| Objective: To investigate the protective mechanism of Polygonatum Polysaccharide(PP) on the apoptosis of primary cultured cerebral cortical neurons caused by hypoxia. Methods: 1. Using the primary culure of cerebral neurons of postnatal rats in serum-free neurobasal medium supplied with 2%B27 supplement. 2. The percentage of neurons which are cultured for 6 days was identified by NSE immunochemical staining techique. 3. The cytotoxicity of various doses of PP which were added to neurons culture system for 48h after 4 days culture was determined by trypan blue exclusion. 4. The relationship of doses of PP with effects which PP prevented neurons apoptosis and necrosis was examined by the Hoechst 33342 and Trypan blue staining, in which PP of 50μg/ml-8mg/ml were added to neurons culture system for 10h after 4 days culture, then neurons were been in hypxia for 12h and again in re-oxygen for 48h. 5. The experimental groups were divided into: (1) the normal control group (the negative control group) in which neurons were cultured in 5%CO2 37℃for 6 days; (2) the apoptotic group (the positive control group) in which neurons were cultured in 5%CO2 37℃for 4 days, then in 5%CO2-95%N2 cabinet (hypoxia) for 12 hours and again returned to normal culture condition (re-oxygen) for 48 hours; (3) the PP experimental groups in which PP were added to culture system for 10h before hypxia in which neurons had been cultured in 5%CO2 37℃for 4 days, then in 5%CO2-95%N2 cabinet (hypoxia) for 12 hours and again returned to normal culture condition (re-oxygen) for 48 hours: the experimental group 1 in which PP was 500μg/ml; the experimental group 2 in which PP was 1mg/ml;the experimental group 3 in which PP was 1.5mg/ml. (4) the PP experimental groups in which PP were added to culture system after hypxia in which neurons had been cultured in 5%CO2 37℃for 4 days, then in 5%CO2 95%N2 cabinet (hypoxia) for 12 hours, after adding PP the culture system was returned to normal culture condition (re-oxygen) for 48 hours: the experimental group 1 in which PP was 500μg/ml; the experimental group 2 in which PP was 1mg/ml;the experimental group 3 in which PP was 1.5mg/ml. 6. The percentage of apoptotic neurons of all groups was determined by tne Hoechst 33342 staining. 7. The ladders of apoptotic DNA fragments of all groups were examined by DNA agarose gel electrophoresis. 8. The percentage of apoptotic and necrotic neurons of all groups was identified by the Annexin/PI Flow Cytometry. 9. The percentage of neurons expressing Bcl-2, Bax and caspase-3 of all groups was determined by immuno-histochemical staining, respectively. Results: 1. There were 93.36%±1.02% neurons in normal neonate rat cerebral cortical neuron culture for 6 days. 2. No cytotoxicity was found in normal neonate rat cerebral cortical neuron culture from 1mg/ml to 6mg/ml PP for 6 days and the death rate of neurons was from 7.96%±0.82% to 8.42%±0.42%, it was P>0.05 comparing with normal control group. But from 7mg/ml to 20mg/ml, the PP expressed cytotoxicity and the death rate of neurons was from 9.72%±0.98% to 43.40%±3.98%, it was P<0.01 comparing with normal control group. 3. The PP could prevent neuronal necrosis from 50μg/ml to 1.5mg/ml in hypoxia and again re-oxygen culture and the necrotic rate of neurons dropped from 21.00%±2.94% to 12.28%±1.37%, it was P<0.01 comparing with apoptotic group (25.31%±2.66%) and normal control group (8.90%±1.50%). But from 2mg/ml to 8mg/ml the capacity of PP preventing neuronal necrosis went down and the necrotic rate of neurons rose from 15.90%±2.66% to 34.32%±7.18%, it was P<0.01 comparing with apoptotic group (25.31%±2.66%) and normal control group (8.90%±1.50%),respectively. 4. The PP could prevent neuronal apoptosis from 50μg/ml to 1.5mg/ml in hypoxia and again re-oxygen culture and the apoptotic rate of neurons dropped from 36.12%±4.12% to 11.80%±1.18%, it was P<0.01 comparing with apoptotic group (41.30%±2.77%). However, when itsconcentration increasing to 2mg/ml or more the capacity of PP preventing neuronal apoptosis dropped and the apoptotic rate of neurons rose from 14.54%±3.76% (2mg/ml) to 28.04%±4.93% (8mg/ml), there was significant statistical difference (P<0.01) in every experimental groups comparing with apoptotic group and normal control group. 