| Objective:Dl-3-n-Butylphthalide(NBP), a synthetic racemic, commonly used in clinical treatment of ischemic cerebrovascular disease, has protective effects on brain neuronal cultures. In clinical, phenytoin sodium(PHT)is commonly used in epilepsy, arrhythmia, trigeminal neuralgia and other diseases, which is most often used in the treatment of antiepileptic. But its therapeutic spectrum is narrow, which is prone to neurotoxicity and many adverse reactions such as dizziness, ataxia, slurred speech and so on. This study mainly researched the impact of Dl-3-n-Butylphthalide on the expression of Bax and Bcl-2 on rat in hippocampus and cerebellar after the effect of phenytoin sodium, and explored the toxicity of phenytoin sodium and the neuroprotective mechanism of NBP.Methods:1 Grouping and administration: 36 healthy clean 8-week-old male SD rats, weighting(170 ± 20g), were randomly divided into NS(Saline+Peanut oil) group, NBP(NBP+Saline) group, high-dose PHT(Phenytoin Sodium) group, low-dose PHT group, NBP combined with high-dose PHT mixed group and NBP combined with low-dose PHT mixed group, and each group had six rats. Using intragastric gavage needle, they were conducted orally at 9: 00-11: 00 Am. PHT of PHT group and NBP combined with PHT group,according to the high and low doses, were given specific doses of 200mg/kg/d and 100mg/kg/d, while the other groups were given the same volume of saline. NBP(dissolved in peanut oil) was given 80mg/kg, while the other groups were received the same volume of peanut oil, once a day, a total of 30 days.2 Drawn and testing: The rats in each group were anesthetized with 10% chloral hydrate(0.36ml/100g), supine fixed and cut the chest to expose the heart, inserted the needle from the apex, at the same time cut the right atrial appendage, rinsed rapidly with 100ml~150ml 0.9% saline and then fixed by 200ml~300ml 4% paraformaldehyde phosphate buffer. After injection, immediately decapitated and removed the cerebrum and cerebellum into 4% paraformaldehyde phosphate buffer, routine graded ethanol dehydrating, xylene, dipping wax, embedding, selectting consecutive coronal slices of the cerebrum and cerebellum, and the thickness of slice was 5um. To observe the neuronal apoptosis and degeneration in each group by HE and Nissl staining; to detect the expression of apoptosis-related protein Bax and Bcl-2 by immunohistochemistry and use the average optical density(AOD) to represent the result of each group.3 Statistical analysis: By SPSS13.0 statistical software for data analysis, using one-way ANOVA comparison between groups. Between the two groups were compared using SNK test; when Ρ<0.05, the difference was considered statistically significant.Results: 1 HE(hematoxylin-eosin)stainingIn NS group and NBP group, the hippocampal and cerebellar neurons of rats were arranged neatly and densely, cell structure clear, cytoplasmic stained red and nuclei stained blue, nucleus round or oval, nucleolus clear, morphology normal, and occasionally a few apoptotic cells: the cellular outlines were vague, the volume of cell shrinked, karyopyknosis stained, eosinophilic cytoplasmic staining enhanced, and vacuolization around. In PHT low-dose group, the hippocampal and cerebellar neurons of rats arranged relatively sparse, outline vague, karyopyknosis stained, vacuolization around and more apoptosis. In NBP combined with low-dose PHT mixed group, the hippocampal and cerebellar neurons of rats were still neatly arranged and the structures were still intact, which compared with the low-dose PHT group had less apoptotic cells. The hippocampal and cerebellar neurons of the high-dose PHT group had a lot of apoptotic cells, the neurons were arranged scattered, sparse, cell shrinkage, nuclear irregularity, lots of nuclear pyknosis, cytoplasm eosinophilic. The hippocampal and cerebellar neurons in NBP combined with high-dose PHT mixed group had less apoptotic cells than in the high-dose PHT group. 2 Nissl staining resultsIn NS group and NBP group,the hippocampal and cerebellar neurons were arranged regularly and closely, morphology complete, nucleoli clear, cytoplasm nissl bodies abundant, no significant loss of neurons. Compared with the NS group and NBP group, the hippocampal and cerebellar neurons of low-dose PHT group and high-dose PHT group lost obviously, and the distribution of cells uneven, showing cell shrinkage, chromatin clumping, pyknosis, the number of Nissl bodies reducing. But the loss of neurons in the high-dose PHT group was more obvious. Compared with the NS group and NBP group, the loss of the hippocampal and cerebellar neurons in NBP combined with low-dose PHT mixed group was no significant. Compared with high-dose PHT group, which in NBP combined with high-dose PHT mixed group was significantly reduced. 