Effect Of Dihydroartemisinin On Pulmonary Metastasis Of Melanoma In Mice And Preliminary Study On Its Mechanism

Background:The onset of melanoma is insidious and invasive,and it is easy to metastasize in the early stage and lose the best treatment period for surgery.Metastasis of melanoma is more common in the lung and brain,and once metastasis occurs,the prognosis is poor.Dihydroartemisinin(DHA)is one of the main derivatives of artemisinin.Studies have confirmed that DHA can exert anti-tumor effects by regulating the host immune response,but the effect and mechanism of DHA on lung metastasis of melanoma are rarely reported.Objective:To verify whether DHA can inhibit the lung metastasis of melanoma in mice and to explore its possible mechanism.Methods:The A375 and B16F10 melanoma cells were treated with different concentrations of DHA for 24 h and 48 h,respectively.The effect of DHA on proliferation of A375 cells was detected by MTT method,and the proliferation of B16F10 cells was detected by CCK-8 method.The safe and effective concentration was screened out to provide concentration basis for subsequent experiments.The effect of DHA on the migration ability of A375 cells and B16F10 cells was detected by wound healing experiment and Transwell method.BALB/c mice were randomly divided into normal group,model group,low-dose DHA group(25 mg/kg/d)and high-dose DHA group(50 mg/kg/d).Except for the normal group,each mouse was injected with B16F10(2 × 106 cell/mL)cells via the tail vein,and then treated by gavage 24 h later.Mice in model group,low dose group and high dose group were gavaged daily.All mice were fed and drank normal diet and water every day.The mice were observed and weighed.The mice were sacrificed after 28 days of continuous gavage treatment.Effect of DHA on liver and kidney function in mice by ELISA;pulmonary histopathological changes of model group and DHA group were observed by H&E staining;changes of melanoma cells proliferation,CD4+T cells,CD8+T cells and Regulatory T cells(Treg)in lung tissue after DHA treatment were observed by immunohistochemistry staining;FasL in lung tissue sections of mice after drug administration was observed by immunofluorescence staining.Western blot assay was used to detect the expression of EMT and MMPs-related proteins in A3 75 cells,B16F10 cells and lung tissues of mice treated with DHA.UHPLC-Q-TOF MS was used to analyze non-targeted metabolomics in lung tissue samples of mice in each group.Results:1.The results of cell proliferation experiment showed that DHA could significantly inhibit the proliferation of A375 cells and B16F10 cells;2.The results of wound healing experiment showed that the transverse migration ability of A375 cells and B16F10 cells was weakened by DHA treatment at different concentrations;3.Transwell chamber invasion assay results showed that the longitudinal migration of A375 cells and B16F10 cells was inhibited after DHA treatment;4.ELISA results showed that DHA had no toxic side effects on the liver and kidney of mice;5.Gross maps of mice showed that DHA significantly reduced pulmonary melanoma nodules and relieved lung wet weight;6.H&E staining showed that in the model group,the nuclei of melanoma cells in the lung tissue were hypertrophic,malformed,stained deeper,mitotic figures were common,and heterogeneity was obvious(red arrows).In addition,under the microscope,alveolar tube and alveolar structure were damaged,alveolar dilatation,atrophy and subsidence,alveolar wall capillary congestion,alveolar wall thickening,alveolar epithelial injury(blue arrows),indicating that the model group was black.Melanoma cells grow actively and invasively.After DHA treatment,the growth of melanoma cells in lung tissue was relatively limited,the pathological mitotic figures were reduced,the heterogeneous cells were reduced,melanoma cells burst,melanin was released,and the nucleus shrunken(red arrows).At the same time,the structure of alveolar tubes and alveoli was clear,alveoli did not expand or collapse,alveolar wall capillaries were not congested,alveolar wall was not seen.Thickening,no damage to alveolar epithelium(blue arrow);7.Immunohistochemical results showed that after DHA treatment,the number of ki-67 positive brown granules was significantly reduced;compared with the model group,the number of CD4+ T cells,CD8+T cells positive brown granules in the lung tissue of DHA group increased,while the number of Foxp3 positive brown granules was significantly reduced;8.The results of Western blot of lung tissue showed that compared with the model group,the expression levels of E-cadherin and p-Ezrin protein in lung tissue of the drug group were significantly increased,while the expression levels of vimentin,MMP-2 and MMP-9 protein were significantly decreased(P<0.05);After DHA treatment of A375 cells and B16F10 cells,the protein expression level of E-cadherin increased significantly,while the protein expression levels of vimentin,Ezrin,MMP-2 and MMP-9 decreased in a dose-dependent manner,the difference was statistically significant(P<0.05);9.Immunohistofluorescence staining results showed that FasL expressed by CD8+ T cells and granzyme B-positive cells secreted were significantly increased after DHA treatment;10.Through untargeted metabolomic analysis,43 differential metabolites were found,with a focus on riboflavin and arachidonic acid,which are closely related to tumors.The results of KEGG pathway enrichment map showed that riboflavin belonged to 2 metabolic pathways and arachidonic acid to 11 metabolic pathways.Conclusion:DHA inhibited proliferation and metastasis of A375 and B16F10 cells;DHA enhanced the anti-tumor effect of CD8+CTLs and inhibited lung metastasis of melanoma;DHA may play an anti-melanoma role by regulating the metabolic pathways involved in riboflavin and arachidonic acid in melanoma lung metastasis mice.