| Background:Asthma is a complex heterogeneous disease characterized by chronic airway inflammation and airway hyperresponsiveness[1].At present,asthma has become the second most prevalent chronic lung disease in the world,and it severely threatens the health and quality of life of the public because of its high incidence rate and poor control rate.Therefore,there is an urgent need to determine the pathological mechanism of asthma so that more effective treatments can be developed.Asthma is generally considered to be a type I allergic airway disease mediated by T helper 2(T2 cells)and immuno-globulin E(IgE),and it is usually accompanied by a dramatic increase in airway and lung eosinophils[2].As reported byrecent studies,in approximately 50%of asthma cases,especially moderate to severe asthma and glucocorticoid-resistant asthma,a significant increase in neutrophils is measured[3,4].Present studies showed that the increased accumulation of neutrophils in airway is primarily due to the delayed or reduced autophagy o neutrophils[5],which could be involved in increased migration of neutrophils to lung[6].However,the underlying mechanism still need to be elucidated.As extracellular vesicle,exosome can promote the intercellular transfer of proteins,nucleic acids and metabolites.The transfer of bioactive substances suggests that exosome may play an important role in underlying mechanisms for diseases.And exosomes probably acts as a porter for asthma-related immunoregulatory cellular interaction[7,8].As we all known,internal ROS can induce the accumulation of iNOS when asthma especially severe asthma attacks,thus influenced the level of NO.Free radicals can promote the maturation of MDRC(myeloid-derived regulatory cells),which is considered as crucial regulatory factors in airway inflammation.NO can lead to the formation of immune-repressive MDRCs,which can inhibit the proliferation of T cells and airway hyperresponsiveness.However,superoxide-induced MDRCs is the pro-inflammatory airway factors[9].Shegan Mahuang Decoction(SMD)is a representative prescription of traditional Chinese medicine(TCM),and it can effectively relieve the clinical symptoms of asthma patients[11].But there is little known for its effect on neutrophilic asthma.Thus,focused on the effect of Shegan Mahuang Decoction on neutrophilic asthma,this thesis takes a deep dive into the biological mechanism by which Shegan Mahuang Decoction treats the neutrophilic asthma.This thesis will elucidate the following medical and scientific questions:1,what about the impact of Shegan Mahuang Decoction on neutrophilic asthma?2,what is the biological mechanism by which Shegan Mahuang Decoction treats the neutrophilic asthma?3,what is working mechanism of irisflorentin,which is major component of Shegan Mahuang Decoction?Methods:1.Establish of a neutrophilic asthma mouse model,and verify the curative effect of Shegan Mahuang Decotion on neutrophilic asthma mice(1)Preparation of lipopolysaccharide(LPS)and ovalbumin(OVA)stock solution:0.03 g LPS powder was dissolved in 0.3 mL of normal saline to make a 100 μg/μL solution.For usage,this solution was diluted 10 times to 10 μg/μL and stored at 4℃.For OVA,0.1 g of powder was dissolved in 1 mL of normal saline to make a 100 μg/μL solution.For usage,this solution was diluted 100 times to 1 μg/μL and stored at 4℃.BALB/c mice in the NA and SMD treatment groups were anesthetized on day 1 and day 7;LPS(4 μg/g,calculated based on mouse body weight)was instilled from the airway,and OVA(100 μL/mice)was injected intraperitoneally.From the 14th day to the 18th day,the mice were challenged by aerosolizing 5 mL of OVA solution every day,thereby establishing a neutrophilic asthma mouse model.The mice in the control group were sensitized with phosphate-bufered saline(PBS)instead of LPS or OVA,challenged with normal saline,and the experiment was performed synchronously with the model group.(2)After model establishment,intragastric administration of drugs was performed for the mice in each group,and the same amount of normal saline(10 μL/g)was intragastrically administered to the mice in the NC and NA groups.The SMD-L and SMD-H groups were treated with relative SMD,for 5 consecutive days,twice a day.All mice were euthanized for analysis 24 hours after establishing the model.(3)Analyze the bronchial structure of the lung tissue and the infiltration situation of peripheral inflammatory cells via pathological section of lung tissue.(4)Analyze the cardiac structure via pathological section of heart.(5)The airway resistance was measured using a small animal pulmonary function measurement system to evaluate the airway reaction of the mice to methacholine.2.Investigate the potential mechanism by which Shegan Mahuang Decoction treats the neutrophilic asthma mice(1)Analyze the changes of cytokines(IL-4,IL-13,IL-33,and TGF-β1)in the BALF via ELISA assays.(2)Analyze the number of CD8/CD11 double positive cells of BALF via FACS(3)Extract the exosomes of MDRC in mice using differential centrifugation,view the morphology of the exosomes using transmission electron microscopy,and analyze the protein level of CD9 and CD63 via western blot,which are the marker proteins for exosomes.