基于体外肝代谢考察大黄素甲醚肝毒性作用
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摘要
以胆红素代谢过程中UDP-葡萄糖醛酸转移酶1A1(UGT1A1酶)介导的胆红素葡萄糖醛酸结合环节为切入点,评价大黄素甲醚潜在肝毒性风险。实验以胆红素为UGT1A1酶底物,以表观抑制常数K_i为评价指标,采用体外肝微粒体孵育法,启动Ⅱ相代谢反应,考察大黄素甲醚原型成分的抑制作用,启动Ⅰ,Ⅱ相代谢反应,考察代谢产物及原型成分的综合抑制作用。结果显示仅启动Ⅱ相反应时,大黄素甲醚以原型形式直接作用于UGT1A1酶,表现为弱抑制,抑制类型为混合型抑制;同时启动Ⅰ,Ⅱ相反应时,大黄素甲醚对UGT1A1酶的抑制作用变为强抑制,抑制类型为混合型抑制,提示大黄素甲醚存在Ⅰ,Ⅱ相代谢过程,并且其代谢产物大黄素葡萄糖苷结合物及大黄素葡萄糖醛酸化物对UGT1A1酶抑制作用较强。该研究所得结果初步证明大黄素甲醚原型对UGT1A1无明显抑制作用,经由Ⅰ,Ⅱ相代谢后抑制作用增强,推测其代谢产物为引发肝毒性的主要成分。
To evaluate the hepatotoxicity risks of physcion on the basis of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1A1(UGT1A1 enzyme). The monomers were added into the rat liver microsomes to test the hepatotoxicity by using bilirubin as UGT1A1 enzyme substrate, with apparent inhibition constant K_i as the evaluation index. Liver microsome incubation in vitro was adopted to initiate phase Ⅱ metabolic reaction and investigate the inhibitory effect of physcion. Then the phase Ⅰ and Ⅱ metabolic reactions were initiated to investigate the comprehensive inhibition of metabolites and prototype components. The results showed that when only the phase Ⅱ reaction was initiated, physcion directly acted on the UGT1A1 enzyme in a prototype form, exhibited weak inhibition and the inhibition type was mixed inhibition; When the phase Ⅰ and Ⅱ reactions were initiated simultaneously, the inhibitory effects of physcion on UGT1A1 enzyme became strong and the inhibition type was mixed inhibition, suggesting that physcion had phase Ⅰ and Ⅱ metabolic processes, and the metabolites had strong inhibitory effect on UGT1A1 enzyme. This experiment preliminarily proved that the metabolites of physcion may be the main components to induce hepatotoxicity.
To evaluate the hepatotoxicity risks of physcion on the basis of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1A1(UGT1A1 enzyme). The monomers were added into the rat liver microsomes to test the hepatotoxicity by using bilirubin as UGT1A1 enzyme substrate, with apparent inhibition constant K_i as the evaluation index. Liver microsome incubation in vitro was adopted to initiate phase Ⅱ metabolic reaction and investigate the inhibitory effect of physcion. Then the phase Ⅰ and Ⅱ metabolic reactions were initiated to investigate the comprehensive inhibition of metabolites and prototype components. The results showed that when only the phase Ⅱ reaction was initiated, physcion directly acted on the UGT1A1 enzyme in a prototype form, exhibited weak inhibition and the inhibition type was mixed inhibition; When the phase Ⅰ and Ⅱ reactions were initiated simultaneously, the inhibitory effects of physcion on UGT1A1 enzyme became strong and the inhibition type was mixed inhibition, suggesting that physcion had phase Ⅰ and Ⅱ metabolic processes, and the metabolites had strong inhibitory effect on UGT1A1 enzyme. This experiment preliminarily proved that the metabolites of physcion may be the main components to induce hepatotoxicity.
引文
[1] Gao F,Peng W,Li X,et al.Emodin is identified as the active component of ether extracts from Rhizoma Polygoni Cuspidati,for anti-MRSA activity [J].Can J Physiol Pharmacol,2015,93(6):485.
[2] Yao W Y,Zhou Y F,Qian A H,et al.Emodin has a protective effect in cases of severe acute pancreatitis via inhibition of nuclear factor kappa B activation resulting in antioxidation [J].Mol Med Rep,2015,11(2):1416.
[3] NTP toxicology and carcinogenesis studies of EMODIN(CAS NO.518-82-1) feed studies in F344/N rats and B6C3F1 mice[J].Natl Toxicol Program Tech Rep Ser,2001,493:278.
[4] Su Y T,Chang H L,Shyue S K,et al.Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway[J].Biochem Pharmacol,2005,70(2):229.
