| Objective:Epilepsy is one of the most common and serious neurological disease,most patients require lifelong medication. Phenytoin Sodium from1938to antiepileptic treatment, its role is to prevent epileptic discharge spread to the surrounding normal tissue,for generalized tonic-clonic seizures, tonic seizures and partial seizures is better, but its narrow therapeutic window prone to toxic reactions. When used long-term high doses or high blood concentrations,cerebellar damage symptoms appeared,may be related to promoting apoptosis of cerebellar granule cells and Purkinje cells.Probucol has the effect of lipid-lowering, anti-inflammatory, anti-oxidant, etc.Recent studies showed that it also has antioxidant effects in the nervous system, and thus play its neuroprotective effects.But whether it would affect apoptosis remains to be further studied.In this study mainly researched the impact of Probucol and Phenytoin Sodium on mice in hippocampus and cerebellar of apoptosis.Discussed the adverse reactions of Phenytoin in nervous system,and whether Probucol play a protective role in nervous system.Methods:1Groupings and administration:50clean and healthy4-week-old male mice, weighing (22±2g), randomly divided into NS (Saline) group, PHT (Phenytoin) group and PHT+PB (Probucol) mixed group. NS group, PHT group each group10,PHT+PB mixed group of30, according to PB dose of low, medium and high doses of three sub-groups, each sub-group of10. Using intragastric gavage needle,PHT group was given a specific dose of100mg/(kg.d) for drug intervention,PHT+PB low, medium and high dose mixed groups were given mixed PHT100mg/(kg.d), and PB250mg/(kg.d),500mg/(kg.d),1000mg/(kg.d);NS group was given normal saline, mixed dose administered twice at intervals of30min; Drug intervention groups once a day; total of10days.2Drawn and testing:Mice in each group anesthetized with10%chloral hydrate (0.35ml/100g),supine fixed and cut the chest to expose the heart,insert the needle from the apex, while cut the right atrial appendage.Flushed with50ml-100ml0.9%saline,and fixed by200ml4%paraformaldehyde phosphate buffer.After injection,decapitated and remove the cerebrum and cerebellum into4%paraformaldehyde phosphate buffer.Graded ethanol dehydration, xylene, dipped wax, embedding, select the cerebrum and cerebellum consecutive coronal slices. To observe the the expression of apoptosis-related protein Bax and Bcl-2by immunohistochemistry;to observe the neuronal apoptosis by TUNEL3Statistical analysis:By SPSS13.0statistical software for data analysis, using one-way ANOVA comparison between groups,between the two groups were compared using the SNK-q test.Results:1HE(hematoxylin-eosin)staining:1.1The hippocampal neurons of HE staining:NS group the hippocampal neurons of mice arranged in neat rows, cell outline clear and complete, transparent cytoplasm, round or oval nucleus, nucleolus clear, normal morphology, no significant apoptosis.PHT group arranged in scattered of neurons, the cell spacing increases, cell vague outline, pyknotic nuclei stained and around vacuolization.PHT+PB low-dose mixed group neurons loosely arranged in hippocampus, the number of apoptotic cells was significantly increased.PHT+PB high-dose mixed group neurons still neatly arranged,apoptotic cells scattered around. PHT+PB dose group apoptosis situation between PHT+PB high-dose group and low-dose group.1.2The cerebellar neurons of HE staining:In the NS group outline clear and complete of cells, transparent cytoplasm, normal morphology, occasional apoptotic cells;PHT and PHT+PB low-dose mixed group the cells were loosely arranged, showing a large number of apoptotic cells; PHT+PB high-dose mixed group can be seen apoptotic cells scattered around;PHT+PB dose group apoptosis situation between PHT+PB high dose group and low dose group.2Bax,Bcl-2protein expression:2.1Bax protein expressionThe Bax protein is pro-apoptotic protein, its positive expression cells were cytoplasmic brownHippocampal neurons:Positive cells were occasional expression in NS group;PHT+PB low, medium and high dose mixed groups with varying degrees of increased expression.Positive cells in PHT+PB low-dose mixed group aggregated distribution; In PHT+PB high-dose mixed group positive cells scattered. Medium expression of Bax could be seen between low and high dose mixed groups.The maximum number of positive cells were in PHT group.By analyzing AO (Average optical density)value of positive cells, concluded that different treatment groups affected the AO value of Bax positive cells.Be drawn between-group analysis:NS group and PHT group differentiated of AO value for Bax positive cells(p<0.05);PHT group compared with PHT+PB low-dose mixed group showed no significant difference(p>0.05);The differences between the PHT group and PHT+PB dose mixed group were statistically significant(p<0.05);The differences between the PHT group and PHT+PB high-dose mixed group were statistically significant(p<0.05);PHT+PB dose group compared with PHT+PB high-dose mixed group showed significant difference(p<0.05).Cerebellar neurons:In NS group Bax protein positive cells expressed occasionally; In PHT+PB low-dose mixed group positive cells aggregated distribution; In PHT+PB high-dose mixed group positive cells scattered. Medium expression of Bax could be seen between low and high dose mixed groups.The maximum number of positive cells were in PHT group.By analyzing AO value of positive cells, concluded that different treatment groups affected the AO value of Bax positive cells.Be drawn between-group analysis:NS group and PHT group differentiated of AO value for Bax positive cells(P<0.05);PHT group