Induction Of Cytochrome P450s(CYPs) Caused By Celastrol And The Role Of The Induced CYPs Played In The Anti-inflammatory Effect Of Celastrol

SECTION ? The induction of CYPs caused by celastrolObjectiveCelastrol is one of the main active components of Tripterygium Wilfordii Hook.f.,which is a traditional Chinese medicine.The traditional Chinsese medicine containing celalstrol may be co-administered when treating rheumatoid arthritis and other diseases.To investigate whether celastrol may induce drug-drug interaction(DDI)when used in combination with other drugs that metabolized by drug metabolic enzymes,the present investigation study the effect of celastrol on CYPs expression.MethodsTwo-step collagen perfusion method was employed to extract rat primary hepatocytes from Wistar rats.The cells were cultured for 12 h before treated with celastrol.Fluorescent quantitative RT aggregate enzyme chain reaction(qRT-PCR)was adopted to detect the changes of CYPs mRNA.Protein immune imprinted(Western blot)was used to detect the protein expression.LC/MS/MS was employed to detect the metabolism activity using of CYP2C,CYP2D and CYP3A specific substrates.The data was analyzed by GraphPad Prism 5.Results1.CYP2C,CYP2D and CYP3A,which are major drug metabolism enzymes,were induced by celastrol.The mRNA of CYP2C12,CYP2D2,CYP3A1 and CYP3A9 were significantly induced by celastrol in rat primary hepatocytes,while the mRNA levels of CYP2C7 and CYP2C11 were not changed.The changes in protein level were also detected.2.After treated rat primary hepatocytes with celastrol,the enzyme activity of CYP2C,CYP2D and CYP3A was also increased.ConclusionsCYP2C,CYP2D and CYP3A,which are the major drug metabolism enzymes,were induced by celastrol.The results indicates that when the traditional Chinese medicine is co-administered with the substrates of these enzymes,the drug-drug interaction may happen and as a result,the metabolic elimination rate and the effect of the co-administered drug may be altered.SECTION ? The mechanisms of the induction effect of celastrol on Cytochrome p450sObjectiveThe results of section one showed that celastrol induced CYPs in rat primary hepatocytes.Thus,further investigated the possible mechanism may provide evidence for the rational use of celastrol.It has been reported that the nuclear receptor PXR and CAR as well as transcriptional factors,such as NF-?B and p53,may be involved in CYPs regulation.In this section,the investigation will explore the mechanism on the inductive effect of celastrol by focusing on nuclear receptor,p53 and NF-?B.MethodsTwo-step collagen perfusion method was employed to extract rat primary hepatocytes from Wistar rats.The cells were cultured for 12 h before treated with celastrol.The mRNA and protein expression of nuclear receptors(PXR and CAR)were detected by qRT-PCR and Western blot assay,respectively.The P-gp mRNA level was detected by qRT-PCR to further illustrate the activity of these nuclear receptors.Western blot assay was employed to detect the protein levels of p53,I?B? and NF-?B.The specific inhibitors of the nuclear receptors was also adapted to affirm the role of transcriptional factors.The data was analyzed by GraphPad Prism 5.Results1.After celastrol treatment,the mRNA and protein expression of CAR and PXR was not significantly induced.Their downstream target gene P-gp was downregulated.The results demonstrated that the activities of nuclear receptors were inhibited by celastrol,indicating that the nuclear receptors may not be involved in the induction of CYPs by celastrol.2.Celastrol showed no significant effect on p53 protein level.The results indicates that p53 may not be involved in the induction of CYPs by celastrol.3.Celastrol inhibited the protein expression of NF-?B in a dose-dependent manner.After co-treatment with NF-?B inhibitor,the inductive effect of celastrol on CYP2C12,CYP2D2 and CYP3A1 were inhibited.ConclusionsNF-?B may be at least partly involved in the inductive effect of CYPs caused by celastrol in rat primary hepatocytes.The nuclear receptor and p53 may not be involved in this induction mechanism.SECTION ? The role of celastrol induced cytochrome P450s played in inflammationObjectiveCYP2C converts arachidonic acid(AA)to epoxyeicosatrienoic acid(EET),which plays an anti-inflammation effect.The results of section one and section two showed that CYP2C was induced by celastrol.According to these information,we assume that CYPs induced by celastrol may play a role on the anti-inflammatory effect of celastrol.MethodsTwo-step collagen perfusion method was employed to extract rat primary hepatocytes from Wistar rats.The cells were cultured for 12 h before treated with drugs.SRB assay adopted to investigate the cell viability after rat primary hepatocytes were treated with LPS,while ELISA assay was employed to investigate the secretion of TNF-a and IFN-y.After cells were treated with celastrol with or without CYPs inhibitor,the secretion of TNF-a and IFN-y was detected by ELISA kit.Results1.5 ?g/ml lipopolysaccharide(LPS)induced inflammation response in rat primary hepatocytes.2.When LPS treated rat primary hepatocytes were treated with celastrol and CYPs inhibitor,the secretion of TNF-a and IFN-? was not altered compared with celastrol alone group.The results indicate that CYPs induced by celastrol may not involve in the anti-inflammatory effect of celastrol in rat primary hepatocytes.ConclusionsCYPs induced by celastrol may not involve in the anti-inflammatory effect of celastrol.