The Protective Effect of Gypenosides on Ethanol-induced Neural Precursor Cells Damage and the Effect of Gypenosides on Proliferation of Neural Precursor Cells
|Keywords||Gypenosides Neural precursor cells Alcoholism Proliferation|
Background: neural precursor cells (neural precursor cells, NPCs) are immature nerve cells at different developmental stages of the general term in the central nervous system, including neural stem cells and neural progenitor cells with self-renewal, proliferation, and differentiation into neural tissue each The potential of the type of cells, is recognized seed cells for nerve repair. Fetal alcohol syndrome (fetal alcohol syndrome, FAS) is the offspring birth defects caused by the mother during pregnancy, excessive alcohol consumption, and its symptoms include a series of central nervous system abnormalities. The studies have shown that alcohol suppressed the proliferation of NPCs, to cause some NPCs apoptosis, reduce the number of nerve cells, is considered to be one of the causes of the disease in FAS. But its mechanism is not yet clear, the current study suggests that alcohol may be by increasing the intracellular free radicals, damage the cell membrane, to break the intracellular ionic homeostasis, interfering with normal signaling pathways leading to apoptosis. Gynostemma gypenosides (gypenosides, GPs) as the Cucurbitaceae perennial herbaceous liana [gynostemma pentaphyllum (Thunb.) makino] The main active ingredient with anti-cerebral ischemia, hypoxia, anti-oxidation, anti-aging, improve nerve cell damage extent and variety of pharmacological activity. The findings of the research group of mature neurons, GPs significantly antagonize glutamate-induced oxidative nerve injury, and inhibition of apoptosis. GPs of alcohol caused NPCs injury is also has a protective effect, currently no relevant reports. In recent years, NPCs nerve repair has aroused attention. Animal experiments showed that rat, monkey brain after ischemia and other noxious stimuli can cause brain tissue proliferation and differentiation of NPCs, but its limited proliferative capacity, not enough to create a self-healing effect. Therefore, to study the NPCs the proliferation mechanism and the discovery can promote NPCs proliferation of material, thus achieving the nerve repair purposes, the important area of ??neurobiology research. GPs proliferative capacity of NPCs, at home and abroad has not been reported. Research purposes: 1, to establish in vitro adherent the cultured NPCs alcohol damage model and GPs protection model to investigate the protective effect of the injury mechanism of alcohol NPCs and GPs, to provide a theoretical basis for the prevention and treatment of fetal alcohol syndrome. Establish vitro passage adherent cultured NPCs proliferation study an experimental model to explore the impact of different concentrations of GPs on the proliferative capacity of NPCs to provide experimental evidence for the role and mechanism of nerve repair in-depth study of GPs. Experimental methods: adherent cultured in vitro pregnancy 14d fetal rat telencephalon cortical NPCs as experimental material, the following experiment: GPs protective effect of alcohol damage NPCs (1) NPCs ① primary culture and identification drawn adherent using an inverted phase contrast microscope after various incubation times Morphological observation; ② by immunohistochemical staining were identified NPCs purity; ③ BrdU incorporation analysis NPCs proliferative capacity; (4) removal of growth factor-induced differentiation 7d identification NPCs natural differentiation capacity; were divided into four concentrations of alcohol (2) alcohol damage model and damage mechanisms ① action group (0, 25, 50 and 100 mmol / L); ② the influence of alcohol after 24h, inverted phase contrast microscope differences in cell morphology; ③ MTT assay different concentrations of alcohol on cell viability; ④ biochemical markers detect SOD activity in the cells in each group, the MDA content; the ⑤ PI/Hoechst33342 fluorescent staining to identify cell death; (3) GPs protection model establish ① Experimental divided into normal control group, alcohol injury group, GPs (the five concentration 25, 50, 100, 200 and 400μg/mL) alcohol group, a total of seven groups; ② at four different times (before the injury 24h before the injury 12h, the damage, while damage after 12h) were added to GPs, the influence of alcohol after 24h, MTT method cell viability in all experimental groups, determine the GPs optimal duration of action and the role of concentration; (4) GPs of the protective effect of (1) an inverted phase contrast microscope observed under normal control group, alcohol injury group, the best GPs protected alcohol injury group cell morphological differences; ② PI/Hoechst33342 fluorescent double staining groups of cell death, changes in the way; ③ flow cytometry: PI single dye for detecting the the rate of apoptosis and cell cycle changes; Rh123 staining groups of mitochondrial membrane potential was measured changes; ④ the use of Fluo-3/AM fluorescent probe, cells Ca2 concentration observed in laser confocal microscope changes; ⑤ Western blot to detect changes in the cells Bcl-2 and Bax expression levels. 