Study On IL-6 Suppresses The Expression And Activity Of HCE1,HCE2 And CYP3A4 In Human Hepatocytes And Theirs Mechanism

In 1985,WHO suggested that "Reasonable drug administration requires that the drug which patients received meets with their clinical requirement,and the dosage is individualized by each patient." The dosage of the drug,the plasma drug level is directly relevant to clinical effects,and this relevance is the basis of drug dosage.The plasma drug level is the most important parameter of pharmacodynamics and is affected by drug metabolizing enzymes.The liver is the major site of drug metabolism and clearance from circulation,and it expresses numerous drug metabolizing enzymes(DMEs),including PhaseⅠenzymes(CYP3A4,HCE1,HCE2...),PhaseⅡenzymes(UGT,GST...) and PhaseⅢenzymes(Transporter).CYP enzymes constitute a superfamily of heme-containing proteins and rank first among the phaseⅠbiotransformation enzymes in terms of catalytic versatility and the number of xenobiotics they metabolize. Therefore,CYP enzymes determine the intensity and duration of action of drugs and play key roles in the detoxication and bioactivation of xenobiotics.CYP enzymes are present in all mammalian tissues with the highest concentration in the liver endoplasmic reticulum.In each mammalian species,four families of P450 enzymes with a total of～20 members are shown to involve xenobiotic metabolism.Among drug metabolizing enzymes,CYP3A subfamily members are the most abundant P450 enzymes and involve the metabolism of 60%of xenobiotics.It is approved that generic polymorphism,age,drugs(including herbal),diet,smoking and drinking can modulate DMEs expression and activity directly and indirectly.Clinically,in some pathological states, such as infection,it is apparent that inflammation can decrease metabolic clearance of CYP substrates by 20%-70%.Obviously,the potential for clinically relevant consequences of these changes will be highest when large changes in clearance occur,and with drugs that have a low safty margin.It is well known that IL6 is the most important cytokines that causes hepatic inflammation.Cancer is the 2^ndword-wide heath threatening disease after cardiovascular diseases.In 2000,new tumor patients reached 1.6 million in China and more than 1.3 million patient died of tumor.Now 3.8 million patients are suffering from cancer and the numbers of tumor incidence and death will be double in 2050.Now the causation and etiology of tumor is still not well known,but it is closely relevant to infection and inflammation.Therefore,the study on IL-6 of the modulation and effect of CYP3A4,HCE1 and HCE2 gives a theortical basis of reasonable clinical drug administration window.It is not only money-saving,socially and economically profitable but also improves effect as well as avoids oversdosage and decreases toxicity of drugs.We examined the IL-6 on CYP3A4,HCE1 and HCE2 mRNA and protein levels,and functional changes in cultured primary human hepatocytes after its treatment using QRT-PCR,Western Bolt and enzymatic assay.We constructed the expression and promoter reporter constructs of those 3 DMEs,and discussed the molecular mechanism of the inhibition of IL-6 on CYP3A4,HCE1 and HCE2.PartⅠFocus on associated plasmids constructsAIM:To construct wild and polymorphism mutant human pregnane X receptor(PXR),human carboxylesterasel(HCE1),carboxylesterase2 (HCE2)expressed constructs;and series of CYP3A4 promoter reporter constructs,and also to build HCE1/HCE2 promoter reporter constructs.METHODS:Constructing the wild and mutant human PXR and HCE1 HCE2 expressed constructs and CYP3A4,HCE1 and HCE2 promoter reporters in clone,subclone and site-mutant ways.Testing the function of these constructs with Western Bolt and reporter assay after transfection the cells with these constructs.RESULTS:Wild and polymorphism human PXR,HCE1 and HCE2 expressed constructs were constructed.Western bolt showed they expressed in mammal cells after they were transfected with these constructs.CYP3A4,HCE1 