Effects Of Phenytoin Sodium And Rannasangpei On Hepatocellular Apoptosis In Rats

Objective: Epilepsy (EP) is a kind of syndrome characterized byparoxysm, transient, repeatable, and usually rigid central nervous systemdysfunction. It is caused by abnormal discharge of highly synchronized brainneurons resulted in different causes. Antiepileptic drug (AED) is the maintreatment for epilepsy in clinical practice. As the preferred medicine forepilepsia gravior, phenytoin sodium (PHT-Na) is a common AED.Rannasangpei (RNSP), which has efficacy on calmness and can be used as atreatment for epilepsy sometimes, is one of typically rare Tibetan medicines.In recent years, reports of hepatic injury caused by AED are increasingespecially combined medication. Hepatocellular apoptosis is considered asone of the basic mechanisms of drug-induced liver injury. In the cascadereaction of cellular apoptosis, caspase-3is called as “apoptosis executioner”,and Fas protein plays a very important role in the pathway mediated by deathreceptor. To explore effects of PHT-Na and RNSP on hepatocellular apoptosisin rats and its possible mechanism, this experiment was to give SD ratsPHT-Na and RNSP by gavage, and measured expression levels of caspase-3and Fas protein in liver by immunohistochemical method. We tried to providebases for researches of drug’s safety, guide clinical medication, reduce drug’sadverse reactions, and improve life quality.Methods:1Animals and division:40clean and healthy male SD rats aged10weeks,weighing200±20g, were divided into4groups randomly. They were named asPHT group, RNSP group, PHT+RNSP group, and NS group. Each groupwhich had10rats was divided into two subgroups,5in a subgroup: Rats of10-day subgroups (PHT-10group, RNSP-10group, PHT+RNSP-10group,NS-10group) were fed for10days, and rats of20-day subgroups (PHT-20 group, RNSP-20group, PHT+RNSP-20group, NS-20group) were fed for20days.2Experimental interventions: Rats of PHT group, RNSP group, andPHT+RNSP group were treated with corresponding drugs by homemadegastric filler two times a day on the dose calculated as25times of averagedose on adult per weight; NS groups were treated with physiological saline inthe same volume two times a day.3Production of hepatic paraffin sections: Having been anesthetized by10%chloral hydrate (0.35ml/100g) within24hours after the end of druginterventions, rats in each group were perfused and fixed by4%paraformaldehyde. We opened the rat’s abdominal cavity and took off the leftlobe of liver in volume of about1cm3, then placed it into4%paraformaldehyde quickly and produced paraffin sections24hours later inprocedures of dehydration, vitrification, paraffin embedding, and sectioning.4HE staining: Paraffin sections were stained with hematoxylin and eosin, andwe observed histological change of liver through optical microscope.5Detection of apoptosis: Expressions of caspase-3and Fas protein weredetected by immunohistochemical method, and we calculated positiveexpression rates of caspase-3and Fas in hepatocytes respectively.6Statistical treatment: The variance were analyzed by SPSS13.0and means ofall groups were compared with each other by One-Way ANOV, it hadsignificant difference when P<0.05, SNK test was used in comparisonbetween two groups.Results:1HE staining1.1Hepatic paraffin sections of rats showed normal structures when observedby optic microscopy except PHT-20group: Surrounding the central vein,hepatocytes were ranged in radiation. Hepatic cords and sinuses were normal.Hepatocytes, with one or more nuclei which were big, round, blue or bluishviolet and had one or two nucleoli, were polygonal in clear boundary. Thecytoplasm was stained evenly in rosiness. 1.2Hepatic paraffin sections of rats in PHT-20group showed that vacuolardegeneration and increased eosinophilic cytoplasm were observed in a smallamount of hepatocytes, but nuclear shrinkage, apoptotic body and othertypical characters of apoptosis were not observed.2Detection of apoptosis: Hepatocytes of positive caspase-3expression werebrown or pale brown, caspase-3mainly expressed in cytoplasm and nucleuspartly. Hepatocytes of positive Fas expression were brown, Fas alwaysexpressed in cellular membrane and (or) cytoplasm, especially around thecentral vein.2.1There was no significant difference in positive expression rates ofcaspase-3and Fas among10-day subgroups.2.2Comparisons of positive expression rates of caspase-3and Fas among20-day subgroups: There was no significant difference between RNSP-20group and NS-20group; PHT-20group were significantly higher thanRNSP-20group and NS-20group (P<0.05); PHT+RNSP-20group weresignificantly higher than RNSP-20group and NS-20group (P<0.05); PHT-20group were significantly higher than PHT+RNSP-20group (P<0.05).2.3Comparisons of positive expression rates of caspase-3and Fas betweentwo subgroups: There was no significant difference between RNSP-20groupand RNSP-10group, NS-20group and NS-10group; PHT-20group weresignificantly higher than PHT-10group (P<0.05); PHT+RNSP-20group weresignificantly higher than PHT+RNSP-10group (P<0.05).Conclusions:1Results of HE staining except PHT-20group showed that hepatic structurescharacterized with normal shape of hepatocytes and neat form had no obviousdifference, it meant that hepatic structures of rats who had taken PHT-Na andRNSP together or only RNSP were not obviously affected. Vacuolardegeneration and increased eosinophilic cytoplasm were observed in a smallamount of hepatocytes in PHT-20group, it meant that hepatic modalities ofrats who had taken PHT-Na for a long time might be affected mildly.2Expressions of caspase-3and Fas in liver 2.1Positive expression rates of caspase-3and Fas in PHT-20group weresignificantly higher than NS-20group and PHT-10group, it meant thatPHT-Na might promote hepatocellular apoptosis as time prolonged. Increasedapoptosis induced by PHT-Na, whose mechanism might be related withpathway of death receptor, might lead to hepatic adverse reactions.2.2Positive expression rates of caspase-3and Fas in RNSP groups had nosignificant difference compared to NS groups, it meant that RNSP might haveno obvious effect on hepatocellular apoptosis.2.3Positive expression rates of csaspase-3and Fas in PHT-20group weresignificantly higher than PHT+RNSP-20group, it meant that RNSP mightweaken the promoting effects of PHT-Na on hepatocellular apoptosis, andplayed a protective role in hepatic adverse reactions induced by PHT-Na.