The Effect And Mechanism Of Dihydroartemisinin On Airway Inflammation In Mouse Model Of Asthma

Background:Artemisinin was isolated from the Chinese Compositae herbal medicine Artemisia carvifolia and has been widely used in the treatment of malaria with high efficiency and low toxicity.The Dihydroaretemisinin(DHA)is a derivative of Artemisinin.It can inhibit the proliferation and promote the apoptosis of many kinds of tumors,and plays an important role in immune regulation.The latest study found that artemisinin is also effective in treating systemic lupus erythematosus.In view of its diverse functions,this study investigated whether dihydroartemisinin is also effective in treating asthma.The present study investigated the mechanism of DHA in treating ovalbumin-induced asthma in BALB/c mice to provide a new treatment plan and theoretical basis for asthma.Materials/Methods:1.Forty BALB/c mice were used to construct mouse asthma model by OVA.Mice were divided into three groups:control group,model group and treatment group.The state of mice in each group was observed,the weight of mice was weighed,the time of death was recorded,and the survival rate was counted.2.The total airway resistance and airway compliance of mice stimulated by Acetylcholine(Ach)were measured by mouse lung function instrument.3.Mice was anesthetized and alveolar lavage fluid(BALF)was collected,and the cells were collected for diff-Quik and Giemsa staining.Total cell number as well as neutrophils,lymphocytes,monocytes,eosinophils and basophils was counted.4.The concentration of IL-17,IL-1,TNFα,IFNγ,IL-17F,IL-17A,IL-22,IL-10,IL21 and GM-CSF in the BALF of mice in the three groups were detected by Elisa kit.5.The proportion of Th17 cells in BALF of mice in three groups was determined by flow cytometry.6.Paraffin sections were prepared from mouse lungs,and H&E and PAS staining were performed to observe the pathological changes of mice lungs in the three groups.7.Western blot was used to detect the expressions of IL-6,STAT3 and STAT3(Tyr705)in the lungs of mice in the three groups.8.Quantitative real-time PCR(qRT-PCR)was used to detect the expressions of il6,tnfa,il17a,il17f,il22,il10 and stat3 in the lungs of mice in the three groups.9.The contents of miRNA-183-5p,miRNA-96-5p and miRNA-182-5p in lung tissues were detected by qRT-PCR.10.The target of miRNA-183C was predicted by online software TargetScan.11.The expression of Rorcand Foxo1 was detected by qRT-PCR,and the expression of Foxol was detected by Western blot.12.The luciferase reporter assay system was used to verify that Foxol is the regulatory gene of miRNA-183C,and to explore whether its regulation is influenced by DHA.Results:1.OVA induced asthma in BALB/c mice successfully.The weight of asthmatic mice decreased significantly with behavioral changes.DHA treatment was effective in increasing weight and improving clinical performance during acute attacks.The lung/body weight ratio in model group was significantly higher than that in control group and in DHA group.DHA treatment was effective in reducing OVA-induced death in mice.2.OVA stimulation significantly increased the number of total cells,neutrophils,lymphocytes,monocytes,eosinophils and basophils in BALF,and the number of cells in BALF was significantly lower after DHA treatment.3.OVA stimulation significantly increased airway resistance in mice,which increased with the increase of Mch concentration.DHA treated mice significantly decreased airway resistance in mice.And had significantly difference with the model group,after Mch concentration 6.25mg/mL.4.In the model group,lung tissue was seriously damaged,alveolar morphology was irregular,and inflammation was increased.In the DHA treatment group,lung tissue destruction was alleviated,and alveolar morphology was partially restored.5.OVA significantly increased concentration of IL-17,IL-1β,and TNFa in BALF,and DHA significantly decreased these cytokines in BALF.The proportion of CD4+IL-17+cells was significantly increased after OVA stimulation,and was significantly decreased after DHA treatment,but the proportion of CD4+IL-17+cells was still significantly higher in DHA treatment group compared with the control group.6.OVA significantly increased the IL-6 and STAT3(Tyr705)expression in lung,while DHA treatment significantly decreased IL-6 and STAT3(Tyr705)expression.7.The levels of il6,tnfa,il17a,il17f,il22 were increased by OVA,while the levels of il10 were decreased.After DHA treatment,the transcription levels of ol6,tnfa,il17a,il17f,il22 were decreased,while il10 was increased.8.OVA significantly increased the levels of IL-17F,IL-17A,IL-22,IL-21and GMCSF in BALF of the model group,and decreased the levels of IFNy and IL-10.After DHA treatment,the levels of IL-17F,IL-17A,IL-22,IL-21 and GM-CSF were significantly reduced,and the levels of IFNy and IL-10 were significantly increased.9.The expression of miRNA-183C was increased by OVA stimulation,and was decreased after DHA treatment.The transcription and translation of Foxo1 were significantly decreased by OVA stimulation and was increased after DHA treatment.10.MiRNA-183C significantly inhibited the transcriptional activity of the 3’-UTR(wildtype),but did not inhibit the transcriptional activity of the 3’-UTR(mutant type).DHA was able to resist the transcriptional inhibition of Foxol by miRNA-183C,but was not effective against the 3’-UTR(mutant type).Conclusions:1.DHA can effectively increase the weight of asthmatic mice,reduce the ratio of lung/body weight,improve the behavior of mice,increase the survival rate,and effectively reduce the airway impedance of model mice,thereby improving the overall symptoms of asthmatic mice.2.DHA effectively improved the lung tissue structure of the model mice,reduced the number of inflammatory cells in the BALF,reduced the inflammatory infiltration and the proportion of Th17 cells,which had a significant inhibitory effect on the inflammatory response.3.DHA can effectively inhibit the IL-6/Stat3 signaling pathway and reduce the transcription and translation of inflammatory molecules downstream of the pathway.4.DHA can inhibit the expression of miRNA-183C,which can bind to the 3’-UTR of Foxol to inhibit its transcription and translation.Therefore,DHA promotes the expression of Foxol by inhibiting miRNA-183C,thus affecting the immune response.