细胞色素P450同工酶与含马兜铃酸中药毒性的关系研究

【作者】 于长安；

【导师】 梁爱华；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2006， 硕士

【摘要】 1993年在比利时妇女因服用含中草药的减肥药发生一种新的、快速进展的间质性肾纤维化,中草药肾病(Chinese herb naphropathy,CHN)因此在西方国家被提出。CHN主要是由中草药中所含的马兜铃酸引起的,故将其命名为马兜铃酸肾病(aristolochic acid nephropathy,AAN)更为确切。AAN引起国内外医学界的广泛关注,很多国家先后禁止或限制含关木通、广防己在内的十几种中草药和中成药的进口。我国国家食品药品监督管理局也对含马兜铃酸中药采取了相应的监管措施,包括取消关木通、广防己、青木香药用标准;凡含马兜铃、寻骨风、天仙藤和朱砂莲的中药制剂,严格按处方药管理。以上7种中药只是含马兜铃酸中药品种的一部分,我国有含马兜铃酸中草药品种30余种,其组成的成药制剂品种相当多。可见含马兜铃酸中药在临床上仍有广泛的应用。因此,科学研究含马兜铃酸中药的毒性机制和寻找防止或减轻毒性的方法仍然具有重要的临床意义。 虽然有关含马兜铃酸中药的毒性研究有很多,但到目前为止其毒性机制仍不完全清楚。除马兜铃酸本身外,其他因素对其。肾脏毒性的影响研究较少,在我国中西药联合应用比较普遍,已知西药在体内的代谢与肝脏细胞色素P450酶关系密切,细胞色素P450酶可以被药物诱导或抑制而导致代谢性药物相互作用,当西药与含马兜铃酸中药联用时是否会影响马兜铃酸的毒性我们尚不清楚,因此本论文中我们根据文献报道选择了几种细胞色素P450酶的诱导剂—苯巴比妥(PB)(典型的CYP总酶诱导剂)、奥美拉唑(OM)(典型的CYP1A诱导剂)和利福平(RFP)(典型的CYP3A诱导剂)与含马兜铃酸中药关木通联用研究这些诱导剂对其毒性的影响,探讨被诱导的细胞色素P450酶与含马兜铃酸中药关木通毒性的关系。本研究得到北京市自然科学基金资助(课题号:7052058)。 方法 一、根据T.C.chou报道的半数效应评价联合用药的拮抗或协同作用的方法,研究关木通提取物(GMT)和P450酶诱导剂单剂量联合一次用药对小鼠的急性毒性。 二、采用序贯用药即先提前3天给予小鼠P450酶诱导剂,以预先诱导小鼠肝脏细胞色素P450酶,于第四天给予诱导剂后约3小时再给小鼠灌胃不同剂量的GMT,研究对小鼠的急性毒性。更多还原

