| Bronchial asthma,which is also referred to as asthma,is a common chronic airway disease affecting approximately 300 million people worldwide.The number of eosinophils in the peripheral blood and lung tissue of asthmic patients remains at a high level and is often related to the severity of asthma.Eosinophils contain cytotoxic granule proteins in their cytoplasm.In addition,they can synthesize nearly 28 cytokines,chemical factors,and growth factors,and can also secrete factors that promote airway mucus secretion and smooth muscle contraction.As a result,eosinophils can cause airway obstruction and narrow.The pro-inflammatory production secreted by eosinophils is one of the main factors that make airway epithelial damaged.When airway epitheial cells are damaged,type 2 T helper(Th2)immune response is induced,inflammatory cytokins are released,and further inflammtory responses are triggered.As a result,airway hyperresponsiveness appears and asthma breaks out.Studies have found that the anti-apoptotic ability of eosinophils in asthmatic patients is enhanced,which is also the key reason for the accumulation of eosinophils in peripheral blood and lung tissue.Former studies suggest that factors such as granulocyte-macrophage colony stimulating factor(GM-CSF)and Interleukin-5(IL-5)are important to the maintenance of the eosinphilic airway inflammation.However,new studies have found that the enhancement of anti-apoptosis ability in eosinophils can only be partially inhibited by anti-GM-CSF and anti-IL-5 theraties.In addition,IL-33,leptin,and the acidic airway pH environments are all factors those can affect the the survival ability of eosinophils.In summary,a combination of multiple factors contributes to the significant increase in eosinophilic activity in asthmatic patients.Current studies have demonstrated that reducing eosinophils can significantly relieve the symptoms of asthma.Therefore,targeting eosinophils is the focus of researches in asthma.Several eosinophil-targeted drugs such as Mepolizumab,Reslizumab and Benralizumab are recommended to used in the severe asthma by the updated GINA guidelines.Targeted drugs have already been used in clinical.However,the use of targeted drugs in many country countries including China has been limited because of the high price.Consequently,the development of drugs with low price and significant efficacy is necessary.Artesunate(ART)is a semi-synthetic artemisinin derivative.Its sodium salt has good water solubility,and it is a first-line antimalarial drug.Chinese pharmacologist Tu Yuzheng was awarded the Nobel Prize in Physiology or Medicine for the discovery of artemisinin in October,2015.Studies have found that artesunate also had many other effects such as anti-tumor and anti-inflammation.It can induce dysfunction of mitochondrial through the p53 and Bcl-2 pathway,thus inducing programmed cell death of cancer cells.Its treatments for breast cancer,rectal cancer and hepatocellular carcinoma have already entered the phase II clinical trials.It has also been found that artesunate can play an anti-inflammatory role through oxidative stress and NF-?B signaling pathway,resulting in good therapeutic effects on autoimmune related diseases and allergic inflammation.The treatment of artesunate in systemic lupus erythematosus has also entered the phase IV clinical trials.Considering the facts that the ability of anti-apotosis in eosinophils enhances in the asthma which is a chronic airway inflammation,and that artesunate can induce apotosis of tumor cells as well as have anti-inflammatory effects,whether artesunate can alleviate eosinophilic airway inflammation in asthmatic patients by inducing apoptosis of eosinophils deserves further study.In addition,computer-aided techniques provide a great deal of convenience in predicting the effects of artesunate on asthma.With the development of sequencing technology,the Genome Wide Association Study(GWAS)provides new ideas for understanding the mechanism of asthma and developing targeted drugs.The GEO(Gene Expression Omnibus)database is one of the NCBI(National Center for Biotechnology Information)online public databases that includes multi-species and multi-disease genome wide information.The GEO database includes sequencing results of asthmatic patients with