EGR2-mediated Artesunate Combined With Photodynamic Therapy Promotion Of Ferroptosis In Cholangiocarcinoma

Purpose:The purpose of this project was to explore whether Artesunate(ART)combined with Pyropheophorbide-a-mediated photodynamic therapy(PYRO-PDT)had synergistic effect on the killing of cholangiocarcinoma cells Hucct-1 and RBE,whether this synergistic effect is via ferroptosis and the specific mechanism.Methods:1.Changes in cell viability(CCK8 method),ROS(DCFH-DA probe),GSH(GSH-GSSG kit),MDA(colorimetric method)and lipid peroxidation(C11-BODIPY probe)were detected after 24 h treatment with different doses of ART(0,50,100,150,200μM)alone.Treated with different doses of PYRO(0,50,100,150,200 n M)alone for 24hours and then illuminated(660 nm,20 J/cm~2),cell viability,ROS,GSH,MDA and lipid peroxidation were detected 6 hours after illumination.2.Cholangiocarcinoma cell proliferation was assessed by clone formation after 7 days of treatment of cells with different doses of ART,PYRO-PDT,and the combination of both.3.ROS produced by ART alone,PYRO-PDT and their combination in HUCCT-1 and RBE cell lines were detected by reactive oxygen species detection kit(DCFH-DA probe).4.The changes of lipid peroxide and ferroptosis-related proteins in HUCCT-1 and RBE cells after ART and PYRO-PDT treatment were detected by C11-BODIPY and Western blot assays.5.The differentially expressed genes in untreated Hu CCT-1 cells and Hu CCT-1 cells treated with ART alone,PYRO-PDT alone and the combination of the two were analyzed by second-generation sequencing.After analysis,q PCR was used to verify and screen EGR2 with actual expression differences.6.The changes of ROS(DCFH-DA probe)and lipid peroxide(C11-BODIPY probe)were detected after ART combined with PYRO-PDT treatment of cholangiocarcinoma cells overexpressing EGR2.7.After ART combined with PYRO-PDT treatment of cholangiocarcinoma cells overexpressing EGR2,the expressions of GLUT1 and GPX4 genes and proteins downstream were detected by Western blot and q PCR experiments.Results:1.ART and PYRO-PDT affect the viability of HUCCT-1 and RBE cells in a dose-dependent manner,and the combination of ART and PYRO-PDT is more effective than PYRO-PDT alone.2.ART and PYRO-PDT inhibited the proliferation of cholangiocarcinoma cells HUCCT-1 and RBE in a dose-dependent manner,and the combined effect of ART and PYRO-PDT was stronger than that of PYRO-PDT alone.3.Both ART and PYRO-PDT can enhance the production of ROS in HUCCT-1 and RBE cells,and the combination of the two can generate more ROS.4.ART and PYRO-PDT could promote the formation of lipid peroxidation in HUCCT-1 and RBE cells,and GPX4 was down-regulated.5.The second generation sequencing showed that in HUCCT-1 cells,EGR2 was up-regulated after ART treatment,and down-regulated after PYRO-PDT treatment.EGR2 was up-regulated after the combination of the two treatments.6.HUCCT-1 and RBE cells overexpressing EGR2 can produce more lipid peroxides and ROS after ART and PYRO-PDT treatment.7.Western blot and q PCR experiments confirmed that overexpression of EGR2 decreased GLUT1 and GPX4.Conclusion:ART enhances the killing effect of PYRO-PDT on cholangiocarcinoma cells by regulating ferroptosis,which may be achieved by up-regulating EGR2 and down-regulating GPX4 and GLUT1.