| Object：To observe the effects of Panax quinquefolium L. saponin(PQLS) onnecrosis and apoptosis of cerebral cortical neurons induced by hypoxia-reoxygenationin fetal rats,then to investigate the therapeutic effect and the detailed protectivemechanism.Methods：1. Primary culture of fetal rat cerebral cortical neurons was performed in vitrofor6days, then the NSE and GFAP expression of the culture system was observed byimmunocytochemistry technique.2. Effects of different doses of PQLS on viability of neurons were investigatedby MTT assay.3. A model of apoptosis of cerebral cortical neurons was established by hypoxiafor12h then reoxygenation for48h.4. Deprived of oxygen for different time(6、12、24hours),the survival rate offetal rat cerebral cortical neurons was investigated by trypan blue exclusion.5. The experimental groups were divided into: the normal control group (thenegative control group): neurons were cultured in5%CO2,37℃for6days; theapoptotic group (the positive control group): neurons were cultured in5%CO2,37℃for4days, then in anaerobic incubator for12hours and re-oxygen for48hours; thePQLS experimental groups:four different final concentration(0.025、0.05、0.1、0.2mg/ml) of PQLS were added to culture system for pharmacologicalpreconditioning in4hours before induced by hypoxia-reoxygenation.6. Effects of different doses of PQLS on viability of neurons of all groups wereinvestigated by MTT assay.7. The percentage of apoptosis and necrosis rate of all groups was analyzedquantitatively by flow cytometry with the Annexin FITC/PI stainning.8. The Bcl-2, Bax and caspase-3protein expression of all groups was observedby immunocytochemistry technique.9. The ladders of apoptotic DNA fragments of all groups were examined by DNA agarose gel electrophoresis.10. The apoptosis rate of all groups was examined under fluorescent microscopywith Hoechst33342staining.Result：1. The expression of NSE,GFAP were respectively (92.8±0.35)%,(5.9±0.32)%when fetal rat cerebral cortical neurons were cultured in vitro for6days.2. The results of MTT assay showed that:0.025-0.2mg/ml PQLS groups couldincrease viability of neurons.Compared with normal control group(0.450±0.027),theviability of neurons was increased significantly in0.05mg/ml(0.494±0.011) and01mg/ml(0.535±0.022) PQLS groups(P<0.01), but0.4mg/ml(0.243±0.022) and0.8mg/ml(0.082±0.007) PQLS groups showed obvious cytotoxicity to neurons (P﹤0.01).3. The results of trypan blue exclusion showed that: the survival rate of neuronsinduced by hypoxia for6,12,24hours was (87.87±3.04)%、(66.80±3.82)%、(28.03±4.45)%respectively.Except6h group, in12h and24h groups, the survivalrate of neurons was decreased significantly compared with normal controlgroup(92.40±3.24)%(P﹤0.01).4. The results of MTT assays showed that: Compared with normal controlgroup(0.488±0.030), the viability of neurons in positive control group(0.324±0.023)was decreased significantly(P﹤0.01).0.025-0.2mg/ml PQLS could increase viabilityof neurons induced by hypoxia. Except0.025mg/ml(0.371±0.074) PQLS group, in0.05mg/ml(0.425±0.024)、0.1mg/ml(0.446±0.050) PQLS groups, the viability ofneurons was increased significantly compared with positive control group(P<0.01).5. The results of flow cytometry with the Annexin FITC/PI stainning showedthat: Compared with normal control group(2.05±0.13)%, the apoptosis rate ofpositive control group(28.77±0.50)%was increased significantly(P﹤0.01). Theapoptosis rates of0.025mg/ml、0.05mg/ml、0.1mg/ml and0.2mg/ml PQLS groupswere (14.47±0.49)%、(9.43±0.10)%、(6.13±0.68)%and (10.85±0.25)%respectively.Compared with the positive control group,the apoptosis rates of allPQLS groups were decreased significantly(P﹤0.01). Compared