Effection And Regulation Mechanism Of Cecropin B On Expression Of CYP3A29

In the past decades,several antimicrobial peptides(AMPs)and derivatives are currently in clinical development,mainly as topical agents.However,there have been no reports on the molecular mechanisms underlying the effects of biological polypeptides like AMPs on drug metabolism enzymes which affect drug safety and efficacy.With the increasing use of Cecropins for various purposes,there is an urgent need for a better understanding of their effects on the P450 metabolic pathways which are often responsible for drug metabolism and drug-drug interaction.CYP3A29 is the predominant isoform in the hepatic-intestinal system and is responsible for metabolizing more than 50% of clinically used drugs in pig.Alterations in CYP3A29 activity may lead to a remarkable change in drug efficacy.The similarity of the primary structures and metabolic characteristics of pig CYP3A29 and human CYP3A4 suggests that pig CYP3A29 is a good experimental model for the metabolic studies of drugs metabolized by human CYP3 A enzymes.In the present study,we have demonstrated that cecropin B regulates the expression of CYP3A29 by interacting with TLRs,leading to NF-κB activation and the modulation of the expression of downstream genes PXR(Pregnane X Receptor)、RXR-α(Retinoid X Receptor α)and CYP3A29.The results of our study could provide theoretical guidance and molecular basis for the rational use of drugs and better use of AMPs in veterinary and human clinical medicines.1.Research of the expression and regulation of CYP3A29,PXR and RXR-α by Cecropin BIn order to test the effect of Cecropin B on expression of CYP3A29 and PXR,RXR-α,We investigated the effects of Cecropin B on the expression of CYP3A29,PXR,RXR-α in primary pig hepatocytes and Hep Li cellsat various concentrations for different durations;Results revealed that: 1,CYP3A29 and PXR levels significantly decreased,and the inhibitory effects of CYP3A29 and PXR was related;2,Cecropin B had no effect on the expression of nuclear receptor RXR-α.To ascertain the effects of Cecropin B via PXR,PXR transient transcription assays,PXR si RNA assayswere performed,results showed that Cecropin B represses the expression of CYP3A29 through repressing the expression of PXR.To ascertain the effects of Cecropin B via RXR-α,RXR-αsi RNA assays and detection of protein distribution of RXR-αwere performed,results have demonstrated that Cecropin B suppressed CYP3A29 via a RXR-α-dependent mechanism.2.Research ofthe effectsby Cecropin B on the transcriptional activity of CYP3A29To analyze the regulation of CYP3A29 expression mediated by PXR and RXR-α at the transcriptional level,the following experiments were perfoemed: The transcriptional inhibition assay was perfoemed and result suggested that Cecropin B downregulated the expression of CYP3A29 by inhibiting its promoter activity;The promoter luciferase reporter gene of CYP3A29 was constructed,and found that Cecropin B dreased the activity of CYP3A29 promoter;Moreover,we used the JASPAR(http://jaspar.genereg.net/)software to predict the binding sites of PXR/RXR-α in the CYP3A29 promoter region,and performedsite-directed mutagenesis assay of the PXR/RXR-αbinding site to screening the correct binding site;At last we performed a Ch IP assay to identify the result from site-directed mutagenesis assay.Our results revealed that Cecropin B can repress the binding ability of PXR/RXR-α to CYP3A29 promoter.The transcriptional regulations of pig CYP3A29 and PXR may help elucidate the regulatory mechanisms of pig drug metabolism.3.Research ofthe NF-κB/PXR pathway induced by Cecropin BIn order to detect the upstream signal of PXR/RXR-α,the role of transcription factor NF-κB was analyzed,results were obtained as follows:We transiently transfected NF-κB p65 overexpression vector into Hep Li cells to determine the role of NF-κB in mediating the suppression of CYP3A29 transcriptional inhibition.Our results revealed that NF-κB inhibited the expression of CYP3A29 and PXR,which were the same as Cecropin B.We transiently transfected the NF-κB p65-driven luciferase reporter gene into Hep Li cells,the expression of p65 was up-regulated by Cecropin B;When specific inhibitor BAY117082 that could inhibit nuclear translocation of NF-k B was used,the suppression ability of Cecropin B on CYP3A29 was significantly attenuated;Furthermore,the activation of NF-κB by Cecropin B was confirmed by IHC for p65 nuclear translocation.These results supported the notion that NF-κB activation was directly responsible for Cecropin B-regulated gene expression.Then we constructed the promoter luciferase reporter gene of PXR,used the JASPAR(http://jaspar.genereg.net/)software to predict the binding sites of NF-Κb p65 in the PXR promoter region,and performed a Ch IP assay to identify the results.Results indicated that NF-κB inhibits the expression of PXR by binding to the promoter of PXR.These results indicated that Cecropin B inhibited CYP3A29 expression via NF-κB/PXR.4.Research ofthe NF-κB/ RXR-αpathway induced by Cecropin BKnown that NF-κB repress the expression of CYP3A29 through repress the promoter activity of PXR and Cecropin B increased the nuclear exportation of RXR-α into the cytoplasm,we porfermed Co-Immunoprecipitation to test the effect of NF-κB to RXR-α.Result indicates that cecropin B inhibited CYP3A29 expression via promoting the physical interaction between RXR-α and the NF-κB p65 subunit,induce the exportation of RXR-α into the cytoplasm,and reducing the interaction of the PXR/RXR-α complex with the CYP3A29 promoter.From these results,we can conclude that Cecropin B regulates CYP3A29 through multiple mechanisms.5.Research ofthe TLRs pathway induced by Cecropin BIn order to detect the transmembrane signal transduction of Cecropin B,si RNA assays of TLRs were performed by transfecting Hep Li cells with TLRs 1,2,4,5 and 6 si RNAs,followed by treatment with Cecropin B.Silencing TLR2/4/5 attenuated the activity of Cecropin B in the expression of CYP3A29.We also assessed the activation of p65 in Hep Li cells co-transfected with TLR si RNA and NF-κB 65 reporter plasmid,followed by Cecropin B treatment.TLR2/4/5 si RNAs reduced the activity of Cecropin B in inducing NF-κB.Further confirm these results,we used 293 T cells;since 293 T cells do not express TLR2/4,confirming that TLR2/4 were required.