Role And Molecular Mechanisms Of Artesunate In Programmed Eosinophilic Death In Asthma

BACKGROUNDBronchial asthma(i.e.asthma)is a common chronic disease which has seriously affected individual health.In the development and progression of asthma,eosinophils take an essential role,so as to be the main target of glucocorticoids and multiple biologics,which however,have brought about various adverse events and heavy economical burders to patients and the community.Therefore,it is urgent to discover a more cost-effective medicine to replace or cooperate with glucocorticoids or biologics.According to the previous findings of our studies,artesunate,one of the main components of Artemisia Annua L.,could decrease the amount of eosinophils in the bronchoalveolar lavage fluid(BALF)possibly by inducing programmed cell death(PCD).METHODSThis study includes four sections.Methods utilized in the first section are network pharmacology and molecular docking.In this section,we first evaluated the general phamacological properties(including physiochemical properties,absorption,distribution,metabolism,excretion and toxicology)of artesunate and its internal active metabolite(dihydroartemisinin,DHA)and then predicted the potential mechanisms and targets of artesunate in treating asthma by acquiring potential targets of artesunate,DHA and asthma from public platforms,constructing compound-target network and protein-protein interaction network,screening out hub genes,and performing enrichment analysis and molecular dockings.In Section 2,we established bronchial asthma mice models,and set four groups to receive different intervention s,then assessed their behaviors,airway responsiveness by spirometer,inflammation infiltration severity by Hematoxylin and Eosin(HE)staining,and the amounts and viability of eosinophils in BALF by flow cytometry so as to evaluate the effects of artesunate on eosinophils in asthma in vivo.In Section 3,we established interleukin-5(IL-5)-activated eosinophil models,and set groups to examine the viabiliy of eosinophils at different drug concentrations and timepoints by cell counting kit.Then the optimal drug concentration and intervention period were determined through fitted equations.Next,different PCD inhibitors were added respectively to the eosinophils and then cell viability was examined to make clear the specific PCD methods artesunate induces eosinophilic death.Finally,in Section 4,we explored the effects of artesunate on the target proteins in the receptor interacting protein kinase 1(RIPK1)-mediated pathways through immunohistochemical(IHC)staining(in vivo)and Western blotting(WB)(in vitro).RESULTS1.Through network pharmacology and molecular docking,we found artesunate had desirable druglikeness and safety for clinical application.The mechanisms it may treat asthma include biosynthesis and metabolism of and response to steroid hormone,response to internal and external stimuli,immune and inflammatory response,gland development and extracellular matrix organization,and cell chemotaxis,survival and death.Potential targets include PTGS2,MAPK3,JAK2,MTOR,CASP8,ERBB2,MAP2K1,CCND1 and EGFR.2.In the in-vivo experiments,when comparing the control group and the asthma model group,we found artesunate could significantly allviate peribronchial(P<0.001),perivascular(P<0.0001)and parenchymal(P<0.01)inflammation infiltration,decrease the amount of eosinophils in BALF(P<0.001),and induce programmed eosinophilic death in BALF(P<0.01).3.From the in-vitro study,we found that above 100μM of artesunate could significantly suppress the viability of IL-5-activated eosinophils at every selected timepoint(P<0.0001);intervention with artesunate for at least 24 hours with every preset concentration could suppress the viability of IL-5-activated eosinophils(P≤0.0001).Half maximal inhibitory concentrations(IC50)were 166.00μM(6h),210.74μM(12h),226.08μM(24h),and 43.51 μM(48h),respectively.Through transmission electron microscopy,we found that the eosinophils treated with artesunate were larger with swelling organelles(especially mitochondria with swelling and fading cristae and blebbing),penetrated cell membrane and intracellular content outflow.As for the effects of different PCD inhibitors,0.5μM(P=0.0376)and 1μM(P=0.0013)Necrosulfonamide(NSA)could significantly reverse the eosinophilic death induced by artesunate;Q-VD-OPh showed no effects on eosinophilc viability even with various concentrations(25nM:P=0.3814,50nM:P=0.3145,100nM:P=0.2448);no differences were found between the eosinophilic viability of NSA-only groups and corresponding NSA-and-Q-VD-OPh groups(NSA:1μM:P=0.9975,2μM:P=0.9120);40nM(P=0.0002)and 80nM(和 P<0.0001)Liproxstatin-1 could inhibit the eosinophilic death induced by artesunate with statistic significance.Meanwhile,when treated with additional 3-Methyladenine,the impaired eosinophilic viability by artesunate was significantly suppressed further(P<0.0001).4.In-vivo IHC analysis showed that when compared with the control group,the expression of RIPK1 in the model group was increased significantly(P<0.0001);in comparison with the asthma group,the expression of RIPK1 in the artesunate group was increased significantly(P=0.0073),caspase-8(P=0.0101)and p-MLKL(P=0.0278)were expressed less,while RIPK3 didn’t change significantly.WB analysis indicated that after treated with artesunate for 24 hours,activated eoxinophils expressed further less RIPK1(P<0.0001)and caspase-8(P<0.0001),but much more p-RIPK1(P<0.0001),RIPK3(P=0.0014)and p-MLKL(P=0.0062)than those without intervention,and the ratio of pRIPK1/RIPK1 was significantly increased(P<0.0001)in comparison with the control group.CONCLUSION1.The in-silico study showed that artesunatecould might treat asthma through multiple targets,pathways,and mechanisms with desirable safety for clinical practice.Artesunate has the potential to be a safe,effective and affordable anti-asthmatic drug.2.The in-vivo experiment suggested that artesunate could decrease inflammation infiltration and the eosinophil amount in lungs by inducing programmed eosinophilic death.3.The in-vitro experiment indicated that artesunate could suppress the viability of in vitro-activated eosinophils through programmed cell death,including necroptosis and ferroptosis.4.The in-vitro and in-vivo experiments showed that artesunate could decrease the expression of RIPK1 and caspase-8 to induce eosinophilic necroptosis through the RIPK1/RIPK3/MLKL signaling pathway.