different subtypes and provides services for online analyzes.Researchers can use GE02R which is an online tool provided by the GEO database,to analyse homogenized sequencing data and to obtain differentially expressed genes.Then with the help of functional enrichment classification and protein-protein interaction analyzes,researchers can understand the pathogenesis of severe asthma better.Docking is a kind of computational chemistry methods.It refers to using softwares to put the ligands to the active sites of macromolecules in order to evaluate the interaction and the efficacy of the ligands based on their geometric properties,energy relationships,and the chemical environment.The development of computational simulation methods such as docking,greatly increase the efficiency of developing new drugs and also provide new ideas for predicting the effects of artesunate on asthma.Based on previous studies,our study focuses on the effects and mechanisms of artesunate on eosinophilic airway inflammation when administrated intratracheally in a mouse model of asthma.Besides,we also study the effects of artesunate on human eosinophils in vitro.Finally,we download the genome wide sequencing data of asthmatic patients provided by the GEO database,and analyse the possibility of artesunate as a therapy for asthma with the help of bioinformatics.ObjectiveTo study the role and mechanisms of artesunate in eosinophilic airway inflammation of the asthmatic mouse model.To explore its mechanism of specific effects on eosinophils.The bioinformatic methods were used to predict whether artesunate can be targeted treatment for asthma.MethodsPart 1The HDM asthmtic mouse model was established by instilling house dust mite antigen(HDM)into the airway of wild-type mice.Different concentrations of artesunate were instilled into the airway of model mice,and the airway eosinophilic inflammation in the mice of each group was detected.Explore the best dose of artesunate.Part 2Construct the HDM mouse model,instill artesunate into the airway,and observe effects of artesunate on eosinophils in vivo.Collect inflammatory cells from bronchoalveolar lavage fluid(BALF)of asthmatic mice,extract peripheral blood eosinophils from IL-5 transgenic NJ.1638 mice.Both were treated with artesunate in vitro to study the effects and mechanisms of artesunate on eosinophils in vitro.Instill artesunate into the mouse model of neutrophilic airway inflammation induced by lipopolysaccharide(LPS).The airway inflammation was detected.Collect BALF cells from LPS model mice.Cells were cultured in vitro and treated with artesunate to study whether artesunate had effects on neutrophils.Part 3Enrich human peripheral blood eosinophils,treat them with Artesunate of different concentrations in vitro and observe the effects.The data set GSE74986 which was a collection of asthma patient's alveolar lavage fluid sequencing data was downloaded from the GEO database.Important genes and key proteins that might be involved in the pathogenesis of severe asthma were analyzed using bioinformatic tools,and the interactions between artesunate and these proteins were predicted.The possible targeted proteins and potential therapeutic effects of artesunate in asthma were predicted according to the its chemical structure.The effects of artesunate on asthma were predicted based on the results.ResultsPart 11.The proportion of eosinophils in BALF of HDM model mice increased to 61.2±3.9%,while the proportion dropped to 3.9±3.4%when 50?g ART/50?l normal saline(NS)artesunate was instiled into the airway of the model mice(P<0.05).The total number of eosinophils also decreased from(32.0±10.3)x104/ml to(0.4±0.3)x104/ml(P<0.05).2.When the dose of artesunate was more than 10?g ART/50Ll NS,the decrease of the eosinophilic ratio in the BALF of the HDM model mice did not change much more.However,the best dose of artesunate was 50?g ART/50?l NS.3.Instillation of 50?g ART/50?l NS artesunate could relieve the airway inflammatory infiltration and mucus secretion.The inflammation score was decreased from 3.7±0.5 to 0.3±0.5(P<0.05),and the mucus score was from 3.2±0.8 to 0.3±0.6(P<0.05).The expression of Th2 inflammatory cytokines like IL-4?IL-10?IL-13?IL-17?IL-25?IL-3 3 and Muc5ac which was associated with mucus secretion was also