The Refinement Of The Nerve Growth Factor Of Guangxi Cobra Venom And Its Influence On HSC-T6 SAPK/JNK Signaling Patheways

Objective:To obtain higher purity and activity of Guangxi cobra venom nerve growth factor (Nerve Growth Factor, NGF), improve the original separation and purification methods. And to explore the separation and purification of the resulting NGF on the expression of JNK in Stress-Activated Protein Kinase/c-Jun N-terminal Kinase (SAPK/JNK) signaling pathway of rat hepatic stellate cells (Hepatic Stellate Cell-T6, HSC-T6), and then clarify the mechanism of NGF inhibiting the hepatic stellate cells by SAPK/JNK signaling pathway.Methods :To use DEAE Sepharose CL-6B anion-exchange column,Sephadex G-50 gel filtration column and Macro-prep High S strong-cation columns to purify the Guangxi cobra venom. The Rat Pheochromocytoma Cells(PC12) was adopted for detecting the activity of NGF after each step of separation and purification, and the minimum concentration of NGF to differentiate PC 12 cells. CCK-8 (Cholecystokinin-octo Peptide) was used to assay the proliferation inhibition effcet of NGF on HSC-T6 cell. Western Blot(WB) was used to detect the expression level of JNK in SAPK/JNK signaling pathway after NGF treated HSC-T6 cells with time (Omin, 5min,10min, 30min, 60min, 120min and 24h)changes, and the expression level of JNK in SAPK/JNK signaling pathway after inhibitor and NGF treated HSC-T6 cells. The inhibitor treatment divided as blank group, inhibitor group, NGF group and inhibitors+NGF group.Results:By DEAE Sepharose CL-6B anion-exchange column,Sephadex G-50 gel column, Macro-prep High S strong-cation exchange column,the component of peak 2 show the activity of the NGF, and the minimum NGF concentration to induced differentiation of PC 12 cells was 0.1μg/ml. Its molecular weight was about 23kDa, and reached electrophoretically homogeneity. NGF did inhibition effects on the HSC-T6 cells, and the best inhibition concentration was 1μg/ml. With 1μg/ml of NGF treated on the HSC-T6 cells at different times, there was no significant difference in the expression level of JNK, while the expression of phosphorylation JNK weakened due to the extension of time. In the role of 10μmol/L inhibitor and 1μg/ml NGF on the HSC-T6 cells, the expression of p-JNK could significantly be inhibited.Conclusions :High activity NGF can be purified to obtain by DEAE Sepharose CL-6B anion exchange column, Sephadex G-50 gel column,Macro-prep High S strong cation exchange column. NGF can inhibit the expression of phosphorylation JNK in SAPK/JNK signaling pathway, and also influence the occurrence and development of hepatic fibrosis by inhibiting SAPK/JNK signaling pathway.