Effects Of Chinese Yam Polysaccharides On Apoptosis Of Cerebral Cortical Primary Neurons Induced By Hypoxia-reoxygenation In Fetal Rats

Objective:To explore the toxic dose of Chinese Yam Polysaccharide into embryonic ratcerebral cortical neurons in vitro,and the effect of nerve cell activity andanti-apoptotic under the condition of hypoxia/reoxygenation,the anti-apoptoticmechanism as well, so as to provide a theoretical basis and experimental basis forYam Polysaccharide clinically used on prevention and treatment of cerebralischemia/hypoxia disease, and also provide experimental basis for the developmentand utilization of Chinese medicine polysaccharide.Methods:1. Embryonic rat cerebral cortex nerve cells in serum free culture.2. Embryonic rat cerebral cortex nerve cells in serum free cultivation ofanoxia/reoxygenation.3. To determine the different doses of Yam Polysaccharide on anoxia/reoxyg-enation and normal cultured nerve cells of embryonic rat cerebral cortex stimulationand toxic effects by MTT assay.4. The experiment was divided into:（1） the normal control group （Con.）, theprimary nerve cells were cultured at37℃and5.0%CO₂saturated humidity incubatorfor6days;（2） the positive control （apoptosis induced）group （Apo.）, the primarynerve cells were cultured at37℃and5.0%CO₂saturated humidity incubator for4days,and then cultured for12h under anoxia,then24h under reoxygenation;（3）Chinese Yam Polysaccharide intervention group （CYPS）, the primary nerve cellswere cultured at37℃and5.0%CO₂saturated humidity incubator for4days, andthen were added to the final concentration of0.025g/L （CYPS1group）,0.05g/L（CYPS2group）,0.1g/L （CYPS3group）,0.25g/L （CYPS4） Yam Polysaccharide topretreat for4h,and also cultured for12h under anoxia,then24h under reoxygenation,respectively.5. Annexin V-FITC/PI double staining to determine the nerve cells in the early apoptosis and late apoptosis and necrosis by flow cytometry.6. Hoechst33342fluorescence staining to detect the morphological changes ofapoptosis nerve cells and experimental groups of apoptosis rate.7. Rhodamine-123fluorescence staining （Rh-123） nerve cells to determine themitochondrial damage, mitochondrial membrane potential changes by flow cytometry.8. DNA agarose gel electrophoresis to detect nuclear DNA degradation （ladderbanding）in late apoptosis nerve cells of the experimental groups.9. Semi quantitative rt-PCR to detect the relative expression of caspase-3,baxand bcl-2mRNA in nerve cells of experimental groups.10. Immunocytochemistry staining to detect the rate of NSE and GFAP positiveexpression cells, identification of cultured nerve cells,the bcl-2,bax and caspase-3protein positive expression in nerve cells as well.Results:1. SD embryo rat cerebral cortical neurons were cultured in vitro for6days,afterthat, the cells NSE positive expression rate was93.7%±0.56%, while the expressionof GFAP-positive rate was8.78%±0.55%by immunocytochemistry staining withanti-NSE, GFAP antibody.2. SD embryo rat cortex neurons were cultured in vitro and under the normaloxygen,by the MTT assay,proved that when the concentration of Chinese YamPolysaccharide was between the0.05g/L and0.5g/L,the nerve cell growth activityincreased than the control group significantly （P<0.05）, while the1.0g/L⁴.0g/L YamPolysaccharide had no significant influence on activity of nerve cells（P>0.05）.Andwhen the concentration was to8.0g/L, Chinese yam polysaccharide had significantlyinhibitory effect on growth of nerve cells （P <0.05）.3. SD embryo rat cortex neurons after Yam Polysaccharide pretreatment werecultured by hypoxia/reoxygenation in vitro,by the MTT assay, proved that the growthactivity of nerve cells in experimental groups was lower than in the normal controlgroup significantly （P<0.05）.The Chinese Yam Polysaccharide of which concent-ration was between0.05g/L and0.1g/L could suppress the nerve cells induced byhypoxia/reoxygenation injury significantly （P<0.05）.while in the0.05g/L and0.25 g/L¹.0g/L,Yam Polysaccharide also had certain protective effect on the nerve cellsinduced by hypoxia/reoxygenation injury. Yam Polysaccharide of2.0g/L couldincrease the nerve cells induced by hypoxia/reoxygenation injury and its effect wasthe same as the apoptosis positive group.4. SD embryo rat cortex neurons after Yam Polysaccharide pretreatment werecultured by hypoxia/reoxygenation in vitro,by Hochest33342fluorescent dyedetection,proved that the apoptosis rate of experiment group of which concentrationwas between0.025g/L and0.25g/L, were respectively32.08%±1.64%,26.74%±0.45%,22.32%±1.81%,28.54%±0.79%,which was lower obviously than the apoptosispositive group37.61%±2.87%（P﹤0.05）,but higher significantly than normal controlgroup6.48%±0.55%（P﹤0.05）.5. SD embryo rat cortex neurons after Yam Polysaccharide pretreatment werecultured by