| Objective: To establish a rat model of asthma,and to observe the effects of different doses of Jianshu Renju Pulmonary Resolving Phlegm Formula combined with dexamethasone on airway inflammation and gene expression of Cyp11a1,Cyp11b1 and St AR steroid hormone synthase specific molecules in adrenal tissues of asthmatic rats,and then to investigate the effects of Jianshu Renju Pulmonary Resolving Phlegm Formula combined with glucocorticoids on the treatment of asthma rats.This study investigated the mechanism of the effect of the combination of glucocorticoids and spleen tonifying lung and resolving phlegm on asthma rats.Methods: Sixty rats were randomly divided into normal control group(ZC group),asthma group(XC group),dexamethasone group(DM group),dexamethasone +low-dose group(DM+D group),dexamethasone + medium-dose group(DM+Z group),dexamethasone + high-dose group(DM+G group)by using RAND function in EXCEL software.Each group consisted of 10 animals.Except for the normal control group,the asthmatic-modeling groups were sensitized by intraderitoneal injection of 10% OVA solution on day 1 and day 8,respectively.Since the 15 d,1%OVA atomization was stimulated,once every other day,30 min each time.At the same time,on the 15 th day from the beginning of the experiment,except ZC group and XC group,the other 4groups were intraperitoneally injected with dexamethasone sodium phosphate injection1mg/kg daily.DM+D group,DM+Z group and DM+G group were given 5.265g/kg,10.53g/kg and 21.06g/kg Jianpi Bufei Huatan formula solution by intragastric administration,respectively.The DM group was given the same amount of normal saline instead.XC group was given intraperitoneal injection of normal saline and gavage.ZC group rats were sensitized,atomized,intrabitoneal injection and gavage with the same amount of normal saline.The experiment lasted for 8 weeks.The general conditions of rats in each group were observed during the experiment.After the experiment,lung tissue samples of rats were collected and the pathological changes were observed by hematoxylin-eosin(HE)staining and Masson staining.The ultrastructure of rat adrenal tissue was observed under electron microscope.The serum levels of IL-17 A,IL-22,ACTH,CORT and bronchoalveolar lavage fluid(BALF)were determined by enzyma-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the expression of IL-22 and STAT3 genes in lung tissue and St AR,Cyp11a1 and Cyp11b1 genes in adrenal tissue.Western Blot(WB)was used to detect the expression of STAT3 and p-STAT3 in lung tissues.The expression of St AR protein in adrenal tissues was detected by immunohistochemistry(IF).Results:1.General: Compared with the ZC group,rats in the XC group showed restlessness,nose scratching,runny nose,abdominal muscle twitching and lagging growth.rats in the DM group showed less symptoms such as nose scratching,runny nose and shortness of breath than those in the asthma group,but obvious wasting,reduced diet,hugging,reduced voluntary activity and hair loss were seen.In the dose groups of dexamethasone plus the formula for strengthening the spleen,tonifying the lung and resolving phlegm,the rats showed different degrees of improvement in symptoms such as scratching the nose,runny nose and shortness of breath compared with the DM and XC groups,and there was no obvious wasting,and the coat had better luster and better energy.2.HE staining of lung tissues: compared with the ZC group,lung tissue sections of the XC group showed narrowed bronchial lumen,thickened and widened alveolar septum,disorganized morphology,accompanied by a large number of inflammatory cells around.In the four groups with drug intervention,the number of microscopic inflammatory cells decreased and the airway structure improved to different degrees,and the combined drug group showed better improvement than the DM group.3.Masson staining of lung tissue: Compared with ZC group,bronchial wall and basement membrane in XC group were significantly thickened.The thickness of airway wall,smooth muscle layer and basement membrane was significantly reduced in the four medicated groups compared with the asthma group.4.Levels of IL-17 A and IL-22 in serum and BALF: Compared with ZC group,the expression levels of IL-17 A and IL-22 in serum and BALF of other groups were significantly increased(P<0.01).Compared with the XC group,the expression levels of IL-17 A and IL-22 in serum and BALF of the four groups were decreased to different degrees(P<0.01).Compared with the DM group,the expression levels of IL-17 A and IL-22 in serum IL-22 and BALF in the combination group were lower than those in the DM group(P<0.01),but there was no significant difference in serum IL-17 A expression