Isolation And Purification Of Cobra Venom Cytotoxin-4N From Guangxi And Its Effect On ERK Signaling Pathway In HSC-T6 Cells

Objective: to obtain the cobra venom cytotoxin-4N of high purity and elucidate the mechanism about CTX-4N induced apoptosis of hepatic stellate cell through ERK signalling pathway by exploring the proliferation suppression affection of cobra venom CTX-4N on HSC-T6 and the influence of CTX-4N on apoptosis of HSC-T6 through ERK signalling pathway.Methods: Separate and purify the cobra venom protein with the methods of DEAE-Sepharose CL-6B anion exchange column,SpehadexG-50 Gel chromatography and Macro-prep High S cation exchange column.Using HSC-T6 proliferation suppression experiment to do activity detecting of protein CTX-4N of every peek after each step of separation.And then collect the protein peek of the best activity and using SDS-PAGE to identify its purity,then using CCK-8 method to detect proliferation suppression effect of CTX-4N of different concentration on HSC-T6.Using flow cytometry to detect the influence of CTX-4N on HSC-T6 cell apoptosis.Then analysing the changes of levels of ERK1/2 phosphorylation in ERK signalling pathway in different time after CTX-4N had effects on HSC-T6 and the changes of levels of ERK1/2phosphorylation in ERK signalling pathway after adding the suppressor PD98059.Results: We obtain a protein of Electrophoresis purity after DEAE-Sepharose CL-6B anion exchange column,SpehadexG-50 Gel chromatography and Macro-prep High S cation exchange column,and we identify it to be CTX-4N using protein mass spectrometry,its molecular weight was about 9.065 KDa.Different concentrations of CTX-4N showed proliferation suppression effect on HSC-T6 cells and the affection was added with the increase of the concentration,the least concentration of suppression was2μg/mL,IC50 was(12.836±0.045)μg/mL,it was showing the correlation of dose affect.The effect of CTX-4N of different concentration on HSC-T6 apoptosis was added with the increase of its concentration,and the least concentration of inducing apoptosis of HSC-T6 was 4μg/mL.After adding CTX-4N of 4μg/mL to HSC-T6 for different time,the expression of ERK1/2 had no changes in different time period,but the expression of phosphorylated ERK1/2 was increased with the increase of affection time,after 10 min its expression level reached the highest level and then decreased,then its expression was lower than the control group(p<0.05)after 60 min.After adding 10umol/L PD98059 and4μg/mL CTX-4N to HSC-T6 cells,we found the expression of ERK1/2 had no changes but the expression of ERK1/2 phosphorylation was significantly supressed.Conclusion: We had obtained the CTX-4N of Electrophoresis purity after DEAE-Sepharose CL-6B anion exchange column,SpehadexG-50 Gel chromatography and Macro-prep High S cation exchange column.CTX-4N could induced the apoptosis of HSC-T6 cells,and it was also showing some suppression on the expression of phosphorylated ERK1/2 in ERK signallingpathway.So CTX-4N could interfere the occurrence and development of hepatic fibrosis by induced the apoptosis of HSC-T6 through ERK signalling pathway.