The Study On Oxymatrine Inhibits Morphine-Induced MDR1 Gene Overexpression

BackgroudOne important factor in the development of MDR is overexpression of P-glycoprotein,encoded by the MDR1 gene.It is an ATP-dependent efflux pump acting at the blood-brain barrier,limits the access of many drugs to the central nervous system.Morphine is the opioid of choice for moderate to severe cancer pain,and is a substrate of P-glycoprotein.Repeated doses of morphine results in the up-regulation the transcription of MDR1 mRNA,leads to up-regulation of P-glycoprotein expression in the brain tissue and capillary endothelial cell membranes.Then,it will reduce the brain concentrations of morphine during treatment and further delay the development of drug resistance.Oxymatrine is an alkaloid extracted from the Chinese herb Sophora japonica L.(Fabaceae),recent studies revealed that oxymatrine can inhibit P-glycoprotein overexpression in multi-drug resistance tumorr cell lines.Our clinical studies have shown potentially quite large effects in terms of maintaining the pain-relieving effect of a combination of oxymatrine and morphine during prolonged use while also reducing their side effects,suggesting that oxymatrine affects the development of morphine tolerance through changing morphine induced up-regulation of P-glycoprotein expression.ObjectiveTo culture the mouse brain capillary endothelial cells,and analyse the concentr-ations of no inhibitory effect of oxymatrine.The mouse brain capillary endothelial cells are treated with different concentrations morphine(10-7-10-11M),detect the level of MDR1 a gene,MDRlb gene and P-glycoprotein expression among morphine-treated groups and control.Combine oxymatrine with morphine and treat cells,then analysis the level of gene and protein expression among morphine-treated cells,coadministration of oxymatrine and morphine,oxymatrine-treated cells and controls.Investigate if there is any effect of oxymatrine on the development of morphine tolerance.Make sure that oxymatrine attenuate the morphine tolerance by abolishing the up-regulation of the morphine-induced P-glycoprotein expression.The study's aim is supply new target in clinical treatment,prolong morphine analgesic effect,at the same time inhibit the development of opioid tolerance.MethodsThe mouse brain capillary endothelial cells are devided into 15 groups which are treated with increasing concentrations of the oxymatrine.Use MTT assay to detect the inhibition rates of different groups,and makesure the concentration of no inhibitory effect of oxymatrine.The morphine concentrations(10-7-10-11M)are used in our study are well within the physiological range and have been used by other investigators in prior studies,RT-PCR and Western blot are performing to detect the mRNA of MDR1a gene and MDR1b gene and the expression of P-glycoprotein in separately.Then we will combine oxymatrine with morphine and treat cells,at the same time,set up morphine/oxymatrine-treated cells and a contrast group which dosen't accept any treatment,to testify the oxymatrine inhibits development of morphine-induced tolerance associated with decreased expression of MDR1a gene and P-glycoprotein,by using RT-PCR and Western blot techniquesResults1.The results of MTT show us that the inhibitory effects of oxymatrine gradually increased at concentrations ? 400ug/ml,cause a dose-dependent inhibition in cell growth and there is no inhibitory effect of oxymatrine at concentrations<400ug/ml,combining clinically achievable concentrations and drug concentrations other people used in experiments,we choose the 200ug/ml as the proper concentration of oxymatrine in this experiment.2.The results of RT-PCR show us that the mRNA of the MDR1a and MDRlb genes are detected from either morphine-treated cells or controls,but morphine only increase the expression of MDRla gene(P<0.05),it has no significant effect on the expression of MDRlb gene(P>0.05).combine oxymatrine with morphine and treat cells,contrast single morphine-treated group,the result show that oxymatrine can reduce morphine-induced MDRla gene expression(P<0.05).3.The results of Western-blot show us that the expression of P-glycoprotein in morphine-treated groups are higher than control(P<0.05),and contrast single morphine-treated group,oxymatrine can reduce morphine-induced P-glycoprotein over expression(P<0.05)in the mouse brain capillary endothelial cells.Conclusions1.There was no inhibitory effect of oxymatrine when its concentrations is<400ug/ml for the mouse brain capillary endothelial cells.2.The mouse brain capillary endothelial cells autoinduce the MDRla gene and MDR1b genes expression,but morphine only increase the expression of MDR1a gene,it has no effect on the expression of MDR1b gene.3.Oxymatrine can inhibit morphine-induced MDR1a gene and P-glycoprotein overexpression in the mouse brain capillary endothelial cells.