between the DM group and the DM+D group(P>0.05).There were significant differences between DM+Z group,DM+G group and DM group(P<0.01).5.Expression of IL-22 and STAT3 genes in lung tissues: Compared with ZC group,expressions of IL-22 mrna and STAT3 mrna in lung tissues of the other five groups were increased(P<0.01).Compared with the XC group,the expressions of IL-22 m RNA and STAT3 m RNA in the lung tissues of rats in the four groups treated with drug were decreased to varying degrees(P<0.01).The expressions of IL-22 m RNA and STAT3 m RNA in lung tissues of the three groups of rats treated with dexamethasone combined with Chinese medicine were statistically significant compared with those of DM group(P<0.01).6.Expression of STAT3 and p-STAT3 in lung tissues: Compared with ZC group,the up-regulated expression of p-STAT3 protein in lung tissues of the other five groups had statistical significance(P<0.05).Compared with XC group,the expression of p-STAT3 protein in lung tissue of rats in the four groups treated with drug intervention was down-regulated(P<0.05).Compared with DM group,the expression of p-STAT3 protein in lung tissues of the three groups treated with dexamethasone combined with Chinese medicine was down-regulated(P<0.05).There was no statistical significance in the expression of STAT3 protein in lung tissues among the six groups(P>0.05).7.Ultrastructure of adrenal cortex: Compared with ZC group,the cell morphology of rats in XC group was similar to that in ZC group,with a large number of mitochondria and lipid droplets.In the adrenal cortical cells of DM group,mitochondrial swelling,unclear ridge structure,lipid droplet number decreased significantly,and fusion phenomenon were observed.After combined administration of Jianpi Bufei Huatan Formula,the cell structure of adrenal cortex fasciculate zone was improved compared with that of DM group.8.Serum ACTH and CORT levels: Compared with ZC group,the serum ACTH and CORT levels of other groups were decreased in different degrees(P<0.05).Compared with XC group,the contents of serum ACTH and CORT in the four groups treated with drug intervention were lower(P<0.01).Compared with the DM group,the serum ACTH and CORT levels in the combined treatment group were increased to a certain extent(P<0.01).9.Expression of Cyp11a1,Cyp11b1 and St AR genes in adrenal tissues: compared with the ZC group,the expression of Cyp11a1 m RNA,Cyp11b1 m RNA and St ARm RNA in adrenal tissues of the remaining five groups of rats were all decreased(P<0.01).Compared with the rats in the XC group,there were significant differences in the expression of St ARm RNA,Cyp11a1 m RNA and Cyp11b1 m RNA in the adrenal tissues of the rats in the four groups after the drug intervention(P<0.01).Compared with the DM group,the elevated expression of St ARm RNA,Cyp11a1 m RNA and Cyp11b1 m RNA in the adrenal tissue of rats in each group of the combined drug was statistically significant(P<0.05).10.IF detection of St AR protein expression in adrenal tissues: the number of St AR fluorescent spots was reduced in the remaining five groups compared with the ZC group,and the difference was statistically significant(P<0.05).Compared with the XC group,the difference in the number of St AR fluorescent spots in the four groups with pharmacological intervention was statistically significant(P<0.05).Compared with the DM group,the number of St AR fluorescence spots appeared to be increased to different degrees in each dose group of dexamethasone plus the formula for strengthening the spleen,tonifying the lung and resolving phlegm(P<0.01).Conclusion:1.The combination of the formula of Strengthening the spleen,tonifying the lung and resolving phlegm and dexamethasone can significantly improve the pathomorphological changes such as inflammatory cell infiltration and collagen deposition in lung tissues of asthmatic rats.2.The combination of Spleen Tonic and Lung Resolving Phlegm Formula and dexamethasone had better effects than dexamethasone alone in reducing the levels of IL-17 A and IL-22 in serum and alveolar lavage fluid,and in decreasing the expression of IL-22 m RNA,STAT3 m RNA and p-STAT3 protein in lung tissues of asthmatic rats.3.There is a certain degree of adrenocortical suppression in asthmatic rats,and dexamethasone treatment aggravates their adrenocortical insufficiency.4.The combination of Jianshu Tonic Lungs and Dexamethasone can improve the adrenocortical insufficiency caused by dexamethasone treatment alone,which may be achieved by up-regulating the expression of steroid hormone synthesis-related enzymes and proteins Cyp11a1 m RNA,Cyp11b1 m RNA and St ARm RNA. |
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