and HCE2 promoter reporters were cloned and their functions were tested with reporters assay after they were transfected.CONCLUSION:Sequence analysis and function test to confirm the constructs are correct.PartⅡStudy on IL-6 suppresses the expression and activity of HCE1 and HCE2 in human hepatocytes and theirs mechanismOBJECTIVE:Study on IL-6 suppresses the expression and activity of HCE1 and HCE2 in human hepatocytes and its molecular mechanism.METHODS:The primary hepatocytes and hepatoma HepG2 cells were treated with IL-6 for 24 hours.The mRNA and protein expressions of HCE1 and HCE2 were determined using real-time PCR(TaqMan)and Western blots,respectively.The hydrolytic activity was assayed with para-nitrophenylacetate.we constructed HCE1 and its mutant promoters with cloning technique,and study the mechanism of IL-6 suppressed the expression with cotransfection with HCE1 and HCE2 promoters and HCE1 mutant promoters. RESULTS:In both primary hepatocytes and HepG2 cells, treatment with IL-6 decreased the expression of human carboxylesterases HCE1 and HCE2 by as much as 60%.The decreased expression occurred at both mRNA and protein levels,and it was confirmed by enzymatic assay.In cotransfection experiment,both HCE1 and HCE2 promoters were significantly repressed,and the repression was comparable with the decrease in HCE1 and HCE2 mRNA,suggesting that transrepression is responsible for the suppressed expression.In addition,pretreatment with IL-6 altered the cellular responsiveness in an opposite manner of overexpression of HCE1 and HCE2 toward various ester therapeutic agents(e.g.,clopidogrel).Transfection of HCE1,for example,decreased the cytotoxicity induced by antithrombogenic agent clopidogrel,whereas pretreatment with IL-6 increased the cytotoxicity.Such a reversal was observed with other ester drugs,including anti-influenza agent oseltamivir.PartⅢStudy on IL-6 suppresses the expression and activity of CYP3A4 in human hepatocytes and its mechanismOBJECTIVE:Study on IL-6 suppresses the expression and activity of CYP3A4 in human hepatocytes and its mechanismMETHODS:The primary hepatocytes and hepatoma HepG2 cells were treated with IL-6 for 24 hours.The mRNA and protein expressions of CYP3A4 were determined using real-time PCR(TaqMan)and Western blots,respectively.The oxidative activity was assayed with P450—Glo Kit.we constructed CYP3A4 promoter and its mutant promoters with cloning technique,and study the mechanism of IL-6 suppressed the expression with cotransfection with a series of CYP3A4 promoters and its mutant promoters.RESULTS:In both primary hepatocytes and HepG2 cells,treatment with IL6 decreased the expression of human CYP3A4 by as much as 99%. The decreased expression occurred at both mRNA and protein levels,and it was confirmed by enzymatic assay.IL-6 significantly inhibits CYP3A4 promoter reporter,and knockdown PXR with SiPXR can abolish IL-6 inhibition of CYP3A4 expression.These data suggested that PXR is involved in IL6 inhibition of CYP3A4.In addition to human PXR(hPXR), IL-6 inhibited rat PXR(rPXR).12 natural variants of PXR are almost as same as wild type PXR in responding to IL-6.In addition,Our date applied that DEC1 may be also involved in IL6 inhibition of CYP3A4.CONCLUSION:(1)Proinflammatory cytokines exert a broad suppression on the expression of various types of drug-metabolizing enzymes.(2)Multiple mechanism support the action of IL-6 and these mechanisms operate independently or cooperatively,depending on a target gene.(3)The suppressed expression of CYP450(CYP3A4)has profound pharmacological and toxicological consequences.In summary,the present study has several very important finds:(1) Elucidate that the molecular mechanisms of IL-6 induce HCE1/HCE2 and CYP3A4 decrease;(2)Provide that the methods to develop molecular-based technologies to predict such interactions.(3)Suggest that multiple mechanism support the action of IL-6 and these mechanisms operate independently or cooperatively,depending on a target gene.