CYP2E1酶在去甲斑蝥素代謝和環(huán)孢素誘導(dǎo)肝損傷中的作用研究
發(fā)布時(shí)間：2018-11-12 10:43
【摘要】：細(xì)胞色素P450酶2E1亞型(CYP2E1酶)可介導(dǎo)多種體內(nèi)、外源性化合物代謝,在藥理學(xué)和毒理學(xué)研究方面作用十分重要,本研究以去甲斑蝥素和環(huán)孢素為切入點(diǎn),分別對(duì)CYP2E1酶在藥物代謝和藥物性肝損傷發(fā)病過(guò)程中的作用進(jìn)行探討。 去甲斑蝥素用于腫瘤治療多年,但其體內(nèi)代謝過(guò)程尚未完全清楚。本研究首先采用MetaPrint2D軟件,根據(jù)去甲斑蝥素及其水解產(chǎn)物去甲斑蝥酸化學(xué)結(jié)構(gòu)中原子指紋特征,基于數(shù)據(jù)挖掘和統(tǒng)計(jì)分析技術(shù),通過(guò)計(jì)算原子作為代謝位點(diǎn)的發(fā)生率,預(yù)測(cè)去甲斑蝥素及其代謝物去甲斑蝥酸代謝位點(diǎn)及可能代謝產(chǎn)物；采用Sybyl軟件分別進(jìn)行CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2D6, CYP2C9, CYP2E1和CYP2C19酶與去甲斑蝥酸對(duì)接；然后選擇其中得分較高者,進(jìn)行人重組CYP450酶體外代謝實(shí)驗(yàn),結(jié)果顯示,CYP2E1、 CYP2C19和CYP2C9酶均參與介導(dǎo)去甲斑蝥酸代謝過(guò)程。人重組CYP2E1酶介導(dǎo)去甲斑蝥素體外代謝呈米氏動(dòng)力學(xué)過(guò)程,Km為23.64μM, CL為0.689ml/mmol CYP2E1/min;最后對(duì)體內(nèi)外代謝產(chǎn)物進(jìn)行分離鑒定,結(jié)果證實(shí),去甲斑蝥素進(jìn)入體內(nèi)后,首先水解為去甲斑蝥酸,進(jìn)而發(fā)生氧橋水解、六元環(huán)不同方式斷裂開(kāi)環(huán)或脫羧,進(jìn)一步發(fā)生氧化代謝,轉(zhuǎn)化為小分子化合物排出體外,或與葡萄糖醛酸、甘氨酸、谷胱甘肽等發(fā)生結(jié)合反應(yīng),增加水溶性,利用消除,軟件預(yù)測(cè)與體內(nèi)、體外代謝實(shí)驗(yàn)結(jié)果基本一致。 CYP2E1酶活性區(qū)域小,有利于小分子化合物進(jìn)入,但分子內(nèi)作用力和分子間范德華力能量較高,易導(dǎo)致酶-藥物復(fù)合物不穩(wěn)定；CYP2E1酶介導(dǎo)內(nèi)外源性化合物代謝時(shí)發(fā)生變構(gòu),易導(dǎo)致氧化應(yīng)激,誘導(dǎo)肝損傷發(fā)生。環(huán)孢素誘導(dǎo)肝損傷發(fā)病率約為30%,但其機(jī)制尚不清楚。本研究對(duì)322名長(zhǎng)期服用環(huán)孢素進(jìn)行抗排異或免疫抑制相關(guān)治療的患者全血樣本進(jìn)行了CYP2E1基因型檢測(cè),首先對(duì)200名患者進(jìn)行13種SNPs檢測(cè),經(jīng)Hardy-Weinberg平衡檢驗(yàn)和遺傳連鎖分析,從中選擇6個(gè)標(biāo)簽SNPs,其余患者僅進(jìn)行標(biāo)簽SNP測(cè)定,采用病例-對(duì)照研究方法,對(duì)全部患者6種標(biāo)簽SNPs與環(huán)孢素誘導(dǎo)肝損傷進(jìn)行關(guān)聯(lián)分析,結(jié)果顯示,患者服用環(huán)孢素后,環(huán)孢素血清谷濃度與肝損傷發(fā)生未呈現(xiàn)明顯相關(guān)性；rs3813866(-1563TA)為環(huán)孢素誘導(dǎo)肝損傷易感基因(OR:2.325,95%CI:1.491-3.626). 進(jìn)一步從細(xì)胞分子水平,采用點(diǎn)突變技術(shù),構(gòu)建rs3813866突變體質(zhì)粒載體,轉(zhuǎn)染至HepG-2細(xì)胞,探索易感SNP如何調(diào)控CYP2E1蛋白表達(dá)或酶活性,結(jié)果顯示,rs381366突變可使CYP2E1啟動(dòng)子活性增強(qiáng)約2.46倍,mRNA水平上調(diào)約1.64倍,導(dǎo)致攜帶rs381366突變基因的患者服用環(huán)孢素后,易發(fā)生誘導(dǎo)肝損傷；環(huán)孢素可使CYP2E1啟動(dòng)子活性增強(qiáng),上調(diào)啟動(dòng)子活性能力與藥物濃度成正比,但環(huán)孢素對(duì)CYP2E1啟動(dòng)子活性的上調(diào)作用遠(yuǎn)小于rs381366突變的影響：環(huán)孢素對(duì)細(xì)胞中CYP2E1mRNA表達(dá)水平的影響與啟動(dòng)子活性相反,環(huán)孢素干預(yù)后,野生型和突變型CYP2E1mRNA均呈現(xiàn)下調(diào)趨勢(shì),且隨環(huán)孢素濃度增加而下調(diào)明顯,20μM環(huán)孢素干預(yù)后CYP2E1野生型mRNA水平下調(diào)約2倍,突變型mRNA下降可達(dá)20倍；環(huán)孢素干預(yù)可使野生型和突變型CYP2E1蛋白表達(dá)和酶活性略微增強(qiáng),環(huán)孢素濃度越高,上調(diào)蛋白表達(dá)和酶活性能力越強(qiáng),20μM環(huán)孢素干預(yù)后CYP2E1蛋白表達(dá)水平提高約1.72倍,酶活性增強(qiáng)27.28%,環(huán)孢素上調(diào)CYP2E1酶轉(zhuǎn)錄表達(dá)過(guò)程是否存在其它反饋調(diào)節(jié)機(jī)制仍待進(jìn)一步研究。
[Abstract]:Cytochrome P450 enzyme 2E1 subtype (CYP2E1 enzyme) can mediate a variety of in vivo, exogenous compound metabolism, and is very important in the research of pharmacology and toxicology. The role of CYP2E1 in the pathogenesis of drug metabolism and drug-induced liver injury was discussed. Normotilin is used in the treatment of tumors for many years, but its in vivo metabolic processes are not complete It is clear that in this study, the metaPrint2D software is used, and based on the characteristics of the atomic fingerprint in the noreptilin and its hydrolysis product, the atomic fingerprint feature in the chemical structure is based on the data mining and the statistical analysis technique, and the atoms are calculated by the calculation of the atoms as the metabolic site. The ratio of CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2D6, CYP2C9, CYP2E1, and CYP2C19 to normaconic acid was determined by using Sybil software, then the higher of the score was selected, and the in vitro metabolism of human recombinant CYP450 was carried out. The results showed that both CYP2E1, CYP2C19, and CYP2C9 were involved in the mediation of the metabolism of norepinephrine. in that process, the human recombinant CYP2E1 enzyme mediate the in vitro metabolism of the noreptilin in the m's kinetic process, Km is 23.64. mu. M, the CL is 0. 