compared with PHT+PB low-dose mixed group showed no significant difference(p>0.05);The differences between the PHT group and PHT+PB dose mixed group were statistically significant (P0.05);The differences between the PHT group and PHT+PB high-dose mixed group were statistically significant(p<0.05);PHT+PB dose group compared with PHT+PB high-dose mixed group showed significant difference(p0.05).2.2Bcl-2protein expressionThe Bcl-2protein is anti-apoptotic,that positive cells cytoplasmic and nuclear membrane staining heavy brown particles.Hippocampal neurons:NS group expressed significantly;PHT and PHT+PB low-dose mixed group expressed a small amount of Bcl-2positive cells; In PHT+PB dose mixed group the positive cells increased;PHT+PB high-dose mixed group expressed obviously.By analyzing AO value of positive cells, found out that different treatment groups affected the AO value of Bcl-2positive cells.Be drawn between-group analysis:NS group and PHT group differentiated of AO value for Bcl-2positive cells(p<0.05);PHT group compared with PHT+PB low-dose mixed group showed no significant difference(p>0.05);The differences between the PHT group and PHT+PB dose mixed group were statistically significant(p<0.05);The differences between the PHT group and PHT+PB high-dose mixed group were statistically significant(p<0.05);PHT+PB dose group compared with PHT+PB high-dose mixed group showed significant difference(p<0.05).Cerebellar neurons:NS group expressed significantly;PHT and PHT+PB low-dose mixed group expressed a small amount of bcl-2positive cells; In PHT+PB dose mixed group the positive cells increased;PHT＋PB high-dose mixed group expressed obviously.By analyzing AO value of positive cells, found out that different treatment groups affected the AO value of Bcl-2positive cells.Be drawn between-group analysis:NS group and PHT group differentiated of AO value for Bcl-2positive cells(P<0.05);PHT group compared with PHT+PB low-dose mixed group showed no significant difference(p>0.05);The differences between the PHT group and PHT+PB dose mixed group were statistically significant(p<0.05);The differences between the PHT group and PHT+PB high-dose mixed group were statistically significant(p<0.05);PHT+PB dose group compared with PHT+PB high-dose mixed group showed significant difference(p<0.05).3Results of TUNELTUNEL positive cells showed nuclear condensation and brown particles or clumps in it.The apoptosis of hippocampal and cerebellar neurons:NS group can be seen apoptosis occasionally, cells are arranged neatly;PHT group showed cell disorder, a large number of apoptotic cells appeared;PHT+PB low-dose group showed a lot of apoptotic cells, cells shrinkage and irregular shape;PHT+PB dose mixed group showed more apoptotic cells gathering;PHT+PB high-dose mixed group apoptotic cells scattered.Through statistical analysis of apoptosis rate,concluded that:Hippocampal neurons:Different groups have an impact on the average rate of apoptosis in hippocampal neurons;Analysis between group found that NS group compared with PHT group the average apoptosis rate of hippocampal neurons showed significant difference(p<0.05);PHT group compared with PHT+PB low-dose mixed group showed no significant difference(p>0.05);The differences between the PHT group and PHT+PB dose mixed group were statistically significant (P<0.05);The differences between the PHT group and PHT+PB high-dose mixed group were statistically significant (P<0.05);PHT+PB dose group compared with PHT+PB high-dose mixed group showed significant difference(p<0.05).Cerebellar neurons:Different groups have an impact on the average rate of apoptosis in cerebellar neurons;Analysis between group found that NS group compared with PHT group the average apoptosis rate of cerebellar neurons showed significant difference(p<0.05);PHT group compared with PHT+PB low-dose mixed group showed no significant difference(p>0.05); The differences between the PHT group and PHT+PB dose mixed group were statistically significant (P<0.05);The differences between the PHT group and PHT+PB high-dose mixed group were statistically significant (P 0.05);PHT+PB dose group compared with PHT+PB high-dose mixed group showed significant difference(p<0.05). Conclusions:1In PHT group the hippocampal and cerebellar neurons in normal Kunming mice the apoptosis-related proteins Bax express increased, reducing the expression of Bcl-2;TUNEL assay discovered the cells disorder, a large number of irregular shape and pyknotic nuclei of apoptotic cells appeared.2PHT+PB low, medium and high dose mixed group the apoptosis related proteins Bax, Bcl-2of hippocampal and cerebellar neurons expressed differently:PHT+PB low-dose mixed group the apoptosis-related proteins Bax express increased, reducing the expression of Bcl-2;PHT+PB high-dose mixed group the apoptosis-related proteins Bax express decreased,increasing the expression of Bcl-2;PHT+PB dose mixed group of Bax, Bcl-2expressed in between.3PHT+PB low-dose mixed group compared with PHT group the hippocampal and cerebellar neurons of apoptosis-related proteins Bax, Bcl-2expressed no significant difference(p>0.05);PHT+PB high-dose mixed group compared with PHT+PB dose mixed group the expression of Bax, Bcl-2was statistically significant(p<0.05),indicate that Probucol may inhibit the expression of Bax and promote the expression of Bcl-2to inhibit excessive neuronal apoptosis,thus exert its neuroprotective effects.And there were statistically significant between PHT+PB dose mixed group and PHT+PB high-dose mixed group, indicating that this protective effect may exist in a dose-related. |
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