| Objective:Asthma is closely related to inflammation and immune response of the body,and let-7a is closely related to the progression of asthma disease.This study intends to systematically and comprehensively clarify the effect of nebulized budesonide on the expression of let-7a and related cytokines of IL-4、IL-5、IL-13、IL-10、IFN-γ、IL-17 in asthmatic mice at the level of cells,proteins,and tissues,and the changes in lung tissue inflammation in mice;At the same time,compare the effects of the original research and the domestic imitation BUD.It provides new experimental basis and theoretical support for the treatment of asthma,new theoretical basis for the effective treatment of asthma by inhaling BUD in clinical practice,and new targets for the treatment of asthma.Methods:Twenty-four Specific disease Free SPF BALB/c mice were randomly divided into normal saline control group(control group),asthma model group(asthma group),BUD 1(domestic budesonide suspension group)and BUD2(imported budesonide suspension group)with 6 mice in each group according to random number table.The asthma group was sensitized by intraperitoneal injection of 0.2 m L of saline suspension of Ovalbumin(OVA)+ aluminum hydroxide gel on days 0,7 and 14.On day 21,1% OVA was atomized for 30 minutes,once a day,for consecutive 14 days.The asthma model group was atomized and inhaled 2 m L of saline 1 hour before each stimulation.Bud1 group and Bud2 group were sensitized and stimulated the same as asthma group,but budesonide suspension was atomized 1 mg(2 m L)before each stimulation(domestic budesonide and imported budesonide drugs,respectively).Control group: Normal saline solution containing only aluminum hydroxide gel was used,the injection site,dose and time were the same as those in asthma group.On day 21,normal saline was atomized for 30 minutes,once a day,for consecutive 14 days,and 2 m L of normal saline was inhaled 1 time before each stimulation.On day 35,mice in each group were treated separately,and the Broncho Alveolar Lavage Fluid Balf was collected,and the lung tissues of mice were extracted.Hematoxylin-eosin(HE)staining was performed on the lung tissue of mice to observe the presence of inflammatory cell infiltration and structural changes in the lung tissue.Periodic acid-Schiff PAS staining of lung tissue was observed to detect goblet cell proliferation and mucus secretion in bronchial lumen.Enzyme-linked Immuno Sorbent Assay ELISA was used to analyze the expression levels of IL-4,IL-5,IL-13,IL-10,IFN-γ and IL-17 in the alveolar lavage fluid.Elisaclac software was used to calculate the concentration of each sample.Real-time quantitative PCR(real-time fluorescence quantitative PCR)was used to detect the expression of let-7a in the lung tissues of mice.The results of the control group were used as a reference,and the relative expression level of let-7a was indicated by 2-△△ Ct.Results SPSS22.0 software was used for statistical analysis,and Graph Pad Prism 8 software was used to draw histograms.Results:1.Mice in the asthma model group showed restlessness,incontinence and scratching their ears and cheeks during the atomization process.Mice in the BUD1 and BUD2 groups were relatively quiet,while mice in the control group had no such phenomenon.2.HE staining: the bronchial wall of the lung tissue of mice in the asthma model group was thickened,the lumen was narrowed,and the infiltration of inflammatory cells around the bronchus and blood vessels was serious.The bronchial wall of lung tissue of mice in BUD1 group and BUD2 group was not thickened,the lumen was normal,and the infiltration of surrounding inflammatory cells was less.The bronchial wall and lumen of the lung tissue of the control group were normal,and there was no obvious infiltration of inflammatory cells.3.PAS staining: in the asthma group,the goblet cells of bronchial epithelium were proliferated,mucus secretion was increased,epithelial cells were exhumed,bronchial wall was thickened,and surrounding inflammatory cells were infiltrated.The goblet cells in the lumen of BUD1 group and BUD2 group were slightly proliferated,and the surrounding inflammatory cells were slightly infiltrated.There was no goblet cell proliferation in the bronchial lumen of the control group.4.ELISA detection of related cytokines(IL-4,IL-5,IL-13,IL-10,IFN-γ,IL-17)in BALF: the expression levels of IL-4,IL5,IL-13,IL-17 in asthma group were significantly higher than those in control group(P<0.05),and the difference was statistically significant.The expression levels of IL-10 and IFN-γ in asthma group were significantly lower than those in control group(P<0.05),and the difference was statistically significant.The expression levels of IL-4,IL5,IL-13 and IL-17 in BUD1 and BUD2 groups were significantly lower than those in asthma group(P<0.05),and the difference was statistically significant.The expression levels of IL-10 and IFN-γ in BUD1 and BUD2 groups were significantly higher than those in asthma group(P<0.05),and the difference was statistically significant.The expression levels of IL-4,IL-5,IL-13,IL-10,IFN-γ and IL-17 in BUD1 group were not significantly different from those in BUD2 group(P>0.05).5.The expression level of let-7a in lung tissue was detected by real-time quantitative PCR: the expression level of let-7a in asthma group was significantly lower than that in control group(P<0.05),and the difference was statistically significant.The expression level of let-7a in BUD1 and BUD2 groups was significantly higher than that in asthma group(P<0.05),and the difference was statistically significant.The expression level of let-7a in BUD1 group was no different from that in BUD2 group(P>0.05).CONCLUSIONS:1.Budesonide can increase the expression of let-7a in asthmatic mice.2.Budesonide can decrease the expression levels of IL-4,IL-5,IL-13,IL-17,and increase the expression levels of IL-10,IFN-γ in asthmatic mice.3.Domestic budesonide and imported budesonide showed no difference in regulating the expression of let-7a and related cytokines IL-4,IL-5,IL-13,IL-10,IFN-γ and IL-17. |
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