689ml/ mmol of CYP2E1/ min, and finally, the in-vivo metabolism product is separated and identified, and the result is confirmed that the nomotilin enters the body, and then is first hydrolyzed to the nomottle acid, and then an oxygen bridge is generated. in that hydrolysis and six-membered ring, the open-loop or deionization is broken in a different way, the oxidation and metabolism are further happen, the small molecular compound is converted into a small molecular compound to be discharged in vitro, or in combination with the glucuronic acid, the glycine, the glutathione and the like, the water solubility is increased, In vivo, the results of in vitro metabolism experiment are basically The activity of the CYP2E1 enzyme is small, which is beneficial to the entry of the small molecule compound, but the internal force of the molecule and the Van der Waals force between the molecules are high, so that the enzyme-drug complex is not stable; the metabolism of the exogenous compound in the CYP2E1 enzyme-mediated metabolism of the exogenous compound is easy to lead to oxidative stress, induction, The incidence of hepatic injury induced by ciclosporin was about 30%. The results of the study of CYP2E1 genotype were carried out in 322 patients with long-term cyclosporine anti-rejection or immunosuppression-related treatment, and 13 SNPs were first tested by Hardy-Weinberg equilibrium test and genetic linkage analysis, and 6 of them were selected. The results showed that the concentration of the serum trough of the ciclosporin and the hepatic injury did not appear after the administration of the ciclosporin. Significant correlation; rs3813866 (-1563TA) was a risk-sensitive gene for ciclosporin-induced liver injury (OR: 2.325, 95% CI: 1.491-3. 626). Further from the cellular molecular level, using the point mutation technique, the rs3813866 mutant plasmid vector was constructed and transfected into HepG-2 cells to explore how the susceptibility SNP could control the expression of the CYP2E1 protein or the enzyme activity. The results showed that the rs381366 mutation can enhance the activity of the CYP2E1 promoter by about 2.46 times, the mRNA level up to about 1.64 times up-regulation, leading to the induction of liver injury after taking the ciclosporin in a patient carrying the rs381366 mutant gene; the ciclosporin can enhance the activity of the CYP2E1 promoter and increase the activity ability of the promoter to be in direct proportion to the drug concentration, but the regulation effect of the ciclosporin on the activity of the CYP2E1 promoter is far less than that of the rs381 The effect of the 366 mutation: the effect of the ciclosporin on the expression level of CYP2E1mRNA in the cells was opposite to that of the promoter. After the intervention of the ciclosporin, the wild-type and the mutant CYP2E1mRNA were down-regulated and decreased with the increase of the concentration of the ciclosporin, and the CYP2E1 wild-type mRN after the intervention of 20. m The level of level A was reduced by about 2-fold, and the expression of mutant mRNA could be up to 20-fold. Cyclosporin intervention could increase the expression of wild-type and mutant CYP2E1 protein and the activity of enzyme, the higher the concentration of ciclosporin, the higher the expression of protein and the stronger the ability of the enzyme, and the expression of CYP2E1 protein after the intervention of 20. m about 1.72-fold increase in enzyme activity, 27-28% enhancement of enzyme activity, and presence of other feedback regulators in the regulation of the transcription of the CYP2E1 enzyme by the cyclosporine
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.3
本文編號(hào)：2326879
[Abstract]:Cytochrome P450 enzyme 2E1 subtype (CYP2E1 enzyme) can mediate a variety of in vivo, exogenous compound metabolism, and is very important in the research of pharmacology and toxicology. The role of CYP2E1 in the pathogenesis of drug metabolism and drug-induced liver injury was discussed. Normotilin is used in the treatment of tumors for many years, but its in vivo metabolic processes are not complete It is clear that in this study, the metaPrint2D software is used, and based on the characteristics of the atomic fingerprint in the noreptilin and its hydrolysis product, the atomic fingerprint feature in the chemical structure is based on the data mining and the statistical analysis technique, and the atoms are calculated by the calculation of the atoms as the metabolic site. The ratio of CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2D6, CYP2C9, CYP2E1, and CYP2C19 to normaconic acid was determined by using Sybil software, then the higher of the score was selected, and the in vitro metabolism of human recombinant CYP450 was carried out. The results showed that both CYP2E1, CYP2C19, and CYP2C9 were involved in the mediation of the metabolism of norepinephrine. in that process, the human recombinant CYP2E1 enzyme mediate the in vitro metabolism of the noreptilin in the m's kinetic process, Km is 23.64. mu. M, the CL is 0. 