The Protective Effect Of Gypenosides On Ethanol-induced Neural Precursor Cells Damage And The Effect Of Gypenosides On Proliferation Of Neural Precursor Cells

Backgroud:Neural precursor cells (NPCs) is the general name of immature nerve cells in different developmental stages in central nervous system, which include neural stem cells and neural progenitor cells. The NPCs has potencies of self-renew, proliferation and differentiating into all kinds of nerve cells. It has been acknowledged as seed cells for the repairing of neural injury. Fetal alcohol syndrome (FAS) is a birth defect oc-curring in some children whose mother consumed too much ethanol during pregnancy. Its symptoms include a series of dysplasia in central nervous system. Studies have found that ethanol can inhibit NPCs proliferation, cause some NPCs apoptosis and reduce the number of nerve cells, which is considered as one reason for the patho-genesis of FAS. But we know very little about the mechanism of how the NPCs in-jured by ethanol. Maybe ethanol leads to apoptosis through increasing oxygen free radicals, injuring cell membrane, changing intracellular ionic homeostasis and inter-fere the regular cell signaling pathways.Gypenosides (GPs) are the main active ingredients extracted from the Gy-nostemma Pentaphyllum Makino which have a wide range of pharmacological effects, such as the inhibitory effect on lacking blood and oxygen of the brain, antioxidant effectiveness, anti-aging and modification the injuring nerve cells. Our group previous studies showed that, GPs can significantly antagonized Glu-induced oxidative neuro-toxicity and inhibite the neuron apoptosis. Whether the GPs have the protective effect on NPCs damage caused by ethanol or not? It has not been reported.In recent years, the study of the effects of NPCs on the injury and repair in cen- tral nervous system has been taken seriously attention. Experiments from animal tests showed that NPCs in the brain of rats and monkeys would proliferate and differentiate after hurting. But they couldn't fully been repaired because the proliferation ability of NPCs is very limited. Therefore, studying about the proliferation mechanism of NPCs and the matter that can promote the proliferation of NPCs became the hot topic of neurobiology research. We haven't sawn any report about the effect of GPs on the proliferation of NPCs.Objective:1,To study the mechanism of how ethanol damage NPCs and the protective effect of GPs on the ethanol-induced NPCs damage by establishing an ethanol damage model and a GPs protect model for adherent NPCs in vitro.2,To study the effect of GPs on proliferation of neural precursor cells by establishing a passaged culture model for adherent NPCs in vitro.Methods:Isolated cells from the cortical neuroepithelial of embryo rat (El4) and cultured them by adherence method in vitro. Using these cells to complete the following experi-ments:1,The protective effect of GPs on ethanol-induced NPCs damage(1) Culturing and identifying the NPCs①Observing morphology of cells with inverted phase contrast microscope;②Identifying the purity of NPCs by immunofluorescence technique;③Analyzing the proliferation ability of NPCs by incorporating BrdU;④Analyzing the differentiation ability of NPCs by subtracting growth factors for 7d;(2) Establishing an ethanol damage model and studying the damage mechanism①Dividing the cells into four experiment groups (0,25,50 and 100mmol/L ethanol);②Observing morphology of cells with inverted phase contrast microscope when they were culturing with ethanol for 24h;③Analyzing the influence of ethanol on the activity of NPCs by MTT assay;④Detecting the activity of SOD and the content of MDA by biochemistry assay kits; ⑤Measuring the way of cell death by PI/Hoechst33342 double staining; (3) Establishing a GPs protect model①Dividing the cells into seven experiment groups:control group, ethanol group, 100mmol/L ethanol and GPs(25,50,100,200 and 400μg/mL) groups;②Adding GPs to the culture medium at four different times(24h before damage, 12h before damage, at the same time with damage,12h after damage)and analyz-ing the activity of NPCs by MTT assay; Making sure which concentration of GPs and when to add it can get the best protective effect;(4) Studying the protective effect of GPs①Dividing the cells into three experiment groups:control group, ethanol damaged group, GPs protective group. Observing morphology of cells with inverted phase contrast microscope;②Measuring the changes of the cellular death pathway by PI/Hoechst33342 double staining;③Analyzing the changes of apoptosis rate, cell cycle and mitochondrial membrane potential by flow cytometry;④According to Flou-3/AM, detecting the changes of intracellular calcium concen-tration using laser scanning confocal microscope;⑤Assaying the expression level of Bcl-2 and Bax by Western-blot assay;2,The effect of GPs on proliferation of NPCs(1) Along with the increasing of the culture days, the NPCs would proliferate. Passage them on the 7th day. Observing morphology of the passaged adherent culture NPCs with inverted phase contrast microscope after culturing for 3 days;(2) Dividing the cells into six experiment groups (0,25,50,100,200 