| Asthma is a chronic inflammatory airway disease with reversible airflow obstruction,persistent airway hyperresponsiveness(AHR),and airway remodeling.Clinical manifestations include wheezing,shortness of breath,chest tightness,and/or cough.It recurs and may worsen over time,leading to respiratory failure.As a public health issue,asthma affects more than 300 million people worldwide and places a heavy economic burden on society.Although asthma has attracted enough attention worldwide,but its treatments are still limited,and inhaled glucocorticoid(IGS)-based treatments do not benefit all asthma patients.A large number of studies on the immune mechanism of bronchial asthma have shown that the differentiation of CD4+T cells(Th cells)and the secretion of various inflammatory mediators and cytokines play an important role in the pathogenesis of asthma.Most asthma phenotypes are associated with Th1/Th2 imbalances,Treg/Th2 imbalances,and Th17 overexpression.Therefore,the treatment of asthma by correcting immune abnormalities has become an avenue that can be explored.TCM has a long history of diagnosis and treatment of asthma.The"Chuan-ming"and "Chuan-he" described in"The Inner Canon of Huangdi"are similar to the characteristics of this disease.In the Yuan Dynasty,Zhu Danxi first wrote "Asthma" as an independent disease monograph in" Danxi’s Mastery of Medicine".The ancients left a lot of effective prescriptions for treating asthma in clinical practice.Nowadays,researchers use modern science and technology to carry out in-depth discussions on these prescriptions and make scientific explanations for their mechanism of action.In this study,Shegan-Mahuang Decoction(SMD)was selected as the intervention,and its mechanism of treatment of asthma was explored from the perspective of immunity.ObjectiveTo establish a mouse model of asthma,using Shegan-Mahuang Decoction(SMD)to intervene,and comparing it with dexamethasone intervention.In order to observe the differentiation of Th cell subgroups(Th2,Th17,Treg)and related cytokines,exploring the effect of Shegan-Mahuang Decoction(SMD)on asthma Mechanisms of immune intervention and airway inflammation.MethodsBALB/c female mice aged 6-8 weeks and weighing 18-22g were selected to establish a mouse asthma model using classical OVA combined with AL(OH)3 sensitization and challenge.Sensitization:Each mouse was injected intraperitoneally with 0.5mL of sensitizing solution containing 50 μg OVA(Grade II)and 2mg AL(OH)3 on day 0,6 and 13 of the experiment.Challenge:From the 21st day of the experiment,the sensitized mice were placed in a mouse atomization inhalation box,and 1%OVA(GradeⅡ)8ml was used for atomization and inhalation,once a day for 60min for 2 weeks.At the same time,the asthma model mice were randomly divided into 5 groups:Control group,Model group,Dexamethasone group(Dex),Low-dose Chinese medicine group(SMD-L)and high-dose Chinese medicine group(SMD-H),with 10 rats in each group.From the 21st day of the experiment,the drug was administered half an hour before the atomization.The Control group was fed normally without intervention.The Model group was intraperitoneally injected with 0.2ml of normal saline.The Dex group was intraperitoneally injected with dexamethasone injection lmg/kg(0.2mL).The SMD-L group was administered with Shegan-Mahuang Decoction 4.7g crude drug/kg(0.15mL).The SMD-H was administered with Shegan-Mahuang Decoction 18.9g crude drug/kg(0.15mL).After 2 weeks of nebulization,mice in each group were subjected to invasive lung function testing to detect airway resistance under anesthesia,and the lungs,spleen,lymph nodes and serum of the mice were collected within 24 hours.The pathological changes of lung tissue were observed by HE staining.The infiltration of CD3+and CD4+T cells in lung tissue was observed by immunohistochemistry.Detection of IL-4,IL-17A,and IL-23 cytokines in serum with the Milliplex(?)MAP Kit.Flow cytometry was used to detect the expression of CD4+Foxp3+Tregs cells in mouse spleen and lymph nodes.Western blot was used to detect the expression of