| Objective:To investigate the protective mechanism of white beans polysaccharide on the hypoxic apoptosis of primary cultured cerebral cortical neurons.Methods:1. Neurons were cultured in serum-free neurobasalTM -A medium supplied with 2%B27 supplement.2. The percentage of neurons and glial cells, cultured in vitro for 6 days, was identified by NSE and GFAP immunochemical staining technique.3. The proliferation of neural cell was determined by MTT after various doses of white beans polysaccharide had added to neural culture system for 48h.4. The activity of neurons was investigated by trypan blue exclusion to determine the effect of hypoxia and reperfusion in different times(6,12,24 hours) on cultured cortical neurons.5. The relationship between doses and effects of white beans polysaccharide on promoting the proliferation of nerve cells was examined by the MTT, in which 0-8mg/ml white beans polysaccharide were added to neurons culture system for 2h after 4 days conventional cultured, then were been in anaerobic incubator for 12h and again re-oxygen for 48h.6. The apoptotic experimental groups were divided into:(1)the normal control group:neurons were cultured in 5%CO2,37℃for 6 days; (2)the apoptotic group: neurons were cultured in 5%CO2,37℃for 4 days, then in anaerobic incubator for 12 hours and again re-oxygen for 48 hours; (3)the experimental groups:0.5mg/ml, 3.5mg/ml and 8.0mg/ml of white beans polysaccharide was added to culture system for 2h before hypoxia in which neurons had been cultured in 5%CO2,37℃for 4 days, then in anaerobic incubator for 12 hours and again re-oxygen for 48 hours.7. The percentage of apoptotic and necrotic neurons of all groups was identified by the Annexin/PI Flow Cytometry8. The ladders of apoptotic DNA fragments of all groups were examined by DNA agarose gel electrophoresis.9. The percentage of neurons expressing Bcl-2, Bax and Caspase-3 of all groups was determined by immunocytochemistry staining, respectivelyResult:1. There was (92.5±2.20)% neurons and (7.24±1.15)% glial cell when fetal rat cerebral cortical neuron cultured in vitro for 6 days;2. It showed proliferation to neurons when the concentration of white beans polysaccharide less than 6.0mg/ml, and the cell relative growth rate was from 0 up to 28.94%. There was no significant difference compared with normal control group when the concentration is 1.0mg/ml and 8.0mg/ml(P> 0.05), but when the concentration at the range of 2.0mg/ml to 6.0mg/ml showed significant difference between normal group. When the concentration reached 16.0mg/ml, the white beans polysaccharide showed inhibition to neurons cultured at the conventional condition. There was significant difference compared with normal group(P< 0.05). Survival3. The survival rate of neurons, cultured in anaerobic incubator for 6,12,24 hours then re-oxygen for 48 hours, was (91.40±3.85)%, (78.80±4.15)%, (19.60±3.71)% respectively, and (95.80±1.30)% for the normal control group. There was striking statistical difference between hypoxic groups of 12h and 24h to the normal control group(P<0.05～P<0.001), but the group of 6h showed no statistical difference4. The white beans polysaccharide showed against inhibition induced by hypoxia at the range of 0.5mg/ml to 8.0mg/ml, to compared with normal group, the cell relative growth rate was rose from 65.04% to 103.79%, There was striking statistical difference comparing with positive control group (P<0.001). and there was the most significant effect when the concentration within 1.0mg/ml to 4.0mg/ml, but when the construction was 0.5mg/ml or 6.0mg/ml, the effect of against inhibition was weaker than other groups.5. The apoptotic rate of each experimental groups was (20.93±0.47)%, (4.60±0.43)%, (24.47±1.99)%, respectively, analyzed by the AnnexinⅤ-FITC Flow Cytometry. There was striking statistical difference comparing with apoptotic group(29.27±1.76)% and normal group(1.51±0.43)% (P<0.01). Besides, it could also show the necrotic rate of each experimental groups as (5.26±1.79)%, (4.25±0.99)%, (7.94±1.90)% tested by the AnnexinV-FITC Flow Cytometry. There was significant difference comparing with apoptotic group(12.67±1.55)% and normal group(0.86±0.31)% (P<0.05).6. The ladder of DNA fragments extracted from neurons could be found in apoptotic group and group 1 and group 3 of experimental groups. But it was hardly observed in group 2 of experimental groups and normal group.7. The results of immunocytochemistry staining showed that the rate of postive neurons of Caspase-3 protein was (35.75±3.08)% in the normal group, and (68.42±4.81)% in the apoptotic group. The rate of postive neurons of expression of Caspase-3 protein in group 1 to 3 of the experimental groups was (51.33±4.14)%,(44.69±3.79)%,(58.45±5.12)%, respectively. Except the groupl of experimental groups, there was statistical difference between other drug intervention groups and the apoptotic group(P<0.05)8. The rate of postive neurons of Bcl-2 protein was (64.35±3.81)% in the normal control group, and (33.05±5.20)% in the apoptotic group by immun-ocytochemistry staining, and the rate of postive neurons of expression of Bcl-2 protein in group 1 to 3 of the experimental groups was (52.49±3.58)%, (61.91±6.46)%, (47.58±3.17)%, respectively. Except the group1 of experimental groups, there was statistical difference between other drug intervention groups and the apoptotic group(P<0.05).9. The rate of postive neurons of Bax protein was (59.52±3.63)% in the normal control group and (70.40±3.25)% in the apoptotic group by immun- ocytochemistry staining, and the rate of postive neurons of Bax protein in the group 1 to 3 of the experimental groups was (63.02±2.23)%, (58.74±4.15)%, (66.19±3.42)%, respectively. The rate of postive neurons of Bax protein has statistical difference between the apoptotic group and the group 2 of the experimental groups(P<0.05).10. The result of Bcl-2/Bax was(1.08±0.02) in the normal control group and 0.47±0.06 in the apoptotic group. The result of Bcl-2/Bax in the group 1 to 3 of the experimental groups was (0.84±0.05), (1.05±0.05), (0.72±0.07), respectively. Except the group 1 of the experimental groups, there was statistical difference between other drug intervention groups and the apoptotic group(P<0.05).Conclusion:1. Cerebral cortical neuron of fetal rats were successfully cultured in serum-free neurobasalA medium supplied with 2% B-27 supplement.2. It didn't have cytotoxicity on the fetal rat cortical neurons cultured in serum-free medium in vitro when the concentration of white beans polysaccharide less than 8.0mg/ml, However, as the concentration reached 16.0mg/ml, it showed apparent cytotoxicity.3. 1.0mg/ml～4.0mg/ml white beans polysaccharide could significantly inhibit the hypoxia-induced apoptosis in fetal rat cortical neurons. Its anti-apoptotic mechanism possibly through up regulating the anti-apoptotic protein Bcl-2 and/or down regulating anti-apoptotic protein Bax, Caspase-3 expression. |
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