with normal controlgroup(0.84±0.08)%, necrosis rate of positive control group(6.57±0.19)%was increased significantly(P﹤0.01). The necrosis rates of0.025mg/ml、0.05mg/ml、0.1mg/ml and0.2mg/ml PQLS groups were(5.47±0.18)%、(2.15±0.13)%、(2.52±0.07)%and (2.4±0.10)%respectively.Compared with positive group,thenecrosis rates of all PQLS groups were decreased significantly(P﹤0.01).6. The results of anti-apoptosis Bcl-2protein immunocytochemistry techniqueshowed that: Compared with normal control group(67.65±4.32)%, the expression ofBcl-2protein in positive control group(35.61±2.16)%was decreased significantly(P﹤0.01).The expression of Bcl-2protein of0.025mg/ml、0.05mg/ml、0.1mg/ml and0.2mg/ml PQLS groups were (44.22±3.73)%、(51.97±1.86)%、(63.76±2.96)%and(58.46±0.71)%respectively. Compared with positive group, the expression of Bcl-2protein of all PQLS group was increased significantly(P﹤0.05).7. The results of apoptosis Bax protein immunocytochemistry technique showedthat: Compared with normal control group(60.33±2.06)%, the expression of Bcl-2protein in positive control group(72.19±3.97)%was increased significantly(P﹤0.01).The expression of Bax protein of0.05mg/ml、0.1mg/ml and0.2mg/ml PQLSgroups were (62.94±1.68)%、(57.97±2.6))%and (60.27±1.26)%respectively.Compared with positive group, the expression of Bax protein of allPQLS groups were decreased significantly(P﹤0.01).8. The results of apoptosis Caspase-3protein immunocytochemistry techniqueshowed that: Compared with normal control group(40.86±1.44)%, the expression ofCaspase-3protein in positive control group(68.62±1.40)%was increasedsignificantly(P﹤0.01).The expression of Caspase-3protein of0.05mg/ml、0.1mg/mland0.2mg/ml PQLS groups were (56.41±2.87)%、(50.34±2.65)%and(56.52±4.30)%respectively.Compared with positive group, the expression ofCaspase-3protein of all PQLS groups were decreased significantly(P﹤0.01).9. Ratio of Bcl-2/Bax: Compared with normal control group(1.12±0.04), theratio of Bcl-2/Bax in positive control group(0.56±0.11) was decreased significantly(P﹤0.01). The ratio of Bcl-2/Bax in0.05mg/ml、0.1mg/ml and0.2mg/ml PQLS groupswere (0.85±0.05)、(1.11±0.04)and (0.98±0.03)respectively.Compared with positivegroup, the ratio of Bcl-2/Bax of all PQLS groups were increased significantly(P﹤0.01). 10. The results of DNA agarose gel electrophoresis showed that: The ladder ofDNA fragments extracted from neurons could be found in positive control group and0.025mg/ml PQLS groups. But it was hardly observed in normal control group and0.05mg/ml、0.1mg/ml and0.2mg/ml PQLS groups.11. The results of Hoechst33342staining observed under fluorescentmicroscopy showed that: Compared with normal control group(8.51±0.68)%, theapoptosis rate in positive control group(39.47±0.92)%was increased significantly(P﹤0.01).The apoptosis rates in0.025mg/ml、0.05mg/ml、0.1mg/ml and0.2mg/ml PQLSgroups were(29.91±1.06)%、(25.37±0.47)%、(16.07±0.71))%and (19.09±1.06)%respectively.Compared with positive group, the apoptosis rates in all PQLS groupswere decreased significantly(P﹤0.01).Conclusion：1. A model of apoptosis of fetal rat cerebral cortical neurons cultured in vitrocould be successfully established by hypoxia for12h then reoxygenation for48h.2.0.025~0.2mg/ml PQLS could inhibit apoptosis of cerebral cortical neuronsinduced by hypoxia-reoxygenation.The therapeutic effect and the detailed protectivemechanism possibly through up-regulation of the anti-apoptosis Bcl-2proteinexpression and/or down-regulation of apoptosis Bax, Caspase-3proteinexpression,and increase the viability of neurons. And0.05mg/ml~0.1mg/ml PQLSshows the best therapeutic effect. |
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