Mechanism Of Dihydroartemisinin Inhibiting Inflammation By Regulating Autophagy

BackgroundRheumatoid arthritis(RA)is a chronic systemic autoimmune disease with inflammatory synovitis as its main manifestation.RA is characterized by symmetry and invasive arthritis which affects multiple joints,and often accompanied by extraarticular organ lesions.In traditional Chinese medicine theory,RA belongs to the category of "arthralgia syndrome",and its principal etiology and pathogenesis are"insufficiency in essence and blood of kidney and liver","exogenous wind-cold and dampness pathogenic factors",and "external sthenia and internal asthenia".Due to the unrevealed pathogenesis and etiology of RA,the specific therapy is lacking in clinical practice.Artemisia annua,a traditional Chinese medicine which is bitter,cold and innocuous,has main effects of "clearing away heat,relieving summer heat,removing steam pathologic factor,and anti-malaria".As described in ancient literature,Artemisia annua is usually used to treat malaria,while its application on arthralgia is rare.Recently,plenty of studies have confirmed that artemisinin,the extract of Artemisia annua,causes profound immunosuppression,besides its definite antimalarial effect,which can be used to treat "arthralgia syndrome" as defined in traditional Chinese medicine.On the other hand,the critical regulatory role of autophagy in immune diseases has gradually become a research hotspot recently,and clinical application of autophagy-related drugs in RA treatment achieved significant advances.Therefore,the regulatory mechanisms of artemisinin and its derivatives in autoimmune inflammation received increasing attention.ObjectiveAutophagy regulates immunity and inflammation through very complicate mechanisms involving multiple pathways.The anti-inflammatory and immuneregulatory effects of artemisinin have been widely recognized,and its effect on autophagy regulation has been confirmed recently.The purpose of this study was to investigate the effect of artemisinin on cellular inflammation model through autophagy,and the effect of autophagy on the expression of IL-37,a newly discovered inhibitory cytokine,and IL-37related inflammatory cytokines and receptors,as well as the possible regulatory mechanisms.This study provided experimental data for understanding the role of autophagy in the pathogenesis and treatment of inflammatory and autoimmune diseases.Moreover,this study investigated the contributions of the above mechanisms in the anti-inflammatory effects of artemisinin and autophagy regulators,and provided a certain theoretical basis for the application of artemisinin in rheumatoid arthritis treatment.MethodUsing PMA-transformed human monocyte line U937 macrophages with or without lipopolysaccharide phosphate(LPS)stimulation,the effect of dihydroartemisinin(DHA)on autophagy was analyzed.Thereafter,in order to investigate the regulatory mechanism of autophagy on IL-37 expression,we used three classical autophagy modulators,3-MA,rapamycin,and chloroquine,to interfere the autophagic progress,and performed the followed assays:(1)Autophagy markers p62/SQSTMl and LC3 were quantified by Western blot and observed by laser confocal analysis;(2)Quantitative PCR was used to detect three kinds of IL-1 family cytokines(IL-37,The expression of IL-18 and IL-1 ?)and two receptors(IL-1R8 and IL-18RA).(3)Proportion of autophagic cells was quantified by flow-cytometry analysis with Cyto-ID autophagy kit,and the expression levels of IL-37 and IL-1 ?mRNA in sorted Cyto-ID+and Cyto-ID-U937 cells were compared.(4)The effect of autophagy-modulators on intracellular IL-37 and IL-1 ?staining in both U937 cells and peripheral blood mononuclear cells(PBMC)from a healthy donor were detected by flow-cytometry.(5)The activation of Erk and NF-? B signaling pathway was analyzed by Western blot analysis,and the effects of MAPK and NF-? B pathway agonists and antagonists were evaluated.After that,the effects of dihydroartemisinin(DHA)on the expression of IL-37 and its related cytokines and receptors,and the participation of MAPK and NF-? B signaling pathways were further studied by using the above culture system and index system.Results:(1)Dihydroartemisinin could induce the increase of LC3 transformation and increase the proportion of Cyto-ID+cells stimulated by LPS.(2)After treatment with two autophagy-regulators,rapamycin and chloroquine,especially the former,the transformation rate of LC3 significantly increased in LPS-stimulated U937 cells while IL-37 mRNA increased significantly,simultaneously,and there seemed to be a correlation between LC3 transformation and IL-37 expression.(3)After flow-cytometry sorting,IL-37 expression in Cyto-ID+U937 cells was significantly higher than that in Cyto-ID-cells.(4)As U937 cells rarely produce IL-37 protein,we used human PBMC and showed that chloroquine could significantly increase IL-37 protein.(5)Both rapamycin and chloroquine increased the phosphorylation of I ? B ?and Erk1/2.Inhibitors of MAPK p38 and Erk1/2 and NF-? B pathway could block the expression of inducible IL-37,while Erk1/2 agonist could enhance the induced expression of IL-37.(6)Rapamycin and chloroquine increased the expression of IL-10 while decreased the expression of TNF-? and IL-6 stimulated by LPS.(7)The expression of IL-1 ? was significantly affected by p38 and Erkl/2 and weakly affected by NF-? B pathway.The expression of TNF-? was regulated by MAPK and NF-? B pathways.(8)Dihydroartemisinin could increase IL-37 mRNA,and decrease the expression of IL-1 ?,TNF-?,IL-6 and receptor IL-18RA mRNA,as well as the production of TNF-? and IL-6 through inhibition of NF-? B and ERK pathways.Conclusion(1)Dihydroartemisinin induces autophagy in LPS-stimulated U937 macrophages,increase anti-inflammatory factor IL-37,and decrease pro-inflammatory factors IL-1 ?,TNF-?,IL-6.(2)Dihydroartemisinin and autophagy regulators rapamycin and chloroquine may activate MAPK cell signaling pathway and increase the level of IL-37 through the accumulation of LC3-?.(3)This study suggests a new therapeutic mechanism which is shared by dihydroartemisinin,chloroquine and rapamycin in autoimmune inflammatory diseases treatment.(4)This study provides a basis for understanding the anti-inflammatory mechanism of artemisinin,and helps us to further understand the role of autophagy in regulation of pro-inflammatory cytokines and suppress inflammation.