3 Immunohistochemistry staining results 3.1 Bax protein expression resultsThe positive expression of Bax protein were brown-yellow or brown particles, which mainly were distributed in the cell membrane and cytoplasm and the nuclei were not colored.In NS group and NBP group, the Bax protein positive cells of the hippocampus and cerebellum expressed occasionally; which in the NBP combined with low-dose PHT mixed group(NP1 group) expressed a few, but in the NBP combined with high-dose PHT mixed group(NP2 group) the expression of Bax protein positive cells increased significantly. In high-dose PHT(P2 group) group and low-dose PHT group(P1 group), a lot of positive cells were expressed, which were distributed densely, cytoplasm brown-yellow, and stained dark. However, the expression of the Bax protein positive cells in high-dose PHT group(P2 group) was particularly significant. NBP group, NS group and NP1 group, between any two groups, the difference was not statistically significant(P>0.05). P1 group, P2 group and NP2 group, between any two groups, the difference was statistically significant(P<0.05): the expression of Bax positive cells was higher in P2 group than in P1 group(P<0.05), which in NP2 group was lower than in P1 group(P<0.05), which was lower in NP2 group than in P2 group(P<0.05). Between P1 group and NP1 group, the difference was statistically significant, the expression of Bax positive cells in NP1 group was lower than in P1 group(P<0.05),and which was higher in NP2 group than in NP1 group(P<0.05). 3.2 Bcl-2 protein expression resultsBcl-2 protein’s positive expression were brown granules, stainning dark, mainly in the cytoplasma and the nuclear membrane, and the nuclei were not colored.In NS group and NBP group, the Bcl-2 protein positive cells of the hippocampus and cerebellum expressed significantly; which in the low-dose PHT group and high-dose PHT group had a little expression, but the expression of Bcl-2 protein positive cells reduced significantly in the high-dose PHT group. The expression of Bcl-2 protein positive cells of the hippocampus and cerebellum in NBP combined with low-dose PHT mixed group was significantly; but which in NBP combined with high-dose PHT mixed group increased a little more. NBP group, NS group and NP1 group,between any two groups, the difference was not statistically significant(P>0.05). P1 group, P2 group and NP2 group, between any two groups, the difference was statistically significant(P<0.05): the expression of Bcl-2 positive cells was lower in P2 group than in P1 group(P<0.05), which in NP2 group was higher than in P1 group(P<0.05), which was higher in NP2 group than in P2 group(P<0.05). Between P1 group and NP1 group, the difference was statistically significant, the expression of BCL-2 positive cells in NP1 group was higher than in P1 group(P<0.05), and which was lower in NP2 group than in NP1 group(P<0.05).Conclusions:1 The different therapeutic doses of PHT could lead the hippocampal and cerebellar neurons of normal rat to apoptosis, the expression of inhibition of apoptotic protein Bcl-2 was reduced, and the expression of pro-apoptotic protein Bax was increased; however, its influence was more evident in PHT high-dose group, showing dose-related.2 NBP combined with high-dose PHT group and NBP combined with low-dose PHT group separately compared with PHT high-dose group and PHT low-dose group, the expressions of protein Bcl-2 of hippocampal and cerebellar neurons were increased, the expressions of Bax were reduced, which could inhibit the neuronal apoptosis. Which showed that NBP possibly played a neuroprotective effect by increasing the expression level of Bcl-2 and reducing the expression level of Bax to inhibit apoptosis.3 Compared with the NBP group, the expression of Bcl-2 and Bax of hippocampus and cerebellum neurons in NP1 group was not statistically significant; but in NP2 group, the expression of Bcl-2 was reduced and the expression of Bax was increased. Which indicated that NBP could suppress the neuronal apoptosis induced by low dose of PHT,but could not completely inhibit the neuronal apoptosis induced by high dose of PHT. |
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