| Purpose:? To improve the separation and purification method of the nerve growth factor(NGF)of Guangxi cobra venom,so as to obtain the nerve growth factor with high activity and pure electrophoresis.? To explore the impact of the nerve growth factor(NGF)of Guangxi cobra venom on the phosphorylated protein expression in ERK 1/2 and p38 signal paths of hepatic stellate cells-T6(HSC-T6),thus providing a theoretical basis for clarifying the mechanism of liver fibrosis generation and development.Methods:? On the basis of separating crude Guangxi cobra venom through the Sephadex G-50 gel chromatographic column and GM-Sepharose CL-6B weak cation exchange column,the refined nerve growth factor is further separated by making use of the DUOFLOW system and the Macro-prep High S prepacked column.The SDS-PAGE gel electrophoresis is applied to measure the purity and molecular weight;? The pheochromocytoma on a rat's adrenal gland(PC 12)is adopted for activity measurement of the nerve growth factor.The minimum concentration that promotes the synapse change of PC 12 cell is determined,and then the acquired purification rate of the nerve growth factor is calculated;? CCK-8 method is used to test the proliferation inhibition effect of the nerve growth factor on hepatic stellate cell(HSC-T6),and the minimum toxic concentration that causes the apoptosis is found out;? The variation of phosphorylated protein in ERK1/2 and p38 signal paths of cell along with time changes(0,5,10,30,60,120min)is detected by western blotting after the effect of the nerve growth factor on hepatic stellate cell;?This part of the experiment is divided into four groups:blank group(no inhibitors and NGF),the inhibitor group(only join inhibitors),inhibitors+NGF group,NGF group(only join NGF),the inhibiors PD98059 processing the inhibitors and inhibitors+NGF group after 1 h,the NGF plays intervention to the inhibitors+NGF group and the NGF group for 1 h,using WB to detect each grouping the expression of phosphorylated proteins of the ERK1/2 signaling pathwaysResults:? The component of Peak I obtained through Macro-prep High S prepacked column separation of the component of Peak III which has been obtained by separation and purification of Sephadex G-50 gel chromatographic column and GM-Sepharose CL-6B ion exchange column is electrophoretic pure and shows the activity of the nerve growth factor.After working on PC 12 cell for 72h,the minimum concentration for the occurrence of synapse change is 100g/ml;The calculated acquiring rate is 1.21%and the molecular weight is 23.54KDa;? The nerve growth factor can facilitate the proliferation of HSC-T6 cell at the concentration of 0?1?g/ml.It can achieve the most cell at 2?g/ml,and there is inhibition effects on the cell growth when the concentration is 4?16?g/ml.In this scope,the cell amount can be reduced along with the increase of concentration;? The nerve growth factor at the minimum toxic concentration(2?g/ml)functions on HSC-T6 cell.At different time period(0,5,10,30,60,120min),the expression of phosphorylated protein of two single paths of ERK1/2 and p38 will both be weakened due to the extension of time;10?M inhibitors PD98059+2?g/ml NGF group had significant inhibition of the expression of ERK1/2 phosphoprotein,other group's influence on its had no statistical significance.Conclusions:? The NGF component with pure electrophoresis and high activity can be obtained by further separation through Macro-prep High S prepacked column in DUOFLOW system;? The nerve growth factor can play an inhibition role in the expression of phosphorylated protein in ERK1/2 and p38 single paths,which may inhibit the ERK1/2 and p38 paths and further influence the generation and development of liver fibrosis. |
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