Perturbation Of Chronic Unpredicted Mild Stress-induced Depression On Pharmacokinetics Via Glucocorticoids And Adrenergic Pathway

Background and purposeOur previous study found that the depression induced by chronic unpredicted mild stress(CUMS)results in the pharmacokinetic agitation in SD rats,but the mechanism remains unknown.In this study the pharmacokinetics of repaglinide in SD rats with depression induced by CUMS was conducted,and the expression profile of drug-metabolizing enzymes(DMEs)in GK rats' liver was explored.The serum and urine metabolic profiles of CUMS induced depression GK rats were applied,and the biomarkers related to depression and drug metabolism were explored.BRL 3A cell line was treated with glucocorticoid or epinephrine to verify the effect of signal pathway on drug metabolizing enzymes.Methods1.Establishment and evaluation of depression rat model:SD rats in the CUMS-induced depression group were lived alone,and exposed to a series of stressors for 8 weeks.The behavior of open-field test,sucrose preference,plasma cortisone and epinephrine levels were used to confirm the establishment of the depression model.2.Pharmacokinetics analysis of repaglinide and the expression of drug metabolizing enzymes in SD rats' liver:Plasma concentration of repaglinide in model group and control group were measured by LC-MS/MS.Pharmacokinetic parameters were calculated.The mRNA expression of Cyp2c11/13 and Cyp2c8/9 were detected by qRT-PCR.3.Establishment and evaluation of depression GK rat model:Five-week-old male GK rats were kept in the cage for 8 weeks in a specific pathogen free(SPF)-grade lab until the emergence of diabetes and were then divided into two groups:control and depression model group.Rats in the CUMS-induced depression group were lived alone and exposed to a series of stressors for 8 weeks.Plasma serotonin and dopamine levels and behavior of open-field test were used to confirm the establishment of the depression model.4.Gene expression profiling and Bioinformatic Analyses of differentially expressed genes:The mRNA from GK rats' livers was isolated using Trizol according to the manufacturer's instruction.RNA arrays including drug metablism genes were used to examine the gene expression profiles.The gene ontology(GO)and KEGG pathway enrichment analysis of differentially expressed genes were applied,and the relationship of different expression genes were explored by GeneSpring software.5.Confirmed the interested genes:qRT-PCR confirmed the expression of Nr1i3,Cyp3al8,Cyp2b1/2,Ugt1al and Ugt2b1 mRNAs in rat liver tissues,and the threshold cycle(Ct)of tested-gene product from the indicated genotype was normalized to the Ct for ?-actin.We also determined constitutive androstane receptor(CAR),Cyp3a1,Cyp2b1/2,Ugtlal and Ugt2b protein level using western blotting analysis in liver tissues.Band densitometry was quantified using Quantity One Analysis software and data of signaling were expressed as relative expression(Mean±SD)normalized to GAPDH or ?-actin bands.6.Metabolic profiling in serum and urine of rats:LC-Q/TOF-MS deteced the metabolites in serum and urine of rats,and Simca-P software was applied for multivariate statistical analysis.Different metabolites between control and depression group were found out,and enriched in KEGG pathway.7.The pharmacokinetics-related pathway in BRL 3 A:The rats of liver cell lines BRL 3 A were cultured in DMED with 10%fetal bovine serum,then the cells were treated with various concentration of Dexamethasone(DEX),Phenylephrine(PE),Dexmedetomidine(DEXT)and Isoprenaline(ISO)alone,or DEX with 8-Br-cAMP for 48hr.The mRNA and protein expression of Nr1i3,Cyp3a1,Cyp17a1,Ugt1a1 and Ugt2b were determined the according to the above methods.Results1.After 8 weeks of CUMS,SD rats in model group exhibited reduced movement and sucrose preference,and increased cortisone and epinephrine plasma level compared with rats in control group.2.In model group,half-life(T1/2),peak time(Tmax),peak concentration(Cmax),area under curve(AUC0-?)mean and residence time(MRT0-?)of repaglinide were reduced compared control group.The expression of Cyp2c11 in the liver of SD rats was significantly increased,and the expression of Cyp2c13,Cyp2c8/9 were not different between the two groups.3.Plasma serotonin and dopamine levels were decreased in the depression GK rats' group.A total of 49 genes were altered in expression by array analysis based on the expression fold change criteria of ?1.5-fold or ?0.5-fold.The Gene Spring software found the Nr1i3 was the center node of the up-regulated gene net,which influenced the other genes expression and the function.The gene ontology(GO)and KEGG pathway analyses discovered the relating to stress and drug metabolism pathway were separately mapped to the KEGG pathway databases.4.The interested mRNA of Nr1i3,Cyp3a18,Cyp17a,Cyp2b1/2,Ugt1a1 and Ugt2bl in depression rats' liver were up-regulated significantly comparing with the control,and the protein expression of CAR and Ugtlal were also up-regulated.5.The 16 different metabolites in CUMS-induced depression GK rats' serum were detected,and these metabolites were matched with the 22 KEGG pathways.At the same time the 28 different metabolites in GK rats' urine were found,and these metabolites were enriched in the 17 KEGG pathways significantly.6.The Cyp3a18,Ugt1a1 and Nr1i2 mRNA were up-regulated significantly with 1umol·L-1 DEX and 8-Br-cAMP treating for 48hr,while RU486 reversed this effect.The DEXT treating BRL 3 A cells for 48hr up-regulated the expression of the Cyp3a18 mRNA,but the PE and ISO didn't have effect on theses genes.ConclusionCUMS-induced depression might up-regulate the expression of drug-metabolizing enzymes via glucocorticoids and adrenergic pathway,and lead to a variety of endogenous substance metabolism changes,which ultimately agitated the pharmacokinetics of repaglinide in vivo.