689ml/ mmol of CYP2E1/ min, and finally, the in-vivo metabolism product is separated and identified, and the result is confirmed that the nomotilin enters the body, and then is first hydrolyzed to the nomottle acid, and then an oxygen bridge is generated. in that hydrolysis and six-membered ring, the open-loop or deionization is broken in a different way, the oxidation and metabolism are further happen, the small molecular compound is converted into a small molecular compound to be discharged in vitro, or in combination with the glucuronic acid, the glycine, the glutathione and the like, the water solubility is increased, In vivo, the results of in vitro metabolism experiment are basically The activity of the CYP2E1 enzyme is small, which is beneficial to the entry of the small molecule compound, but the internal force of the molecule and the Van der Waals force between the molecules are high, so that the enzyme-drug complex is not stable; the metabolism of the exogenous compound in the CYP2E1 enzyme-mediated metabolism of the exogenous compound is easy to lead to oxidative stress, induction, The incidence of hepatic injury induced by ciclosporin was about 30%. The results of the study of CYP2E1 genotype were carried out in 322 patients with long-term cyclosporine anti-rejection or immunosuppression-related treatment, and 13 SNPs were first tested by Hardy-Weinberg equilibrium test and genetic linkage analysis, and 6 of them were selected. The results showed that the concentration of the serum trough of the ciclosporin and the hepatic injury did not appear after the administration of the ciclosporin. Significant correlation; rs3813866 (-1563TA) was a risk-sensitive gene for ciclosporin-induced liver injury (OR: 2.325, 95% CI: 1.491-3. 626). Further from the cellular molecular level, using the point mutation technique, the rs3813866 mutant plasmid vector was constructed and transfected into HepG-2 cells to explore how the susceptibility SNP could control the expression of the CYP2E1 protein or the enzyme activity. The results showed that the rs381366 mutation can enhance the activity of the CYP2E1 promoter by about 2.46 times, the mRNA level up to about 1.64 times up-regulation, leading to the induction of liver injury after taking the ciclosporin in a patient carrying the rs381366 mutant gene; the ciclosporin can enhance the activity of the CYP2E1 promoter and increase the activity ability of the promoter to be in direct proportion to the drug concentration, but the regulation effect of the ciclosporin on the activity of the CYP2E1 promoter is far less than that of the rs381 The effect of the 366 mutation: the effect of the ciclosporin on the expression level of CYP2E1mRNA in the cells was opposite to that of the promoter. After the intervention of the ciclosporin, the wild-type and the mutant CYP2E1mRNA were down-regulated and decreased with the increase of the concentration of the ciclosporin, and the CYP2E1 wild-type mRN after the intervention of 20. m The level of level A was reduced by about 2-fold, and the expression of mutant mRNA could be up to 20-fold. Cyclosporin intervention could increase the expression of wild-type and mutant CYP2E1 protein and the activity of enzyme, the higher the concentration of ciclosporin, the higher the expression of protein and the stronger the ability of the enzyme, and the expression of CYP2E1 protein after the intervention of 20. m about 1.72-fold increase in enzyme activity, 27-28% enhancement of enzyme activity, and presence of other feedback regulators in the regulation of the transcription of the CYP2E1 enzyme by the cyclosporine
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.3
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