decreased.Part 21.After treated with 50?g ART/50?l NS artesunate,the inflammatory cells in BALF of HDM model mice had vacuolar degeneration,and the expression of Bcl-2 decreased while the expression of Cleaved Caspase-9 increased.Besides,the expression of Bcl-2 in lung tissue also decreased.2.The inflammatory cells from BALF of HDM model mice were extracted and cultured in vitro.The survival rate of leukocytes was decreased after treated with 10?M artesunate for 12h.The number of eosinophils decreased from(20.5±2.4)x103/ml to(9.8±1.5)x103/ml(P<0.05).When the concentration of artesunate were increased to 50?M and 100 ?M,the survival rate of leukocyte and the number of surviving eosinophils decreased further(P<0.05).The morphology of the treated cells changed when observed under microscope.The results of immunofluorescence showed that the expression of Cleaved Caspase-9 in BALF cells increased after treatment.3.Eosinophils in the peripheral blood of NJ.163 8 mice were extracted and cultured in vitro for 24h.The number of survived eosinophils in the control group was(57.7±1.8)×104/ml,while the number of those treated with 10 ?M,50 ?M,and 100?M artesunate decreased to(31.9±1.7)x104/ml,(24.8±1.0)x104/ml,and(21.3±2.4)x104/ml(P<0.05).The morphology of eosinophils was also destroyed after the treatment of artesunate.The results of immunofluorescence showed that the expression of Cleaved Caspase-9 in eosinophils increased.Besides,the results of western blotting suggested that the expression of activated Caspase-9 was increased.4.Instil 750?g ART/50?l NS artesunate into the airways of LPS model mice and detect the inflammation in BALF.The results showed no significant decrease in the proportion and number of neutrophils.BALF cells of LPS model mice was extracted,cultured and treated with artsunate for 12h in vitro,there was no significant difference in neutrophil survival rate.Part 31.Eosinophils in the human peripheral blood were enriched and cultured for 24h in vitro.The percentage of eosinophils treated with 10 ?M,50 ?M,and 100 ?M artesunate was decreased.In the control group,the percentage was 60.9±2.8%.However,in the experimental groups,the percentage decreased to 38.8±8.1%,29.7±7.7%,and 27.3±1.8%(P<0.05).2.The analyzes of the data set GSE74986 from the GEO database showed that there were 1121 differentially expressed genes associated with severe asthma,of which 508 genes were up-regulated and 613 genes were down-regulated.3.The results of enrichment analyzes suggested that the up-regulated genes were related to processes and signaling pathways such as lymphocytes activation,apoptosis signaling pathway,cytokine production,humoral immune response,and systemic lupus erythematosus.The down-regulated genes were mainly related to processes and signaling pathways such as endosomal parts,vacuolar organization,regulation of immune effector process,phagosome.4.The results of protein-protein interaction analyzes suggested that TCF7,LEF and SMC1A were the key proteins in up-regulated proteins,while the key proteins among those down-regulated were UBC,DHX9 and POLR2B.5.Protein data bank(PDB)was used to screen the key proteins selected according to the protein-protein interaction analyzes.Seven proteins with well-defined chemical structure and known active sites were selected,they were CDC16,DHX9,FCH02,FUS,SISH1,SYNJ1 and UBA3.Dock them with artesunate and corresponding natural ligands to predict the interactions.The results showed that artesunate scored high and interacted better with those proteins.6.According to the chemical structure and the active sites of artesunate,the potential targeted proteins were predicted,including CAT,VEGFA,NFE2L2,TLR4,MMP,GSTM2,CASP3,GCLM,NACA,RPL41.Further prediction suggested that artesunate might be targeted treatment for asthma.ConclusionArtesunate can alleviate eosinophilic airway inflammation in a mouse model of asthma induced by HDM,by down-regulating Bcl-2 and up-regulating Cleaved Caspase-9 to induce eosinophil apoptosis.Artesunate can also induce apoptosis of eosinophils from human peripheral blood.Bioinformatics analyses suggest that artesunate may interact certain asthma-related proteins,thereby serving as a targeted treatment strategy for asthma. |
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