| Objective: Carbamazepine (CBZ) is similar to tricyclic antidepressantsin structure, but it doesn’t have antidepressant activity. As one of thebroad-spectrum anti-epileptic drugs, it was used in the clinic in the1970s andconsidered as the drug of first choice to cure psychomotor seizures, and alsohas a certain effect on other systemic clonic-tonic seizures as well as mixedseizures of epilepsy. Because of its low price and significant effect, it is widelyused in the clinic. However, carbamazepine may lead to adverse drug reactionswhich are quite different among individuals, and may affect multiple bodysystems and organs which take on a variety of clinical manifestations. Higherincidence of adverse reactions may occur in the nervous system, such asdrowsiness, poor articulation, unsteady gait, language barriers, disturbance ofconsciousness, cerebellar ataxia, dizziness, poisoning to status epilepticus,partial body convulsions-hemiplegia syndrome, etc. In addition, the drug alsohas an impact on systems including the cardiovascular system, blood system,digestive system, liver and gallbladder as well as urinary system, and mightcause death in a serious adverse reaction.Rannasangpei is one of the representative precious Tibetan medicines,which is based on25prescriptions. It is made out of70herbs such as ZuoTaiwan, nine stone, agate, coral, saffron, Tianzhu Huang, licorice, dalbergia,sandalwood, pearls and and so on. It has the function of restoringconsciousness and resuscitating, regulating of qi and blood, clearing andactivating the channels and collaterals, soothing and calming the nerves. It isone of the commonly used medicines to treat such neurological illnesses asepilepsy, infantile convulsions, autonomic dysfunction, and such acute andchronic cerebrovascular diseases as stroke, cerebral hemorrhage as well as hemiplegia. Rannasangpei is very effective in treatment with a certain degreeof neuroprotective effect, and has no serious adverse reactions.This experiment aimed to study the side effects of carbamazepine oncentral nervous system and the possible mechanism of rannasangpei fortreatment of central nervous system diseases, through the observation of nervecell apoptosis in the hippocampal CA1area and parietal lobe cortex of ratswhich were fed with regular doses of carbamazepine and rannasangpei, so asto provide a theoretical basis for better clinical treatment and reduce theincidence of adverse drug reactions.Methods:1Experimental animals and groups:40SPF healthy male (10weeks old)Sprague-Dawley rats, weighing (200±20) g. These rats were randomly dividedinto four groups, each group divided into two subgroups with5in eachsubgroup, as follows:①the physiological saline groups: the physiologicalsaline short treatment group(N1); the physiological saline long treatmentgroup(N2);②the CBZ groups: the CBZ short treatment group(C1); the CBZlong treatment group(C2);③the RNSP groups: the RNSP short treatmentgroup(R1); the RNSP long treatment group(R2);④the CBZ and RNSPcombined treatment groups: the CBZ and RNSP combined treatmentshort-course group(M1); the CBZ and RNSP combined treatment long-coursegroup(M2).2Drug interventions: The CBZ groups were irrigated with carbama-zepine by400mg/kg/d deliquescing in physiological saline (1ml/100g) for15and30days respectively. The RNSP groups were irrigated with rannasangpeiby250mg/kg/d deliquescing in physiological saline (1ml/100g) for15and30days respectively. The CBZ and RNSP combined groups were irrigated withcarbamazepine by400mg/kg/d and rannasangpei by250mg/kg/d deliquescingin saline (1ml/100g) for15and30days respectively. The saline groups wereirrigated with equal dose of normal saline (1ml/100g/d).3Preparation of samples: The rats were anesthetized by10%chloralhydrate, supine fixed and exposed the heart. The perfusion needle was passed through the apex into the left ventricle-aortic valve-ascending aorta.Simultaneously the right atrial appendage were cut open and the rats wereinfused the normal saline from left ventricular until the colorless liquidoutflew, then fixed in4%paraformaldehyde. After perfusion was complete,the rats were decapitated and placed in4%paraformaldehyde for fixing.Specimens were dehydrated, transparent, embedded in paraffin and sliced forindex determination.4Apoptosis detection: The paraffin sections of hippocampal CA1regionand parietal cortex were detected to observe the morphological changes underlight microscopy by hematoxylin-eosin staining, to observe the expression ofapoptosis-related genes bax and bcl-2by immumohistochemical staining, andto observe the apoptosis of nerve cells by TUNEL.5Statics: The experimental data were statistically analyzed with SPSS13.0software, the One-Way ANOVA test was applied to compare thedifferences between groups, pairwise comparisons were applied and tested bythe SNK, P <0.05were considered statistically significant.Results:1Results of hematoxylin-eosin staining:1.1Hippocampal CA1region: Brain structure of the rats in the saline groupsand the RNSP groups was clear and complete, cells were dense and arrangedin neat rows. The