| Objective:By observing changes of the ultrastructural of the hippocampal neurons,and detecting activity,damage degree,apoptosis level,and expression levels of Bcl-2 and Bax of the hippocampal neurons before and after intervention with serum containing tea,to investigate the effect of the serum containing tea on hippocampal neuronal apoptosis induced by high glucose.Methods:1.Blank serum and tea serum were prepared.Determine the content of 10 components in serum based on methodological establishment.Use medium to dilute low,medium and high concentration blank serum and serum containing tea(25%,50%,100%).2.Primary culture and purity of hippocampal neurons:Primary cultured SD neonatal rat hippocampal neurons were cultured until 5 day,and the purity of cell was detected by NSE immunohistochemistry.3.Cell division and drug intervention:Cells were randomly divided into 8 groups:control group,high glucose group,low,medium and high concentration blank serum groups,low,medium and high concentration serum containing tea groups.The glucose concentration was 25 mmol/L in the control group,the glucose concentration was 45 mmol/L in the high glucose group,and low,medium,and high concentration blank serum group were added to have being prepared low,medium,and high concentration blank serum at 10% of the total volume and the glucose concentration were 45 mmol/L,low,medium and high concentration serum containing tea groups were added to have being prepared low,medium,and high concentration serum containing tea at 10% of the total volume and glucose concentration were 45 mmol/L,after continuing to cultivate cells 48 hours.The ultrastructure of hippocampal neurons were observed by transmission electron microscopy.4.The activity of hippocampal neurons were detected by CCK-8 test.5.The damage degree of hippocampal neuronas were detected by lactate dehydrogenase test.6.The apoptotic rate of hippocampal neurons were detected by Flow cytometry.7.The expression levels of Bcl-2 and Bax of hippocampal neurons weredetected by Western blot.Results: 1.The six components were detected in serum containing tea were gallic acid,catechin,caffeine,epigallocatechin gallate,epicatechin and epicatechin gallate,and their mass concentrations were 3.6?g/mL,4.2?g/mL,54.6?g/mL,22.8?g/mL,3.8?g/mL,4.8?g/mL,respectively.2.Successfully cultivated hippocampal neurons,and the cell purity was over 90%.3.In the control group,the nuclear morphology of hippocampal neurons was regular,and the nuclear chromatin was abundant and evenly distributed,the cytoplasm contained more normal mitochondria and endoplasmic reticulum;compared with the control group,the nucleus morphology of the hippocampal neurons was irregular,the chromatin distribution was uneven,and the cytoplasm contained swelling mitochondria and cystic dilated endoplasmic reticulum in the high glucose group;Compared with the high glucose group,the ultrastructure of hippocampal neurons had not improve significantly in low,medium,and high concentration blank serum and low concentration serum containing tea,but the nuclear morphology of hippocampal neurons was completed,and the structure of cytoplasmic mitochondria and endoplasmic reticulum were improved in medium and high concentration serum containing tea,and has a dose dependence.4.Compared with the control group,the activity of hippocampal neurons was decreased in the high glucose group(P<0.01);compared with the high glucose group,the activity of the cells were not statistically improved in the low,medium,and high concentration blank serum group(P>0.05),the activity of cells were increased in low,medium and high concentration serum containing tea groups(P<0.05,P<0.01),and has a dose dependence.5.Compared with the control group,the damage degree of hippocampal neurons was significantly increased in the high glucose group(P<0.01);Compared with the high glucose group,the damage degree of cells had not significant improved in low,medium and high concentration blank serum groups(P>0.05),but the damage degree of cells were decreased in low,medium and high concentration serum containing tea groups(P<0.05,P<0.01),and has a dose dependence.6.Compared with the control group,the apoptotic rate of cells were increased in the high glucose group(P<0.01);compared with the high glucose group,apoptotic rate of cells had nosignificant difference in the low,medium,and high concentration blank serum groups(P>0.05),However,the apoptotic rate of cells were decreased in low,medium and high concentration serum containing tea groups(P<0.01),and has a dose dependence.7.Compared with the control group,the expression levels of Bax of hippocampal neurons were increased,and the expression of Bcl-2 were decreased(P<0.01)in high glucose group;Compared with the high glucose group,the expression levels of Bax and Bcl-2 were not statistically variety in the low,medium,and high concentration blank serum groups(P>0.05),but the expression levels of Bax were decreased,and the expression levels of Bcl-2 were increased in low,medium,and high concentration serum containing tea groups(P<0.05,P<0.01),and has a dose dependence.Conclusion: 1.The increasing of the glucose concentration can make the ultrastructure of hippocampal neurons abnormal,and it can improve the ultrastructure of cells after the intervention of the serum containing tea.2.the activity of hippocampal neurons can be decreased and the damage degree of cell has been increased with the increasing of glucose concentration,The activity of hippocampal neurons have been increased and the damage degree of cells have been decreased after intervention with the serum containing tea.3.The expression levels of Bcl-2 of hippocampal neurons can be decreased,the expression levels of Bax and the apoptotic rate of cells can be increased with the increasing of glucose concentration,while the expression levels of Bcl-2 of hippocampal neurons can be increased,the expression levels of Bax and the apoptotic rate of cells can be increased after intervention with the serum containing tea. |
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