寒证大鼠细胞色素P_(450)亚酶的表征及肉桂的调控
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摘要
目的:考察寒证大鼠肝脏细胞色素P_(450)(CYP_(450))亚酶的表征特点及热性中药的调控效果。方法:选取知母-石膏-黄柏-龙胆草制备寒性造模药,对SD大鼠诱导21 d后复制寒证模型;另取肉桂提取物对寒性大鼠进行干预,制备观察组;用生理盐水处理为对照组。钙盐沉淀法制备肝脏微粒体,与混合探针药物温孵,用高效液相色谱(HPLC)法测定探针药物的代谢消除百分率,评价CYP_(450)亚酶活性变化。提取肝脏总RNA,实时荧光定量PCR法测定CYP_(450)亚酶的mRNA表达。结果:寒性中药对大鼠CYP3A1酶的活性和mRNA具有显著的抑制作用,热性肉桂具有一定的调控作用,并且这种调控发生在mRNA水平。结论:本研究从多角度系统评价和验证寒证机体CYP_(450)亚酶的表征特点及热性中药的调控效果。
Objective: To study the characterization of five kinds of CYP_(450) in the liver of cold syndrome rats and the regulative effect of heat traditional Chinese medicine. Methods: Cold syndrome model was induced in SD rats for 21 days by gavage of Anemarrhena asphodeloides-Gypsum Fibrosuum-Cortex Phellodendri-Gentiana scabra. In addition,cold syndrome rats were treated with Cinnamomum cassia extract to prepare the treatment group. Saline was used as the control medicine. The liver microsomes were prepared by calcium precipitation method,incubated with mixed probe drugs,and the percentage of metabolic elimination of the probe drugs was determined by HPLC method to evaluate CYP_(450) activity. Total RNA was extracted from the liver and mRNA expression of CYP_(450) s was determined by real-time fluorescent quantitative PCR. Results: The cold medicine can inhibit the activity and mRNA of CYP3 A1 in rats. Cinnamomum cassia has a certain regulative effect which took place at mRNA level. Conclusion:This study should be further combined with protein expression experiments to evaluate and verify the characteristics of CYP_(450) enzyme in cold syndrome and the regulation effect of heat traditional Chinese medicine from multi-angle system.
Objective: To study the characterization of five kinds of CYP_(450) in the liver of cold syndrome rats and the regulative effect of heat traditional Chinese medicine. Methods: Cold syndrome model was induced in SD rats for 21 days by gavage of Anemarrhena asphodeloides-Gypsum Fibrosuum-Cortex Phellodendri-Gentiana scabra. In addition,cold syndrome rats were treated with Cinnamomum cassia extract to prepare the treatment group. Saline was used as the control medicine. The liver microsomes were prepared by calcium precipitation method,incubated with mixed probe drugs,and the percentage of metabolic elimination of the probe drugs was determined by HPLC method to evaluate CYP_(450) activity. Total RNA was extracted from the liver and mRNA expression of CYP_(450) s was determined by real-time fluorescent quantitative PCR. Results: The cold medicine can inhibit the activity and mRNA of CYP3 A1 in rats. Cinnamomum cassia has a certain regulative effect which took place at mRNA level. Conclusion:This study should be further combined with protein expression experiments to evaluate and verify the characteristics of CYP_(450) enzyme in cold syndrome and the regulation effect of heat traditional Chinese medicine from multi-angle system.
引文
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[3]周红光.代谢组学在中医基础理论研究中的应用[J].中国中医药信息杂志,2013,20(9):101-104.
[4]陈烁,王秀凤.肾阳虚证代谢组学研究进展[J].广东药科大学学报,2015,31(6):829-832.
[5]Slaughter RL,Edwards DJ.Recent advances:the cytochrome P450enzymes[J].Ann Pharmacother,1995,29(6):619-624.
[6]王子晨,张建军,朱映黎,等.阿萨伊对虚热和虚寒证大鼠的脂肪代谢、免疫物质和内分泌激素的影响[J].中国中药杂志,2017,42(13):2552-2557.
[7]秦路平,张汉平,张卫东,等.蛇床子素和蛇床子总香豆素对肾阳虚大鼠血清甲状腺激素和促甲状腺激素的影响[J].中国中西医结合杂志,1996,16(9):552-553.
[8]成秀梅,杜惠兰,路帅,等.寒凝血瘀模型大鼠下丘脑神经递质的变化及其对生殖内分泌的影响[J].北京中医药大学学报,2012,35(10):670-672.
[9]闫波,孙国祥,孙万阳,等.中药寒热温凉四性研究方法与思路[J].中南药学,2016,6(6):572-580.
[10]赵阳阳,许智慧,刘妍,等.细胞色素P450基因多态性与药物代谢研究进展[J].临床药物治疗杂志,2017,14(4):6-11.
[11]田义超,周静,徐凌云.银杏内酯B注射液对大鼠体外肝微粒体CYP1A2活性的影响[J].中国临床药理学杂志,2014,30(3):204-207.
[12]奚丽君.以半夏药对研究七情配伍对细胞代谢酶P450的影响[D].南京:南京中医药大学,2009.
[13]梁月华,李薪萍,任红.寒证热证时中枢、内脏、尿内儿茶酚胺及5-羟色胺的变化[J].中医杂志,1991,32(12):38-40.
[14]韦灵玉,张玉杰,魏宝红,等.黄连黄芩及配伍诱导对大鼠肝微粒体5种CYP450亚酶活性的影响[J].中国中药杂志,2013,38(9):1426-1428.
[15]爱德华.类药性质-概念、结构设计与方法,从ADME到安全性优化[M].钟大放译.北京:科学出版社,2011:186.
[16]陈西敬.药物代谢动力学研究进展[M].北京:化学工业出版社,2008:100.