| BackgroundBronchial asthma(asthma)is a chronic respiratory inflammatory disease characterized by reversible airflow limitation and high airway reactivity.Current studies generally show that airway remodeling is the pathologic structural basis for the occurrence of reversible airflow restriction and airway hyperreactivity.As the disease progresses,the airway remodeling continues to increase and the bronchial asthma patients' response to conventional treatment drugs and treatment options continues to decrease,and even drug resistance appears in some asthma patients.In the above process,airway smooth muscle cell(ASMC)hyperplasia and hypertrophy are considered to be the main mechanisms of airway remodeling.Transforming growth factor(TGF)-? 1/Smad signaling pathway is an important signal transduction pathway of airway smooth muscle cells involved in the pathogenesis of asthma,which is closely related to the proliferation and protein expression of airway smooth muscle cells.In the Smad protein family,although the structure of Smad2 and Smad3 is very similar,some amino acid structure of Smad3 protein can directly bind to the DNA sequence to regulate gene transcription,that is,Smad3 protein can participate in the regulation of target gene caused by TGF-?1.Interleukin(IL)-33 is considered to be an asthma susceptibility gene and plays a key role in the exacerbation of bronchial asthma induced by rhinoviruses.Previous studies have shown that airway smooth muscle cells secrete IL-33,and the correlation between IL-33 and TGF-?1/Smad3 signaling pathway has not been reported.Studies at home and abroad have shown that 1,25-(OH)2D3(calcitriol)can alleviate airway remodeling in vivo and in vitro,and the specific mechanism affecting airway smooth muscle cells is still under investigation.Based on the latest research at home and abroad,we speculated that TGF-?1 can stimulate the expression of IL-33 in airway smooth muscle cells through the TGF-?1/Smad3 signaling pathway,thereby promoting the inflammatory response in asthma.Calcitriol can be combined with budesonide to more effectively inhibit airway inflammation and airway remodeling and play its role in the treatment of asthma.The aim of this research is to find new therapeutic targets for asthma.Objective1.To investigate the effects of different concentrations of TGF-?1 on the expression and secretion of IL-33 inmouse airway smooth muscle cells cultured in different concentrations of TGF-?1 medium;2.TGF-?1/Smad3 signaling pathway blockers(SIS3)were used to observe the effect of TGF-?1/Smad3 signaling pathway on the expression and secretion of IL-33 by airway smooth muscle cells.3.To study the therapeutic effect of calcitriol combined with budesonide on bronchial asthma and its possible mechanism.Methods1.The mouse airway smooth muscle cells were divided into groups with different TGF-? 1concentration[0ng/mL(blank),lng/mL,10ng/mL,100ng/mL].The supernatant and total protein were obtained.The IL-33 concentration in supernatant of each group was detected by ELISA method,and the expression of IL-33 protein in each group was detected by Western blotting method.2.TGF-?1/Smad3 signaling pathway blocker(SIS3)was added to treat mouse airway smooth muscle cells,which were divided into blank group,pretreated TGF-?1 group,unpretreated SIS3 group and pretreated SIS3 group.The IL-33 concentration in supernatant of each group was detected by ELISA method,and the expression of Smad3,pSmad3 and IL-33 protein in each group was detected by Western blotting method.3.The mouse airway smooth muscle cells were divided into blank group,TGF-?1 group,SIS3 group,budesonide group,calcitriol group and drug co-treatment group.The IL-33 concentration in supernatant of each group was detected by ELISA method,and the expression of Smad3,pSmad3 and IL-33 protein in each group was detected by Western blotting method.Results1.ELISA showed that IL-33 concentration in the supernatant of cell culture was different after TGF-?1 stimulated mouseairway smooth muscle cells.10ng/mL TGF-?1 worked best,while 1 ng/mL TGF-? and 100 ng/mL TGF-?1 did not differ significantly.At the same time,Western blotting showed that after TGF-? 1 stimulated mouseairway smooth muscle cells with different concentrations,cells in each group expressed IL-33 to different degrees.The IL-33 protein expression was highest in 10ng/mL TGF-? 1group.2.After the addition of SIS3,the concentration of IL-33 in the cell culture supernatant of each group was detected by ELISA.The results showed that the concentration of IL-33 in the cell culture supernatant of the pretreated SIS3 group was significantly reduced compared with the pretreated TGF-?1 group.Western blotting showed that after the addition of SIS3,TGF-?1/Smad3 signaling pathway was inhibited,Smad3 protein phosphorylation level decreased,Smad3 protein and pSmad3 protein expression decreased,and IL-33 protein expression also decreased significantly.3.After the drug treatment of airway smooth muscle cells in each group,ELISA showed that the concentration of IL-33 in the supernatant of cell culture in budesonide group was lower than that in the calcitriolgroup,and the concentration of IL-33 in the drugco-treatment group was lower than that in any single drugtreatment group.It showed that the expression level of Smad3 protein,pSmad3 protein and IL-33 protein of western blot in the drug co-treatment group were significantly decreased.Conclusions(1)Different concentration of TGF-?1 stimulates airway smooth muscle cells to secreteandexpressIL-33 to different degrees,suggesting that IL-33 and TGF-?1 have a correlationinsomeway.10ng/mLTGF-? 1is the optimal concentration for this action.(2)TGF-(?1/Smad3 signaling pathway can regulate the expression and secretion of IL-33 in airway smooth muscle cells,which plays an important role in the pathogenesis of asthma.(3)Calcitriol combined with budesonide can effectively decrease the expression and secretion of IL-33 in mouseairway smooth muscle cells byactivatingTGF-(? 1/Smad3 signaling pathwaytoinhibit airway inflammation,and thus treat asthma. |
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