(4)Analyze the changes of protein levels of mitochondrial fusion protein(MFN1),mitochondrial membrane protein(MIRO1),mitochondrial transcription activator(NRF1)and the expression changes in mitophagy-related proteins LC3B and Beclinl in the exosomes via western blot.3.Investigate the working mechanism of irisflorentin,which is the major component of Shegan Mahuang Decoction(1)Analyze that irisflorentin is the major component of Shegan Mahuang Decoction,and verify the curative effect of irisflorentin on neutrophilic asthma mice.(2)Analyze the changes of cytokines(IL-4,IL-10,IL-13,IL-33,IL1RL1,IFN-γ,and TGF-β1)in the BALF via ELISA assays.(2)Analyze the number of CD8/CD11 double positive cells of BALF via FACS(3)Analyze the bronchial structure of the lung tissue and the infiltration situation of peripheral inflammatory cells via pathological section of lung tissue.(4)Analyze the changes of protein levels of mitochondrial fusion protein(MFN1),mitochondrial membrane protein(MIRO1),mitochondrial transcription activator(NRF1)and the expression changes in mitophagy-related proteins LC3B and Beclinl in the exosomes via western blot.Results:1.Establish of a neutrophilic asthma mouse model,and verify the curative effect of Shegan Mahuang Decotion on neutrophilic asthma mice(1)Succeeded in constructing neutrophilic asthma mouse model.(2)The results from pathological section of lung tissue showed that compared with the NA group,in the SMD-L and SMD-H groups,there was significantly less damage to the lung tissue structure,and less bronchial mucosa edema and inflammatory cell infiltration,with clearly more normalized features in the SMD-H group.(3)The results from pathological section of heart showed that there is no significant difference between each groups of samples.(4)The results from small animal pulmonary function measurement system showed that high dose of Shegan Mahuang Decoction had much more efficient treatment for neutrophilic asthma mice.2.Investigate the potential mechanism by which Shegan Mahuang Decoction treats the neutrophilic asthma mice(1)Compared to the NA group,IL-33(P<0.05),TGF-β1(P<0.05),IL13(P<0.01),and IL-4(P<0.05)significantly decreased in the SMD-H group via ELISA assays,which suggested that high doses of Shegan Mahuang Decoction could reduce the expression level of pro-inflammatory cytokines of BALF in neutrophilic asthma mice.(2)The results from FACS showed that the percentage of MDRCs of BALF in neutrophilic asthma mice decreased after the treatment of Shegan Mahuang Decoction.(3)CD63 was highly expressed in the exosomes of the control group,while CD63 expression was sharply decreased in the model group,and CD63 decreased further after SMD treatment.On the contrary,the expression of CD9 in the control group was lower than that in the model group and recovered after SMD treatment.(4)The results showed that the expression of MFN1,MIRO1,and NRF1 in the exosomes of the control group was higher than that of the NA group and the SMD-H group.However,the expression of mitophagy-related proteins LC3B and Beclinl in the exosomes of the control group was relatively low but significantly increased in the NA group.After SMD treatment,the expression of LC3B and Beclinl decreased.3.Investigate the working mechanism of irisflorentin,which is the major component of Shegan Mahuang Decoction(1)Analyze that irisflorentin is the major component of Shegan Mahuang Decoction.(2)The ELISA results showed that the levels of cytokines(IL-4,IL-10,IL-13,IL-33,IL1RL1,IFN-γ,and TGF-β1)in the BALF significantly decreased after irisflorentin treatment.And the curative effect of high-dosage group was better than that of low-dosage group.(3)The results from pathological section of lung tissue showed that compared with the NA group,there was significantly less damage to the lung tissue structure,and less bronchial mucosa edema and inflammatory cell infiltration,with clearly more normalized features after irisflorentin treatment.(4)The expression levels of LC3B and Beclinl in the exosomes decreased after irisflorentin treatment via western blot.Conclusions:(1)Combined literature survey with multiple detection technology including ELISA,pathological section of lung tissue,FACS and WB,this thesis found that Shegan Mahuang Decoction had an effective curation for neutrophilic asthma mice.(2)Further mechanism analysis indicated that Shegan Mahuang Decoction may regulate the function of the mitochondria in MDRCs-derived exosomes by reducing the expression levels of mitophagy-related proteins and then regulate downstream T cells,thereby affecting the secretion of BALF cytokines in the mouse model of neutrophilic asthma and decreasing the occurrence of inflammation.(3)Through analyzing the major component of Shegan Mahuang Decoction,this thesis found that irisflorentin is probably the key component of Shegan Mahuang Decoction for efficient curation of neutrophilic asthma. |
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