hypoxia/reoxygenation in vitro,by the Annexin V-FITC/PI doublestaining,flow cytometry detection showed that the early apoptosis rate of normalcontrol group,apoptosis positive group and CYPS1^CYPS4group was respectively2.46%±0.58%,51.50%±2.44%,36.67%±2.53%,26.17%±3.80%,13.87%±2.67%,43.63%±0.75%.The early apoptosis rate of CYPS1^CYPS4was higher than normalcontrol group significantly （P<0.05）,but was lower than apoptosis induced groupsignificantly（P<0.05）.And within the0.05g/L to0.1g/L,the early nerve cellsapoptosis rate reduced with the increase of the concentration of yam polysacch-aride,but the Yam Polysaccharide had no significant effect on late nerve cell apoptosisand necrosis（P>0.05）.6. SD embryo rat cortex neurons after Yam Polysaccharide pretreatment werecultured by hypoxia/reoxygenation in vitro,by the Rhodamine-123fluorescencestaining,flow cytometry detection showed that neuronal mitochondria meanfluorescence intensity in normal control group, apoptosis positive group andCYPS1^CYPS4group were respectively232.33±17.62,34.30±13.00,54.87±8.95,159.33±4.51,180.33±13.43,45.90±3.53.And Chinese Yam Polysaccharide of which concen-tration was between0.05g/L and0.1g/L could significantly inhibit hypoxia inducedneuronal mitochondria injury （P<0.05）,which showed that Chinese yam polysac-charide was able to in a certain reduce mitochondrial injury of hypoxia nerve cells and had a dose dependent effect.7. SD embryo rat cortex neurons after Yam Polysaccharide pretreatment werecultured by hypoxia/reoxygenation in vitro,DNA agarose gel electrophoresisshowed that apoptosis induced group had obviously DNA ladder bands,but normalcontrol group and0.1g/L Chinese Yam Polysaccharide group did not see obviousDNA ladder bands, other groups had weakened bands.8. SD embryo rat cortex neurons after Yam Polysaccharide pretreatment werecultured by hypoxia/reoxygenation in vitro,by rt-PCR semi quantitative determine-ation,found that,in normal control group, apoptosis positive group, CYPS1^CYPS4groups,the caspase-3mRNA expression rate was respectively0.62±0.02,1.24±0.03,1.17±0.10,0.87±0.10,0.78±0.05,0.97±0.03;the bax mRNA expression rate wasrespectively0.28±0.04,0.70±0.02,0.65±0.07,0.52±0.06,0.38±0.06,0.52±0.11; Thebcl-2mRNA expression rate was respectively0.88±0.13,0.49±0.09,0.45±0.06,0.67±0.01,0.71±0.05,0.63±0.11. Yam Polysaccharide could decrease caspase-3mRNA expression significantly within0.1g/L⁰.25g/L,and could decrease signific-antly bax mRNA expressionin within0.05g/L⁰.25g/L as well, while couldsignificantly increase the bcl-2mRNA expression within0.05g/L⁰.1g/L （P <0.05）.9. SD embryo rat cortex neurons after Yam Polysaccharide pretreatment werecultured by hypoxia/reoxygenation in vitro,by immunocytochemistry staining,foundthat in normal control group, apoptosis positive group and CYPS1^CYPS4groups,thecaspase-3protein positive cells rate was respectively37.03%±0.38%,58.89%±0.45%,55.65%±0.24%,53.54%±0.79%,45.76%±0.53%,52.42%±1.06%; the bax prot-ein positive cells rate was respectively53.32%±0.77%,65.08%±1.02%,61.70%±2.12%,57.34%±2.53%,53.74%±1.72%,58.38%±1.11%; The bcl-2protein positivecells rate was respective59.36%±2.23%,37.83%±1.08%,43.54%±0.93%,46.82%±0.63%,52.82%±1.33%,45.88%±1.38%. Yam Polysaccharide within0.025g/L⁰.25g/Lcould significantly decrease the caspase-3protein expression, and could significantlydecrease bax protein expression within0.1g/L⁰.25g/L, while could significantlyincrease the bcl-2protein expression within0.05g/L⁰.25g/L（P<0.05）. Conclusion:1. SD embryo rat cerebral cortex nerve cells in serum free culture content ofneurons was93.7%±0.56%.2. Yam Polysaccharide within0.05g/L⁰.5g/L could significantly improve thegrowth activity of nerve cell.Yam Polysaccharide within0.05g/L⁰.1g/L couldsignificantly suppress the nerve cell damage induced by hypoxia/reoxygenation.3. Yam Polysaccharide within0.025g/L⁰.25g/L could inhibit the earlyapoptosis of nerve cells. Yam Polysaccharide within0.025g/L⁰.25g/L could signific-antly inhibit hypoxia induced neuronal mitochondrial damage.4. Yam Polysaccharide within0.1g/L⁰.25g/L could significantly down-regulation the caspase-3mRNA expression in nerve cells induced by hypoxia,andcould significantly down-regulation bax mRNA expression within0.05g/L⁰.25g/Las well,while could significantly up-regulation the bcl-2mRNA expression within0.05g/L⁰.1g/L. Yam Polysaccharide within0.025g/L⁰.25g/L could significantlydown-regulation caspase-3protein expression, and within0.1g/L⁰.25g/L coulddown-regulation bax protein expression significantly as well, while couldsignificantly up-regulation the bcl-2protein expression within0.05g/L⁰.25g/L.YamPolysaccharide maybe inhibit nerve cells apotosis through down-regulation caspase-3and bax expression and up-regulation bcl-2expression,and improve the proportion ofBcl-2/Bax protein.