nerve cells in the CBZ groups were arranged in a disorderedway, reduced in size, separated from the surrounding cells, had densecytoplasm and deeper eosinophilic staining, nucleus and chromatin gatheredinto lumps, this phenomenon was more evident in the CBZ long treatmentgroup. Scattered apoptotic cells could be seen in the CBZ and RNSPcombined groups.1.2Parietal cortex: Nerve cells of different experimental groups were arrangedin neat rows. It could be seen occasionally that cell shrinkage and chromatinwere gathered into lumpy. Compared with the saline groups, there were noobvious morphological differences.2Results of immunohistochemisty: 2.1Expression of Bax:2.1.1Hippocampal CA1region: High expression of Bax could be seen in theCBZ groups, this phenomenon was particularly evident in the CBZ longcourse of treatment group. Medium expression of Bax could be seen in theCBZ and RNSP combination groups. A low Bax expression could be seen inthe saline groups and the RNSP groups. Statistically analyzed the averageoptical density value (AO value) of the Bax-positive cells and found out: theAO value of the CBZ groups were significantly higher than that in the salinegroups; the AO value of the CBZ and RNSP combination groups were lowerthan that in the CBZ groups and higher than that in the saline groups; thedifferences between the groups were statistically significant (P<0.05). Theexpression of Bax in the CBZ long course of treatment group was higher thanthe short course, the difference was statistically significant (P<0.05). TheRNSP groups compared with the saline groups, the differences were notstatistically significant (P>0.05).2.1.2Parietal cortex: The expression of Bax could be seen in every group, butthe expression was low. Compared the AO value of the Bax-positive cells indifferent groups, the differences were not statistically significant (P>0.05).2.2Bcl-2expression results:2.2.1Hippocampal CA1region: There was an evident expression ofBcl-2-positive cells in the saline groups and the RNSP groups, a mediumexpression in the CBZ and RNSP combined groups, and a low expression inthe CBZ groups. Statistically analyzed the AO value of the Bcl-2-positive cellsand found out: the AO value of the CBZ groups was significantly lower thanthat in saline groups; the AO value of the CBZ and RNSP combination groupswas higher than that in the CBZ groups and lower than that in the salinegroups; the differences between the groups were statistically significant(P<0.05). The value of the CBZ long course of treatment group was lowerthan the short course of treatment group, the difference was statisticallysignificant (P<0.05). Compared the RNSP groups with the saline groups, thedifferences were not statistically significant (P>0.05). 2.2.2Parietal cortex: Bcl-2expression could be seen in all the groups.Compared the AO value of Bcl-2-positive cells in different groups, thedifferences were not statistically significant (P>0.05).3Results of the TUNEL:3.1Hippocampal CA1region: Apoptotic cells were increased in the CBZgroups, apoptotic cells could be seen scattered in the CBZ and RNSPcombined groups, and were occasionally seen in the saline groups and theRNSP groups. Statistically analyzed the positive rate of apoptotic cells: theapoptotic rate of the CBZ groups was significantly higher than that in thesaline groups; the apoptosis rate of the CBZ and RNSP combination groupswas lower than that in the CBZ groups and higher than the saline groups; thedifferences between the groups were statistically significant (P<0.05). Theapoptotic rate of the CBZ long course of treatment group was higher than theshort course of treatment group, the difference was statistically significant(P<0.05). The RNSP groups compared with the saline groups, the differenceswere not statistically significant (P>0.05).3.2Parietal cortex: Each group showed sparse and scattered apoptotic cells.Compared the apoptotic rate of each group, the differences were notstatistically significant (P>0.05).Conclusions:1Feeding healthy adult Sprague-Dawley rats with regular doses ofcarbamazepine and rannasangpei have little effect on the apoptosis of theneurons and positive expression cells of Bax and Bcl-2in the parietal cortex.2Feeding healthy adult Sprague-Dawley rats with carbamazepine canlead to an increase of positive expression of bax and decrease of Bcl-2in theneurons of hippocampal CA1region, and excessive apoptosis of nerve cells.Besides, the degree of apoptosis is in proportion to medication time span.Feeding rats with rannasangpei has little effect on the apoptosis of the neuronsand positive expression of Bax and Bcl-2in the hippocampal CA1region.3Feeding rats with a combination of carbamazepin and rannasangpeican reduce the effects that carbamazepine has on the increase of the positive expression of bax and decrease of bcl-2in the neurons of hippocampal CA1region, rannasangpei can suppress excessive apoptosis of nerve cells. Itsuggests that neuroprotective mechanism of rannasangpei may relate to thesuppression of excessive apoptosis. |
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