Research Of Tetrandrine Reversing Drug Resistance In K562/ADM Cells And Rats Of Refractory Epilepsy

Epilepsy is a severe illness and the prevalence increased gradually in the past decades according to large epidemiological studies. Although a series of new antiepileptic (AEDs) have been launched in the last two decades,drug-resistance remains a major problem.About30%of the epilepsy patients are resistant to the treatment of more than one AEDs,which is called medically intractable epilepsy. It has poor prognosis with increased morbidity and mortality.Therefore it is far-reaching important to investigate the drug resistance mechanism of refractory epilepsy epilepsy,and develop new strategies for the treatment.An important characteristic of the AEDs resistance is that most patients are resistant to one drug at the beginning,and it will be found that the other AEDs will also be noneffective,even though these drugs act by different mechanisms,which is called multidrug resistance(MDR). In the research of MDR,a series of studies focused on the P-glycoprotein,which was coded by gene MDR1.Many studies have shown that P-gp can efflux drugs out of the brain actively by using ATP,which maybe reduce the concentration of the drugs.There was accumulating evidence that P-gp was expressed in capillary endothellal cells and astrocytes in the normal blood-brain barrier,at the same time,P-gp was expressed in the neurons, capillary endothellal cells and astrocytes of cortex and hippocampus.Some studies showed that most of the AEDs was the natural substrates of P-gp,for example,PHT and VPA.P-gp efflux AEDs out of the epilepsy brain actively by using ATP,then,it reduced the concentration of the AEDs that reached the epilepsy tissues,which maybe one explanation for pharmacoresistance of epilepsy.Many researches had shown that we could reserve the multidrug resistance by blocking the multidrug resistant transports. Ca2+entry blockers is one of the hostest inhibitors of P-gp,which could reserve the MDR by inhibit the expression or activity of P-gp.But these inhibitors had other pharmacory activity that maybe interfere other drugs’metabolism,otherwise,they maybe have the non-specific cell toxicity, which limited the use in the clinical researches.So it is nessary to find an inhibitor with high selective and low cell toxity to act as the adjunctive treatment for medically refractory epilepsy.Tetrandrine is a bis-benzylisoquinoline alkaloid drived from the Chinese medicinal herb Stephania tatrandra. Some researches investigated that tetrandrine was a natural putative Ca2+entry blocker.It has been widely used in China for the treatment of angina and hypertension from1980s,which had been proved having the good effect.Many researches had shown that tetrandrine can reserve the multidrug resistance. Can tatrandrine reserve the MDR of AEDs? There was few studies.In this study,we investigated in two sections:1.We investigated the changes of AEDs resistance in the MDR cell after the tetrandrine’s intervention.2.We investigated the effect of tetrandrine on seizure behavior in rats of refractory epilepsy and the expression in the cortex and hippocampus of the rats after tetrandrine’s intervention.Materials and methods:We divided this research into two sections:In the first section, the resistance to the AEDs of MDR cell line K562/ADM (overexpression of P-gp) and normal cell line K562was observed.After tetrandrine was applied to the two cell lines,the resistance change of valproate (VPA) and phenytoin (PHT) in MDR cell was observed.In the second section, the model of chronic epilepsy rats was established by peritoneal injection with pentrazol, The maximal human adult dosage of carbamazepine(CBZ), VPA and PHT were administered to the chronic epilepsy rats,the rats of refractory epilepsy were screened out by the changes of Racine and EEG.Then, the rats of refractory epilepsy were divided into the tetrandrine group and the control group. We observed the seizure behavior after intraperitoneal injection with the tetrandrine.At last, we investigated, the expression of MDR1mRNA and protein of P-gp in the rats’ cortex and hippocampus after peritoneal injection with tetrandrine.Results:The results of the first section:1. Compared with normal cell line K562,IC50with the PHT and VPA were significantly increased in MDR cell line K562/ADM.(P<0.05)2. Compared with normal cell line K562,IC50with the PHT and VPA were significantly decreased in MDR cell line K562/ADM after tetrandrine’s intervention. (P<0.05)The results of the second section:1. The success rate of induction of the refractory epilisy rats was64%2. Compared with the control group,the behavior grades of Racine in the tetrandrine group were low.(P<0.05)3. Compared with the normal group, the expression of MDR1mRNA and P-gp in cortex and hippocampus of refractory epilepsy groups increased significantly.(P<0.01)4. Compared with the refractory epilepsy groups, expression of MDR1mRNA and P-gp in cortex and hippocampus of the tetrandrine groups (high dose group and low dose group)decreased obviously.(P<0.05)5. Compared with the low tatrandrine group, expression of MDR1mRNA and P-gp in cortex and hippocampus of the high tetrandrine group decreased obviously,but it had no statistical significance.(P>0.05)Conclusions:1.Tetrandrine can reserve the resistance of PHT and VPA in the MDR cell line K562/ADM.2. Tetrandrine can help the AEDs to descend the grades of Racine3. With the treatment of tetrandrine,the expression of MDR1mRNA and P-gpwas significantly reduced in cortex and hippocampus of the rats of refractory epilepsy.