male SD rats were divided into control group,model group,High dose of Manke prescription group(MKPH group),Medium dose of Manke prescription group(MKPM group),Low dose of Manke prescription group(MKPL group),and Suhuang group(SH group),12 rats in each group.Ovalbumin(OVA)was used to sensitize and stimulate the CVA model of rats.From the 15 th day of the experiment,all dose groups of Manke prescription and Suhuang group began to be given related drugs by gavage,while the control group was given normal saline,continuous administration for 14 days.After the last gavage,the change of cough times of rats in each group was observed,and the materials were taken within 24 hours.HE staining was used to observe the pulmonary inflammation;the levels of TNF-?,IL-1?,TGF-?1,MMP-9 and TIMP-1 in serum of rats in each group were detected by ELISA;Western Blotting was used to detect the expression levels of NF-?B and I?B proteins in the lung tissues of rats in each group;and q RT-PCR was used to detect the expression levels of NF-?B and I?B genes in the lung tissues of rats in each group.Results 1 Clinical research In investigation of 86 cases of CVA patients,34 cases of wind heat attacking lung syndrome(39.53%),16 cases of phlegm dampness accumulation lung syndrome(18.6%),12 cases of deficiency of kidney yang(13.95%),10 cases of deficiency of lung qi(11.63%),5 cases of deficiency of spleen qi(5.81%),4 cases of wind cold attacking lung syndrome(4.65%),3 cases of syndrome of blood stasis blocking lung collaterals(3.49%),2 cases of liver fire attacking lung syndrome(2.33%).2 Experimental study 2.1 Statistics of cough times Compared with the control group,the times of cough in the model group increased significantly(P< 0.01).Compared with the model group,the times of cough in the MKPH,MKPM,MKPL and SH group decreased(P< 0.01).The times of cough in the MKPH group and SH group were less than that in the MKPM group and MKPL group(P< 0.01).There was no significant difference between the MKPH group and SH group(P> 0.05).2.2 HE staining results of pathological tissues In the control group,the morphology of lung tissue was basically normal,the structure of each layer was clear,no obvious inflammatory cell infiltration was found in bronchi and peripheral blood vessels,no obvious abnormality was found in tracheal mucosa epithelium,and no obvious stenosis was found in airway.In the model group,the tissue structure of alveoli and bronchi were disordered,epithelial cells were proliferated and exfoliated,and a large number of inflammatory cells were infiltrated,most of which were eosinophils,goblet cells,neutrophils;the blood vessel wall was thickened,the airway was narrowed,the wall of the tube was thickened,and mucus thrombus was seen.Compared with the model group,eosinophils,goblet cells,neutrophils and other inflammatory cells in the MKPL group decreased,the air cavity wall thickened,and the lumen was narrow.The MKPM group was better than the MKPL group.The lung tissue structure was improved,inflammatory cell infiltration was reduced,submucous edema was obviously reduced,and airway stenosis was rare.In the MKPH group,the infiltration of inflammatory cells was less,no obvious thickening of the tube wall was found,and the airway epithelial cells were more complete.The lung histopathology of Suhuang group was similar to that of the MKPH group.2.3 ELISA test results Compared with the control group,the levels of IL-1?,TGF-?1,TNF-?,MMP-9,TIMP-1 and the ratio of MMP-9/TIMP-1 in the model group were significantly higher(P<0.01).Compared with the model group,the contents of IL-1?,TGF-?1,TNF-?,MMP-9,TIMP-1 and the ratio of MMP-9/TIMP-1 in the MKPH,MKPM,MKPL and SH group were significantly lower(P<0.01,P<0.05).Compared with other treatment groups,the effect of MKPH on the level of IL-1?,TGF-?1,TNF-?,MMP-9,TIMP-1 and the ratio of MMP-9/TIMP-1 was more significant(P<0.01,P<0.05).2.4 The results of q RT-PCR Compared with the control group,the expression of NF-?Bp50 and NF-?Bp65 m RNA in the model group increased(P<0.01),and the expression of I?B m RNA decreased significantly(P<0.01).Compared with the model group,the expression of NF-?Bp50 and NF-?Bp65 in the MKPH,MKPM,MKPL and SH group decreased(P<0.01,P< 0.05),and the expression of I?B in the MKPH,MKPM and SH group increased significantly(P<0.01),I?B m RNAexpression in the MKPL also increased compared with the model group,but there was no statistical difference(P>0.05).Compared with other treatment groups,the MKPH reduced NF-?Bp50 and NF-?Bp65 m RNA levels,and the effect of increasing I?B gene level was the most significant. Compared with the control group,the expression of NF-?Bp50 and NF-?Bp65 protein in the model group increased(P<0.01),and the expression of I?B protein decreased(P<0.01).Compared with the model group,the expression of NF-?Bp50 and NF-? Bp65 decreased in the MKPH,MKPM,MKPL and SH group(P<0.01),and the expression of I?B increased(P<0.01).Compared with other treatment groups,the MKPH reduced the levels of NF-?Bp50 and NF-?Bp65 protein,and increased the level of I?B protein most significantly(P < 0.01,P < 0.05).2.5 The results of Western BlotConclusion 1 Among the 86 CVA patients investigated,wind,phlegm and deficiency were the main factors.2 Manke prescription can reduce the level of TNF-?,IL-1?,TGF-?1 in rats,reduce the airway inflammatory response,and thus inhibit CVA in rats.3 Manke prescription can inhibit cough variant asthma by down regulating the level of MMP-9 in rats,regulating the balance of MMP-9/TIMP-1 and reducing the deposition of extracellular matrix.4 Manke prescription can down regulate the expression of NF-?B in lung tissue,up regulate the expression of I?B,inhibit NF-?B signal pathway,and inhibit CVA in rats,especially in the high dose group of Manke prescription.5 Manke prescription may reduce the expression of TGF-?1 and MMP-9 in the downstream of NF-?B signal pathway,reduce the deposition of extracellular matrix,and thus inhibit CVA in rats. |
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