2, GPs proliferative capacity of NPCs (1) the original cultured NPCs 7d passage after passage adherent culture after the first generation of NPCs morphological observation; the GPs role of group (2) were divided into six concentrations (0, 25, 50, 100, 200 and 400μg/mL), after 48h indicator detection; (3) by immunofluorescence staining identified passages and Canadian GPs NPCs purity; (4) MTT assay the growth of the above-mentioned group of NPCs vitality situation; (5) of the group of NPCs cell count, cell growth curve; (6) BrdU incorporation analysis GPs proliferative capacity of NPCs; (7) Western blot detection of the cells proliferating cell nuclear antigen (PCNA) of Expression. NPCs were round or oval-shaped, experimental results: 1 GPs to alcohol damage NPCs protection role (1) of NPCs morphological observation and identification: primary adherent culture cells in three-dimensional projections, a small colony distribution. As the incubation time, the cell colony radially outwardly extending. Cell culture 5d nestin-positive cells in the rate of up to 75.08%, the proliferation index was 30.18%; removal of the growth factor-induced differentiation 7d, neurons, astrocytes and oligodendrocytes ratio were 11.02%, 23.74 % and 4.15%; (2) of NPCs alcohol damage model and mechanism of injury research (1) Morphological observation: the allied NPCs injury treated with different concentrations of alcohol (25, 50 and 100mmol / L) for 24h, NPCs cell body round, protruding retraction, three-dimensional; significantly reduced the number of the high concentration of alcohol (100mmol / L) cells, more apoptotic cells; ② MTT test: four experimental groups (with 0, 25, 50, 100 mmol / L alcohol) cell viability 100%, respectively, 85.37%, 72.18% and 66.42%; the ③ biochemical detection: the four experimental groups intracellular SOD activity for 24.77,25.39,23.65,17.42 U / mgprot MDA content were 2.53,3.02,3.93 , 6.56 nmol / mgprot; ④ PI/Hoechst33342 fluorescent double staining: normal group, the nucleus is round, oval or kidney-shaped, blue fluorescence; gradually reduced with increasing alcohol concentration, cell number, the phenomenon of karyopyknosis gradually obvious blue fluorescence intensity increased, with a blue fluorescent apoptotic bodies, and a small number of necrotic cells, red fluorescence; (3) GPs protection model and protective effect detected ① MTT: 24h injury before adding 100μg / mL GPs protective effect, can significantly increase the damage of alcohol NPCs survival, the survival rate of 85.12%; the following experiment GPs protection group are carried at the time and the role of the concentration of the best role of GPs; ② morphological observation: GPs protection full group cell body protruding rare apoptosis, growth state alcohol injury group, close to the normal control group; ③ PI/Hoechst33342 fluorescent double staining: GPs protection group, the number of cells is not significantly reduced, most of the blue fluorescent nucleus shape Rules, close to the normal control group, occasionally red fluorescent nucleus, (4) flow cytometry analysis: a. apoptosis rate analysis: alcohol injury rate of apoptosis (9.36%) is higher than the normal control group (0%) and GPs protection group (2.12%); b. analysis of cell cycle: cells in G0/G1 phase compared with the normal group, the influence of alcohol after a significant increase in S phase cells decreased, while GPs improve this cycle. (5) of Rh123 staining mitochondrial membrane potential: normal control group, alcohol injury group, the fluorescence intensity of the GPs protection group were 146.96,63.47,124.75; the Ca2 concentration analysis ⑥ cells: normal control group, alcohol injury group, GPs protection group Ca2 fluorescence intensity were 7.44,26.07,13.74; ⑦ Western blot: Compared with the normal control group, alcohol damage cells apoptosis inhibitory protein Bcl-2 expression level was significantly reduced, and promote apoptotic protein Bax expression levels were significantly increased, GPs The protection group can significantly antagonize this effect of alcohol; GPs proliferative capacity of NPCs (1) morphology: passage 1 cultured the 3d of NPCs rounded or oval cell body full of obvious projections evenly distributed ; (2) identifying the purity: After passaged and GPs role nestin-positive cell rate of each experimental group (0,25,50,100,200 400μg/mL) in more than 97%; (3) MTT detection: The each group NPCs activity were 100% 106.44% 120.65% 134.07% 127.47% 107.73%; (4) Growth curve analysis: above the growth curve of the cells in each group are the \/ mLGPs group cell growth rate faster than the normal control group, which 100μg/mL GPs were most significant; (5) the proliferation of the ability to detect: the above groups NPCs proliferation index were 33.36%, 35.11%, 39.21%, 45.59%, 42.26% and 34.27%; (6) Western blot detection: the groups PCNA / α-Tubulin the density ratio of the electrophoretic bands were 0.45,0.46,0.74,0.99,0.92 0.50. Conclusion: 1, primary adherent culture get NPCs is an ideal material for nerve injury protection experiments; 2, the high concentration of alcohol (100mmol / L) cells can decline in SOD activity, MDA content increased, ultimately leading to cell apoptosis; injury before adding GPs alcohol damage the NPCs have a protective effect, improve the survival rate of the cells to injury before 24h joined the 100μg/mL GPs protective effect; 4, GPs by reducing Ca2 concentration within the NPCs elevated, raised the antiapoptotic protein Bcl-2 expression and lowered promote apoptotic protein Bax affects mitochondrial apoptosis signaling process to prevent a decline in the mitochondrial membrane potential caused by alcohol, reducing cell withered apoptosis; passaged culture can improve the purity of NPCs, is an ideal material of NPCs proliferation studies; the 6,25 ~ 400μg/mL GPS after 48h does not affect NPCs purity; GPS by raising the amount of DNA synthesis associated protein PCNA expression to promote the proliferation of NPCs, BrdU-positive rate of cell activity to enhance the growth speed the 100μg/mL the GPs role of the most significant.