Study On Expression Of Drug-Metabolizing Enzyme CYP2C33 Regulated By Pig-Derived IL-6

Cytochrome P450(CYP450)which consists of many isoenzymes is a superfamily of vital drug metabolic enzymes that are required for metabolism of endogenous and exogenous substances,drugs and its derivatives in human and animals.However,endogenous and exogenous substances are also involved in expression of CYP450.In pigs,CYP2C33 is a class of drug metabolic enzymes which serve as a model to investigate functions of human CYP2 C.Interleukin-6(IL-6)is an important cytokine that causes inflammation in livers and tumor lesions.In the industries of livestock and poultry breeding,IL-6 usually functions as immunological adjuvant and immune enhancer,because it can regulate immune response in bodies.But whether IL-6 affects the expression of drug metabolic enzymes is not clear.Changing the expression of drug metabolic enzymes seriously affects the efficiency of clinical drugs,toxic effect and the interaction between drugs and drugs.This study is the first time to explore the mechanism of expression of porcine CYP2C33 regulated by IL-6 derived from pigs on molecular level and provides a sight on CYP450 expression influenced by IL-6.Meanwhile,this research also provides theoretical guidance for veterinary medicine and clinical medicine.1.Distribution of several drug-metabolizing enzymes in different tissues of pigsReal-time PCR was used to detect the distribution of several drug-metabolizing enzymes by indirectly measuring their m RNAs in different porcine tissues which include liver,kidney,small intestine,muscle,spleen,skin and lung.The results showed that the expression of CYP1 A,CYP2C,CYP2 D,CYP2E and CYP3 A in liver is significantly higher than other tissues.And the most abundant expression in liver is drug metabolism enzyme CYP3 A,followed by CYP2 C.The results also revealed that phase I of drug metabolism mainly occur in the liver.So we chose primary hepatocytes and passaged hepatocytes to investigate drug-metabolizing enzymes.Additionally,the highest abundance of expression of drug-metabolizing enzyme CYP3 A and CYP2 C can provide a certain foundation for the following studies.2.Isolation and culture of primary porcine hepatocytesIn order to obtain primary porcine hepatocytes with growth-well condition,we applied three-step perfusion methods that were followed by PBE,PB and PBC to isolate the cells.Cell dying by 0.1% trypan blue showed the survival rate of the cells was nearly99.9% and the cells were well adherent in the plates coated with rat tail collagenase.Primary porcine hepatocytes are appropriate for subsequent studies.3.Expression of several drug-metabolizing enzymes affected by IL-6To investigate function of IL-6 on the expression of several key drug-metabolizing enzymes in pigs,we treated primary hepatocytes and Hep Li cells with IL-6 at different concentrations for different times.Expression of CYP3A29 and CYP2C33 affected by IL-6 in hepatocytes was detected by using real-time PCR and Western-blot.Our results demonstrated that IL-6 had no significant effect on expression of CYP3A29 while CYP2C33 expression was up-regulated in hepatocytes treated with IL-6,and abundance of expression relys on treatment times and concentrations of IL-6.4.Expression of CYP2C33 regulated by IL-6 at transcriptional levelTranscriptional inhibition assay demonstrated that IL-6 can regulate the expression of CYP2C33 at transcriptional level.We transiently transfected the reporter plasmids which contain CYP2C33 promoter into He PLi cells and we measured luciferase activity.We found the core region of CYP2C33 promoter was located at-2424 bp to-1593 bp upstream of CYP2C33’s transcription initiation site,and the sequence of first intron of CYP2C33 can increase the promoter activity.IL-6 affected expression of CYP2C33 by involving the core region of CYP2C33 promoter.It suggests that IL-6 regulates the expression of CYP2C33 in the transcriptional level.5.Effects of IL-6 on expression of PXR,CAR and RXRαPrimary hepatocytes and HepLi cells were treated with IL-6 at different concentrate for times,before we analyzed expression of PXR,CAR and RXRα by real-time PCR and western blot.The results showed that IL-6 had no significant effect on the expression of RXRα,whereas IL-6 significantly increased the expression of CAR and PXR which correlated with CYP2C33 expression.6.Investigating the mechanism of CYP2C33 expression involved by IL-6We supposed that IL-6 upregulates expression of CYP2C33 through changing expression of PXR,CAR and RXRα.Because the full-length RXRα is unknown,we firstly obtained the end sequences of RXRα m RNA by 3′RACE and 5′RACE to get the the full length sequence of RXRα.To investigate whether CYP2C33 expression is upregulated by IL-6,we transfected eukaryotic expression plasmids or interfering RNA into cells,respectively.The results showed that overexpression of PXR or interfering PXR expression by si PXR could not change CYP2C33 expression significantly,but the expression of CYP2C33 was changed correspondingly by overexpressing CAR and RXR-α or interfering CAR and RXR-α,separately.Collectively,the expression of CYP2C33 up-regulated by IL-6 is associated with nuclear receptors CAR and RXRα.