P70s6k,P-P70s6k,RelB and P65 protein in lung tissue.Quantitative Real-time PCR(qRT-PCR)was used to detect the mRNA expression of the transcription factors of GATA-3,ROR γt and T-bet.Results1.Experimental mice invasive lung function results:Airway resistance in the Model group was higher than that in the Control group(P<0.001),indicating that we successfully established a mouse model of asthma.Compared with the Model group,the SMD-L group(P<0.05)and the SMD-H group(P<0.01)can reduce airway resistance in asthmatic mice,and the SMD-H group has almost the same effect as the Dex group.2.HE staining results of lung tissue:The inflammation of the lung tissue was the most obvious in the Model group,with inflammatory cell infiltration around the trachea and blood vessels,as well as thickening of the alveolar wall and extravasation of fluid.Compared with the Model group,the SMD-L group and the SMD-H group can significantly reduce inflammatory cell infiltration and alveolar wall destruction,and there are fewer eosinophils and mucus plugs in the lung.3.Lung tissue immunohistochemical results:Compared with the Model group,the SMD-L group and the SMD-H group can reduce the infiltration of CD3+T cells in the lung tissue of asthmatic mice(P<0.01).At the same time,the SMD-H group also showed inhibition of CD4+T cell infiltration(P<0.01),while the SMD-L group had no statistical difference compared with the Model group(P>0.05).Compared with the Model group,CD3+T cell and CD4+T cell infiltration were reduced in the Dex group(P<0.01).4.Milliplex(?)MAP multifactor detection serum results:Compared with the Control group,OVA-induced Model group increased the production of IL-4,IL-17A and IL-23 in the serum(P<0.05).The SMD-H group can reduce the production of IL-4,IL-17A and IL-23 in serum(P<0.05).However,there was no significant difference between the SMD-L group and the Model group(P>0.05).5.Mice spleen and lymph node flow cytometry results:In the OVA-induced Model group,CD4+Foxp3+Treg in spleen and lymph nodes of asthmatic mice was reduced(P<0.001).Compared with the Model group,the SMD-H group increased the proportion of CD4+Foxp3+Treg cells in the spleen of asthmatic mice(P<0.01).On the other hand,the SMD-H group also increased the frequency of CD4+Foxp3+Treg cells in lymph nodes of asthmatic mice(P<0.05).However,there was no statistical difference between the SMD-L group and the Model group,whether in the spleen or lymph nodes(P>0.05).The Dex group as a positive control showed a significant increase in the proportion of CD4+Foxp3+Treg cells in the spleen and lymph nodes(P<0.001).6.Expression of mTOR and NF-κB pathway-related proteins in lung tissues:Both the SMD-H and the SMD-L groups inhibited the expression of P70s6k(P<0.001)and phosphorylated P70s6k(P-P70s6k)(P<0.05)in the lung tissue.Compared with the Control group,the expression of P65 in the Model group was higher(P<0.001),and the expression of RelB was also increased(P<0.05).Compared with the Model group,the expressions of RelB and P-65 were reduced in the SMD-H group(P<0.05).Compared with the Model group,the effect of SMD-L group on the expression of RelB showed no statistical difference(P>0.05).The Dex group also showed a inhibitory effect on mTOR and NF-κB pathway related proteins(P<0.05).7.The mRNA expression results of the transcription factors about Th cell:Compared with the Control group,the Model group showed that mRNA expression of ROR γt(P<0.001)and GATA-3(P<0.01)increased,while T-bet decreased(P<0.01).After treatment with SMD-H,SMD-L and Dex,the mRNA expression of GATA-3 and RORγt in the lung tissue of mice decreased,and the mRNA expression of T-bet increased(P<0.05).ConclusionShegan-Mahuang Decoction inhibits Th2 and Th17 differentiation,promotes CD4+Foxp3+Treg production,and alleviates airway hyperresponsiveness and airway inflammation by regulating the abnormal immune environment in asthmatic mice.In addition,Shegan-Mahuang Decoction also inhibited mTOR and NF-κB activity and reduced airway inflammation. |
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