[5] Sun H,Yang J P,Mao Y,et al.Involvement of Fas-dependent pathway in rhein-induced apoptosis of HK-2 cells[J].J China Pharm Univ,2015,16(4):469.
[6] Srinivas G,Anto R J,Srinivas P,et al.Emodin induces apoptosis of human cervical cancer cells through poly(ADP-ribose) polymerase cleavage and activation of caspase-9[J].Eur J Pharmacol,2003,473(2):117.
[7] 赖潇潇,吴俊标,陈设,等.基于回顾性研究的何首乌分析因素分析与安全应用建议[J].中国中药杂志,2018,43(15):3205.
[8] 林艳,肖榕,尹林子,等.基于粗糙集理论构建“谱毒”关系研究何首乌特异质肝毒性物质基础[J].中国中药杂志,2019,44(3):509.
[9] Vitek L,Ostrow J D.Bilirubin chemistry and metabolism metabolism;harmful and protective aspects[J].Curr Pharm Des,2009,15(25):2869.
[10] Wang Q,Dai Z,Zhang Y J,et al.Study on the hepatotoxicity of dianthrones in Polygoni Multiflori Radix [J].Chin J Pharm Anal 2018,38(2)):268.
[11] Scriver C R.The metabolic & molecular bases of inherited disease[M].8th ed.New York:McGraw-Hill,2001.
[12] Song R,Xu L,Xu F G,et al.Metabolic analysis of rhubarb extract by rat intestinal bacteria using liquid chromatography-tandem mass spectrometry[J].Biomed Chromatogr,2011,25:417.
[13] Wang Q,Dai Z,Wang Y D,et al.Hepatotoxicity of eight components of Radix Polygonum multiflorum Thumb.in rat liver microsom system in vitro[J].Chin Pharm J,2018,53(80):589.
[14] Quan Z Y,Sun Z X.Study on hepatotoxicity of aqueous extract of Polygoni Multiflori Radix and its main components based on zebrafish[J].Chin J Pharmacov,2018,15(1):1.
[2] Yao W Y,Zhou Y F,Qian A H,et al.Emodin has a protective effect in cases of severe acute pancreatitis via inhibition of nuclear factor kappa B activation resulting in antioxidation [J].Mol Med Rep,2015,11(2):1416.
[3] NTP toxicology and carcinogenesis studies of EMODIN(CAS NO.518-82-1) feed studies in F344/N rats and B6C3F1 mice[J].Natl Toxicol Program Tech Rep Ser,2001,493:278.
[4] Su Y T,Chang H L,Shyue S K,et al.Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway[J].Biochem Pharmacol,2005,70(2):229.
[5] Sun H,Yang J P,Mao Y,et al.Involvement of Fas-dependent pathway in rhein-induced apoptosis of HK-2 cells[J].J China Pharm Univ,2015,16(4):469.
[6] Srinivas G,Anto R J,Srinivas P,et al.Emodin induces apoptosis of human cervical cancer cells through poly(ADP-ribose) polymerase cleavage and activation of caspase-9[J].Eur J Pharmacol,2003,473(2):117.
[7] 赖潇潇,吴俊标,陈设,等.基于回顾性研究的何首乌分析因素分析与安全应用建议[J].中国中药杂志,2018,43(15):3205.
[8] 林艳,肖榕,尹林子,等.基于粗糙集理论构建“谱毒”关系研究何首乌特异质肝毒性物质基础[J].中国中药杂志,2019,44(3):509.
[9] Vitek L,Ostrow J D.Bilirubin chemistry and metabolism metabolism;harmful and protective aspects[J].Curr Pharm Des,2009,15(25):2869.
[10] Wang Q,Dai Z,Zhang Y J,et al.Study on the hepatotoxicity of dianthrones in Polygoni Multiflori Radix [J].Chin J Pharm Anal 2018,38(2)):268.
[11] Scriver C R.The metabolic & molecular bases of inherited disease[M].8th ed.New York:McGraw-Hill,2001.
[12] Song R,Xu L,Xu F G,et al.Metabolic analysis of rhubarb extract by rat intestinal bacteria using liquid chromatography-tandem mass spectrometry[J].Biomed Chromatogr,2011,25:417.
[13] Wang Q,Dai Z,Wang Y D,et al.Hepatotoxicity of eight components of Radix Polygonum multiflorum Thumb.in rat liver microsom system in vitro[J].Chin Pharm J,2018,53(80):589.
[14] Quan Z Y,Sun Z X.Study on hepatotoxicity of aqueous extract of Polygoni Multiflori Radix and its main components based on zebrafish[J].Chin J Pharmacov,2018,15(1):1.