The Role And Mechanism Of Artesunate In Macrophage Polarization In Asthma

BackgroundAsthma is a common chronic inflammatory disease of the airways,always accompanied with airway remodeling.Glucocorticoids,beta-2 receptor agonists,anticholinergic drugs,and biological therapies are the mainstays of asthma care currently.The above drugs cannot completely control airway remodeling,and severe patients suffered more adverse reactions who were treated with large dose of glucocorticoids.Artesunate(ART)is a semi-synthetic water-soluble artemisinin extracted from Artemisia annua.Studies have shown that Artesunate can reduce inflammation and airway remodeling in asthma.However,the mechanism has not been clarified.Eosinophils dominated allergic asthma accounts for a large proportion.The alternatively activated macrophage(AAM,also known as M2)is often associated with allergic asthma.M2 is a polarizing phenotype of macrophage,which corresponds to the classically activated macrophage(CAM,also known as M1),could enhances airway remodeling.Some other studies have shown that Artesunate can affect macrophage polarization,but the role of ART on macrophage polarization in asthma has not been clarified.Therefore,the effects and molecular mechanisms of ART intervening asthma and macrophage polarization were investigated in this study,providing theoretical basis for the treatment of asthma by Artesunate.MethodsIn the first part,ovalbumin(OVA)induced BALB/c female mice were used to establish an asthma model.The mice were randomly divided into control group,asthma group,ART+asthma group and ART+control group(8 mice per group).The airway hyperresponsiveness(AHR)changes of mice to acetylcholine chloride stimulation test were observed.And the effect of ART on pulmonary inflammation,was assessed by Haematoxylin and Eosin(H&E)staining.The effect of ART on trachea goblet cell proliferation was evaluated by Periodic Acid-Schiff(PAS)staining and Masson staining was used to analyze airway collagen deposition.Differently expressed genes(DEGs)between control,asthma and ART+asthma groups were analyzed by RNA sequencing(RNA-Seq).Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and protein-protein interaction network(PPI)analysis were analyzed subsequently.In the second part,in vitro,to determine the effect of ART on macrophage polarization,a model M2 macrophages(RAW264.7 cell line)was constructed.Western blot(WB)and immunofluorescence(IF)were used to detect the symbolic proteins found in inflammatory zone 1(FIZZ1),Arginase-1(Arg-1)of M2,and inducible nitric oxide synthase(iNOS)of M1 after drug stimulation.In the third part,in vivo,to evaluate the effect of ART on macrophage polarization and airway remodeling and explore the possible mechanism of action in asthma,real-time quantitative PCR(RT-qPCR)and WB were used separately to test expression of Arg-1 and iNOS.Paraffin slices of mouse lung tissue were stained for IF to detect the expression of F4/80,Arg-1 and iNOS protein.The proportion of M2 macrophages(Arg-1&F4/80 positive cells)and M1 macrophages(iNOS&F4/80 positive cells)was observed.Meanwhile,to understand the effects of ART on airway remodeling by regulating M2 macrophages,RT-qPCR,immunohistochemistry(IHC)and WB were used to evaluate the expression of FIZZ1 after ART intervention.Finally,the expression level of related proteins of phosphoinositide 3-kinase(PI3K)/protein kinase B(PKB,also AKT)pathway was detected by WB.ResultsFirstly,compared with the OVA asthma group,ART could significantly reduce the infiltration of airway inflammatory cells,airway goblet cell metaplasia and collagen deposition around the airway.The above difference was statistically significant.RNA-seq showed that ART could reverse 863 up-regulated genes and 928 down-regulated genes in asthma group.The GO of DEGs mainly focuses on B cell receptor signaling pathway,extracellular matrix tissues and immune system processes.KEGG is concentrated in cytokine receptor interaction,PI3K/AKT signaling pathway and so on.Secondly,in vitro studies,ART decreased the expression of FIZZ 1 and Arg-1 protein and increased the expression of iNOS protein,when compared with M2-type macrophages.The difference was statistically significant.Thirdly,in vivo experiments showed that the expression levels of M2(Arg-1,FIZZ1)and M1 markers iNOS mRNA in asthmatic group were higher than those in control group.After ART intervention,the above indexes decreased.WB showed that the expression of Arg-1 increased significantly in asthma group,but decreased significantly after ART treatment(P<0.001).There was no significant difference in the expression level of iNOS between the three groups.IF showed that compared with the control group(6.335±4.091%),the proportion of M2 type macrophages in asthmatic mice was significantly increased(39.04±7.19%)(P<0.001).After Artesunate intervention,the M2 ratio(24.2±4.503%)was lower(P<0.05).There was no significant difference in the proportion of M1 macrophages among the three groups(P>0.05).Meanwhile,the expression of FIZZ1 was reduced after ART intervention in RT-qPCR,immunohistochemistry(IHC)and WB tests(the difference was statistically significant).At last,WB results showed that the expression level of protein p-PI3K and p-AKT on PI3K/AKT signaling pathway decreased in the ART intervention group,compared with the asthma group,and the difference was statistically sigificant.Conclusion1.RNA-seq data analysis showed that ART can reduce airway hyperresponsiveness,inflammatory infiltration,goblet cell metaplasia and airway remodeling in asthmatic mice by multi-targets and multi-pathways.2.ART might relieve M2 polarization of macrophages in vitro.3.ART could reduce M2 polarization and suppress the production of FIZZ1 in vivo,alleviating airway remodeling.The PI3K/AKT signaling pathway may be involved in the function.