5. In normal control group, the apoptotic group, PP groups adding 500μg/ml,1mg/ml and 1.5mg/ml before hypoxia the apoptotic rate of neurons was 6.60%±1.06%, 41.30%±2.77%, 13.00%±4.52%, 12.72%±2.15%, 11.80%±1.18%, respectively. Each group was P<0.01 comparing with apoptotic group or normal control group. However, in PP groups adding 500μg/ml, 1mg/ml and 1.5mg/ml after hypoxia the apoptotic rate of neurons was 36.77%±1.45%, 36.60%±1.61 %, 36.37%±2.02%, respectively, and there was not any statistical difference (P>0.05) comparing with apoptotic group (38.03%±1.05%). 6. For normal control group, apoptotic group, PP groups adding 500μg/ml, 1mg/ml and 1.5mg/ml the apoptotic rate of neurons was 1.85%±1.26%, 24.38%±4.15%, 18.30%±4.50%, 13.29%±3.61%, 7.94%±2.67% respectively, tested by the Annexin/PI Flow Cytometry. The apoptotic rate of neurons was P>0.05,P<0.01 and P<0.01 in PP groups of 500μg/ml,1mg/ml and 1.5mg/ml comparing with apoptotic group, and was P<0.01,P<0.01 and P>0.05 in PP groups of 500μg/ml, 1mg/ml and 1.5mg/ml comparing with normal control group. Besides, for normal control group, apoptotic group, PP groups adding 500μg/ml,1mg/ml and 1.5mg/ml the necrotic rate of neurons was 4.56%±0.45%, 14.27%±1.22%, 11.15%±1.48%, 7.89%±1.48%, 4.69%±1.37% respectively, tested by the Annexin/PI Flow Cytometry. The necrotic rate of neurons was P<0.05, P<0.01 and P<0.01 in PP groups of 500μg/ml,1mg/ml and 1.5mg/ml comparing with apoptotic group, and was P<0.01,P<0.05 and P>0.05 in PP groups of 500μg/ml,1mg/ml and 1.5mg/ml comparing with normal control group. 7. For normal control group, apoptotic group, PP groups adding 500μg/ml, 1mg/ml and 1.5mg/ml the positive rate of neurons expressing caspase-3 was 35.66%±3.75%, 68.10%±3.99%, 58.50%±1.76%, 51.20%±3.17%, 45.52%±3.81%, respectively, and statistical difference was P<0.01 in all PP groups comparing in apoptotic group and in normal control group. 8. For normal controlgroup, apoptotic group, PP groups adding 500μg/ml, 1mg/ml and 1.5mg/ml the positive rate of neurons expressing Bcl-2 was 68.22%±3.37%, 38.70%±6.59%, 46.96%±4.10%, 54.32%±4.13%, 61.48%±3.56%, respectively, and statistical difference was P<0.01 in all PP groups comparing in apoptotic group and in normal control group. 9. For normal control group, apoptotic group, PP groups adding 500μg/ml, 1mg/ml and 1.5mg/ml the positive rate of neurons expressing Bax was 61.92%±2.80%, 73.44%±1.67%, 69.40%±2.23%, 65.46%±1.33%, 63.14%±1.01%, respectively, and statistical difference was P<0.01 in all PP groups comparing in apoptotic group, and was P<0.01, P<0.05, P>0.05 in all PP groups comparing in normal control group. 10. For normal control group, apoptotic group, PP groups adding 500μg/ml, 1mg/ml and 1.5mg/ml the ratio of Bcl-2 to Bax (Bcl-2/Bax) was 1.10±0.09, 0.53±0.09, 0.68±0.07, 0.83±0.06, 0.97±0.05, respectively, and statistical difference was P<0.01 in all PP groups comparing in apoptotic group, and was P<0.01, P<0.01, P<0.05 in all PP groups comparing in normal control group. 11. The ladder of DNA fragments extracted from neurons could be found in apoptotic group and PP group of 500μg/ml. But the ladder of DNA fragments was hardly observed in normal control group and PP groups of 1mg/ml and 1.5mg/ml. Conclusion: There were 93.36%±1.02% neurons in normal neonate rat cerebral cortical neuron cultured by Neurobasal and B27 Supplement for 6 days. No cytotoxicity was found in normal neonate rat cerebral cortical neuron culture for PP from 1mg/ml to 6mg/ml for 6 days. The PP of 500μg/ml-1.5mg/ml could prevent neuronal apoptosis induced by hypoxia in primary neonate rat cerebral cortical neurons culture. The effects of PP preventing neuronal apoptosis induced by hypoxia were realized by a way of raising Bcl-2 expression and suppressing Bax and caspase-3 expression. If PP of 500μg/ml-1.5mg/ml was added after hypoxia for 12h, it had no effect of anti-apoptosis.
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