| Objective: To purify high purity cytotoxin-1(CTX-1)and cytotoxin-2(CTX-2)with high biological activity from Guangxi cobra venom by chromatography and to explore whether CTX-1 and CTX-2 selectively inhibit human hepatic stellate cells(HSC-LX2)compared with normal human liver cells(HL7702).In addition,the effects of CTX-1 on the cell cycle,morphology and ultrastructure,and the PI3K/AKT signaling pathway of HSC-LX2 cells were studied to clarify the mechanism by which CTX-1 inhibits the proliferation of HSC-LX2 cells by blocking cell cycle and induces the apoptosis of HSC-LX2 cells by PI3K/AKT signaling pathway.Methods: Guangxi cobra venom CTX-1 and CTX-2 were separated and purified by DEAE-Sepharose CL-6B anion exchange chromatography,Spehadex G-50 gel chromatography and Macro-prep High S strong cation exchange chromatography.The purity of protein peak was determined by SDS-PAGE electrophoresis and the protein composition and molecular weight were determined by NanoLC-ESI-MS/MS protein spectrometry.Theproliferation inhibition rates of HSC-LX2 and HL7702 cells treated with different concentrations of cytotoxins was detected by CCK-8 method.Flow cytometry was applied to detect the changes in the cell cycle of HSC-LX2 cells after the intervention of CTX-1 and the effect of CTX-1 on the apoptosis of HSC-LX2 and HL7702 cells.The changes of morphology and ultrastructure in HSC-LX2 cells treated with CTX-1 were observed by transmission electron microscopy.Western blot was used to analyse the changes in AKT protein phosphorylation levels in the PI3K/AKT signaling pathway when CTX-1 acted on HSC-LX2 cells at different time(0,3,6,9,12 h).The changes in AKT protein phosphorylation levels in HSC-LX2 cells treated with CTX-1 of minimum effective concentration,LY294002(inhibitor of PI3K/AKT signaling pathway,50 μmol/L)and CTX-1 combined with LY294002 was also analyzed by western blot.Results: After separation and purification of DEAE-Sepharose CL-6B anion exchange chromatography,Spehadex G-50 gel chromatography and Macro-prep High S strong cation exchange chromatography,electrophoretically pure proteins with high biological activity were obtained from the crude Guangxi cobra venom,which were identified as CTX-1 and CTX-2 via NanoLC-ESI-MS/MS protein spectrometry.The molecular weight of CTX-1 was about 9.499 KDa and its yield was 1.76 %.The molecular weight of CTX-2 was about 9.548 KDa and its yield was 3.73 %.As the results of CCK-8showed,after the treatment with cytotoxins at different concentrations for 24 h,the proliferation of HSC-LX2 and HL7702 cells was all inhibited and the proliferation inhibition rate increased with the increase of concentration,showing a dose-effect relationship.The minimum effective concentration of CTX-1 to inhibit the proliferation of HSC-LX2 cells was 0.5 μg/ml and theIC50 was 2.50 μg/ml,while its minimum effective concentration to inhibit the proliferation of HL7702 cells was 8 μg/ml and the IC50 was 18.97 μg/ml.The minimum effective concentration of CTX-2 to inhibit the proliferation of HSC-LX2 cells was 0.5 μg/ml and the IC50 was 1.213 μg/ml,and its minimum effective concentration to inhibit the proliferation of HL7702 cells was 0.5μg/ml and the IC50 was 1.253 μg/ml.After 24 hours’ treatment of CTX-1,with the increase of drug concentration,the percentage of S phase in HSC-LX2 cells increased,while the percentage of G1 and G2 phase decreased.The results of flow cytometry showed that CTX-1 induced apoptosis in both HSC-LX2 and HL7702 cells,and the apoptosis rate increased with increasing dose,showing a dose-effect relationship.The minimum effective concentration of CTX-1 to induce apoptosis of HSC-LX2 cells was 2 μg/ml,while the minimum effective concentration for inducing apoptosis of HL7702 cells was 4 μg/ml.Under transmission electron microscope,the morphology of HSC-LX2 cells in the control group was normal and there was no obvious apoptosis,while the treated HSC-LX2 cells with CTX-1 presented typical apopotic features such as the decreased number of cells,cytoplasmic vacuolation,chromatin condensation and margination,nuclear fragmentation and the formation of apoptotic bodies.When 2 μg/ml of CTX-1 was applied to HSC-LX2 for different time,the expression of AKT protein in each time period did not change significantly,while the expression of phosphorylated AKT protein decreased with the extension of action time.After treatment with LY294002 at 50 μmol/L and CTX-1 at 2 μg/ml in HSC-LX2 cells,the expression of AKT protein did not change,while the expression of phosphorylated AKT protein was significantly inhibited,and the combination of the two showed the best effect.Conclusions: The electrophoresis pure CTX-1 and CTX-2 with high biological activity were obtained from the crude Guangxi cobra venom by three-step separation method.Compared with HL7702 cells,CTX-1 is more cytotoxic to HSC-LX2 cells and selectively inhibit the proliferation of HSC-LX2 cells and induce its apoptosis.CTX-1 may inhibit the proliferation of HSC-LX2 cells by blocking their cell cycle in S phase,and induce the apoptosis of HSC-LX2 cells by reducing the expression of phosphorylated AKT protein and inhibiting the activation of PI3K/AKT signaling pathway. |
PDF Full Text Request