The Effects And Mechanism Of RINm5F Insulinoma Cells Apoptosis Induced By Photoactivation Of Hypericin

Hypericin is one of the most bioactive substances from Hypericumperforatum L.Studies have shown thathypericin has antidepression,antitumor,anti HIV,antihepatitis B virus,antiherpes simplex virus and antiavian influenza virusactivities.In recent years,its attention and demand has increased dramatically.In this dissertation,3kg commercially available H.perforatum extract was pretreated by silica gel column chromatography to get 4.5g semi finished product,which was further purified using high-speed countercurrent chromatography by hexane-ethyl acetate-methanolwater solvent system(3-5-3-5),the upper phase as the stationary phase,the lower phase as the mobile phase,with the host speed 800 r/min,the flow rate of 2.5 ml/min,and the detection wavelength of 590 nm.For the large-scale preparation,40 mg semi-finished product was injected every time,and a total of 12 injectionswere made,2ghypericin was finally obtained with the purity up to 98% detected by HPLC,its structure was confirmed by 1H-NMRand 13C-NMRspectrum.Insulinomas cause neuroglycopenic symptoms,permanent neurological damage and even death.Current available therapies cannot satisfactorily treat malignant insulinomas and some benign insulinomas.The promising phototherapeutic results and harmless side effects of hypericin in some cancer treatments prompted us to explore possible anti-growth activity of photoactivatedhypericin against RINm5 F insulinoma cells and underlying mechanisms.We now show that detectable and maximal internalization of hypericin in RINm5 F insulinoma cells occurred in 20 and 60 min respectively.Hypericin was considerably associated with the plasma membrane,appreciably localized in the sub-plasma membrane region and substantially accumulated in the cytoplasm.Fluorescence labeling and confocal fluorescent microscopyindicatedthathypericincouldco-localized to theendoplasmic reticulum,Golgi apparatus and mitochondria in thecytoplasm.And cell morphological changes were accompanied by the light activation of hypericin.Photoactivatedhypericin decreased the viability of RINm5 F insulinoma cells due to its anti-proliferative and apoptotic actions.Photoactivation of hypericin inhibited cell proliferation reflected by decreased expression of the proliferation marker Ki-67 and cell-cycle arrest in the G0/G1-phase.The anti-proliferative effect resulted from down-regulation of phosphorylation of JNK and ERK.Photoactivatedhypericin triggered apoptosis through activation of caspase-9 and caspase-3 and elevation of the Bax-to-Bcl-2 ratio.The IlluminaNextseq 500 RNA-Seq arrays were first used to analyze the mRNA expression exposed to hypericin in RINm5 F cells.Consistent with the previous results,the data indicated that hypericin could influence the proliferation and apoptosis in RINm5 F cells.The findings lay a solid foundation for implementation of hypericin-mediated photodynamic therapy in the treatment of insulinomas.