and 400μg/mL GPs) and treating them for 48h before testing;(3) Identifying the purity of NPCs which had been passaged and cultured with GPs by immunofluorescence technique;(4) Analyzing the NPCs activity of every group by MTT assay;(5) Obtaining growth curves for the NPCs in every group by counting the cells;(6) Analyzing the proliferation ability of NPCs in every group by incorporating BrdU; (7) Assaying the expression level of proliferating cell nuclear antigen (PCNA) by Western-blot assay.Results:1,The protective effect of GPs on ethanol-induced NPCs damage(1) Culturing and identifying the adherent NPCs:The cells had round or ellipse solid bodies. Some of them had short processes. They grew as many colonies. Along with the increase of the days we cultured, they would extend out radially with the original colonies as their center; After primary culturing for 5 days, the ratio of NPCs was about 75.08%, the proliferation index (PI) of NPCs was 30.18%; When subtracting growth factors for 7 days, NPCs could differentiate into neurons, astrocytes and oligodendrocytes, the ratios of them were 11.02%,23.74%and 4.15% separately;(2) Establishing an ethanol damage model and studying the damage mechanism①Morphological observation:After the adherent NPCs were damaged by different ethanol concentrations (25,50 and 100mmol/L) for 24 hours, the cell bodies would turn round and the processes would retract at different levels. In the 100mmol/L ethanol damaged group, the numbers of NPCs were obviously re-duced, and we could see many apoptotic cells;②MTT assay:The relative survival rates of four groups (0,25,50 and 100mmol/L ethanol) were 100%,85.37%,72.18% and 66.42% separately;③The SOD activity and MDA content of four groups were 24.77,25.39,23.65, 17.42 U/mgprot and 2.53,3.02,3.93,6.56 nmol/mgprot separately;④PI/Hoechst33342 double staining:Nucleus in control group were round, ellipse or reniform and dyed into blue. With the increasing of ethanol concentration, the cell numbers were reduced, some nucleus crimpled apparently, apoptotic bodies ap-peared and some cells were dyed into red;(3) Establishing a GPs protect model and studying the protective effect of GPs①MTT assay:GPs has the protective effect on ethanol-induced NPCs damage, 100μg/mL GPs working for 24h before damage showed this effect most signifi-cantly. Its relative survival rates can reach 85.12%. We ues this group as the GPs protective group in the later tests;②Morphological observation and PI/Hoechst33342 double staining both showed that the NPCs in GPs protective group remained favorable growth, distinctly con-trasted to the condition of ethanol damaged cells;③Flow cytometry methods:The apoptosis ratio of ethanol damaged group (9.36%) was higher than both control group (0%) and GPs protective group (2.12%), and the cell cycle also changed. Also, the mitochondrial membrane potential of etha-nol damaged group (63.47) was lower than both control group (146.96) and GPs protective group (124.75);④Intracellular calcium concentration analysis:Under the laser scanning confocal microscope, the mean fluoresence intensity of ethanol damaged group (26.07) was higher than both control group (7.44) and GPs protective group (13.74);⑤Western-blot assay:Compared with the control group, there was a higher expression of apoptosis-associated protein Bax, but lower of Bcl-2 in ethanol damaged group. However, condition was reversed in GPs protective group.2,The effect of GPs on proliferation of NPCs(1) Morphological observation:The NPCs cultured for 3 days after passaged for the first time distributed more evenly in the medium than primary culture;(2) The purity of NPCs of six groups (0,25,50,100,200 and 400μg/mL GPs) are all above 97%;(3) MTT assay:The relative survival rates of six groups were 100%,106.44%, 120.65%,134.07%,127.47% and 107.73% separately;(4) Drawing of the growth curve:Growth curves for the NPCs of six groups were all shaped as "S". Cells in 50,100,200μg/mL GPs groups grew more quickly than control group,100μg/mL GPs group was the most significantly one;(5) The proliferation index (PI) of NPCs of six groups were 33.36%,35.11%,39.21%, 45.59%,42.26% and 34.27% separately;(6) Western-blot assay:The value of PCNA/α-Tubulin of six groups were 0.45,0.46, 0.74,0.99,0.92 and 0.50 separately.Conclusion: 1,Adherent NPCs are useful material for nerve cells injuries research;2,100mmol/L ethanol can cause NPCs damaged and even to apoptosis through decreaseing the activity of SOD and increasing the content of MDA;3,GPs have the protective effect on ethanol-induced NPCs damage,100μg/mL GPs treating for 24h before damage showed this effect most significantly;4,In ethanol damaged NPCs, GPs can prevent the increasing of intracellular calcium concentration, prevent the decreasing of mitochondrial membrane potential, change the expression level of Bcl-2 and Bax, and then raise NPCs' survival;5,Passaged culture can increase the purity of NPCs. It is useful for proliferation research;6,Cultureing with 25～400μg/mL GPs for 48h can not change the purity of NPCs;7,GPs can promote the proliferation of NPCs in vitro through increasing expression level of PCNA,100μg/mL GPs showed this effect most significantly.