【Abstract】 In 1993 the rapidly progressive interstitial renal fibrosis in young women associated with Chinese herb (Aristolochia fangchi) included in slimming reagent was found in Belgium, This type of naphropathy was designated to Chinese herb naphropathy(CHN) in western countries. It was aristolochic acid contained in Aristolochia fangchi that caused the naphropathy, reseachers in China designated this type of naphropathy to aristolochic acid naphropathy (AAN). AAN causes much attention in the medical domain. Many countries ban the import and distribution of Chinese herbs products which included aristolochia fangchi and(or) caulis aristolochiae manshuriensis (guanmutong) et al. Some steps had been taken by SFDA to ensure the herbs used safely:1 Aristolochia fangchi, caulis aristolochiae manshuriensis (guanmutong) and Raidx Aristolochice in the Chinese herbs products or formula must be replaced, 2 Fructus Aristolochiae, aristolochiae mollissimae, kaempfer and Herba Aristolochiae containing aristolochic acid must be used under the doctor’s prescription. The aristolochia plants have a widespread use in herbal medicine. It is very important to study the mechanism of the toxicity of the herbs containing aristolochic acid.Although many studies about the toxicity of aristolochic acid have been done,the mechanism of the toxicity is still not identified. The factors that affect the toxicity of aristolochic acid is known little. Almost no studies have been reported about the relationship between the CYP450 and the toxicity of the herbs containing aristolochic acid. The present study had an investigation on the relationship between several CYP450 enzymes and the toxicity of aristolochic acid. This research was supported by Beijing natural science foundation. 1 Acute toxicity of aristolochiae manshuriensis extraction combined with phenobarbital sodium, rifampicin, omeprazole respectively inmice.Two methods were taken to study the acute toxicity of aristolochiae manshuriensis extraction combined with the inducers of P450 isoenzymes. Thefirst method was treating the animals with the extraction and the inducers in single dose simultaneously according to the median effect principle. The second method was that with the inducers in advance for 3 days and in the forth day after giving the inducers for about 3 hours gavaging the extraction. Then we calculated the LD50 value to analyze the combined effects on the acute toxicity.The acute toxic effects were observed in mice of both sexes. Drugs were gavaged to mice in a single dose. The LD50 value of the extraction under different protocols was calculated . By analyzing the LD50 value, we found that the toxic levels of the extraction under different situation were not in concordance. The LD50 value of the extraction alone(3. 37g/kg) was in accordance with the result that we got before. The acute toxic effects of phenobarbital sodium and rifampicin happened to mice quickly. The death of mice usually took place in 3 days. Urea nitrogen (BUN) and creatine(Cr) in the serum of the vital mice were examined to evaluate the renal function and the value were in normal range. It suggested that phenobarbital sodium and rifampicin did not cause acute renal toxicity to the vital mice.The death of the mice usually took place from the first to the third day when the extraction was combined with phenobarbital sodium and rifampicin respectively with the first method. However, the death of the mice treated with the extraction alone usually took place after 3 days.It was concluded that the death was caused primarily by phenobarbital sodium and rifampicin respectively and secondary by the extraction. When the mice were treated with the second method, the time course of the death was in accord with that treated with the extraction alone. It suggested that the death was mainly caused by the extraction.We found that the first method was suitable to assess the effects (summation, synergism and antagonism) of drugs combined, but could not reasonably could induce CYP4 reflect the effects of the extraction acted by the P450 isoenzymes induced, the second method was fit to analyze the effects of the extraction acted by P450 isoenzymes induced.Omeprazole reported at the dose 400mg/kg 501A isoenzyme. The toxicity of the extraction when combined with omeprazole with the second method wasAbstract 7significantly reduced compared to that of the extraction alone. The value ofurea nitrogen (BUN) and creatine(Cr) in the serum of the vital mice reduced , too. We presumed that the CYP4501A enzyme might be involved in the metabolism of aristolochic acid and reduce the renal toxicity of aristolochic acid in mice. And likewise phenobarbital sodium combined with the extraction with the second method could reduce the toxicity of the extraction. Rifampicin could induce CYP450 3A4 isoenzyme. The toxicity of the extraction was increased when combined with rifampicin with the second method. That suggested CYP450 3A4 isoenzyme might increase the toxicity of aristolochic acid. 2 Cytochrome P450 isoenzymes induced and its effect on manshuriensis extraction.The rats were treated with GMT(1. 1, 2. 2g/kg) combined with phenobarbital sodium, rifampicin, omeprazole respectively with the second method, The renal function was examined and the cytochrome P450 enzymes in liver microsome was assayed to analyze whether the interaction between the inducer and GMT existed , and to analyze the relationship between the induced cytochrome P450 enzymes and the toxicity of GMT. After the rats were treated for one week, the GMT (2. 2g/kg) group showed renal function damage: 1. The value of urea nitrogen (BUN)and creatine(Cr) in the serum increased significantly;2. Renal tubular epithelial cells degenerated and got necrosis. Although the value of urea nitrogen (BUN) and creatine(Cr) in the serum of GMT (1. lg/kg) group did not increase significantly, hydropic degeneration still happened in renal tubular epithelial cells over 50% rats. The toxic effect in the female was more serious than that in the male. No significant renal damage was found in PB, 0M and RFP groups afer treated for one week. The toxic effect was more serious in the GMT combined with PB, RFP respectively groups, the value of BUN and Cr in serum raised significantly , and the renal pathologic changes were more obviously than those in GMT alone groups. In GMT combined with 0M group the value of BUN and Cr in serum decreased , but still beyond normal range. This result indicated that the degree of renal pathologic changes were lightened.2.1 The variation of the renal toxicity between the male and the female was due to the the variation of the activity of the CYP enzymesNADP: P450 reductase and CYP isoenzymes are relevant to the drug metabolism. In the procedure of assaying the activity of the AMD, EDM and 7-EORR enzymes, the activity of the CYP2E, CYP3A4 and CYP1A enzyme were reflected respectively.The CYPs activity of the control group showed some difference between female and male rats, the activity of the totle CYP enzymes, CYP3A and CYP1A in male was higher than in female. The treatment with GMT alone had shown different renal toxicity between the male and the female, too. and the toxicity in female was more serious. So we concluded that the variation of the renal toxicity was due to the variation of the activity of the CYP enzymes. 2. 2 Different CYP450 enzymes induced could cause different effect on the toxicity of GMTThe result of the renal toxicity after GMT combined with the inducers to the rats had shown that RFP could increase the renal toxicity of GMT but OM reduce the toxicity. There was no significant change found in the toxicity in the rats treated with PB combined with GMT.2. 3 The relationship between the cytochrome P450 enzymes and the renal toxicity of GMTThe activity of CYP3A4 isoenzyme in the female rats were induced by RFP and the activity of CYP1A isoenzyme in the male by OM. The activity of other P450 enzymes was induced to different levels. The renal toxicity of GMT affected by RFP and OM was completely distinct. RFP cause the toxicity of GMT increased, but OM inversely.By analyzing the activity of the P450 enzymes induced by RFP and OM we inferred that CYP3A4 was the primary enzymes related to the increased renal toxicity of GMT. PB could induce the totle P450 enzymes and NADPH:P450 reducase, NADPH:P450 reducase was concerned with the toxicity. OM could induce CYP1A enzyme, but in the present experiment we could not identify the enzyme related to the reduced renal toxicity.3 The changes of cytochrome P450 enzymes expression in hepatic cells in miceNo CYP2E were found expressed in hepatic cells in each group. But CYP1AK CYP3A4n NADP:P450 reductase had expressed in different levels . The distribution of the positive cells was regular. The positive cells distribute around the hepatic lobule central vein, interlobular vein and some portal area. The result that CYP1AK CYP3A4> NADP:P450 reductase protein expression increased in different degree in the groups treated with GMT indicated that GMT was the inducer for these enzymes, when the aristolochic acid combined with other drugs, we should consider that the drug interaction affected the other drugs.Discussionwe concluded that the individual variation in the activity of the CYP enzymescaused the individual variation in renal toxicity. This result is in accord with the actuality in clinicl. Based on uncomplete statistic the patients who had taken the medicine containing aristolochic acid the incident rate of AAN was about 5%. However some patients who had taken the medicine containing aristolochic for long time , there was no renal toxicity found. P450 enzymes could be induced or inhibited by drugs and produce the interaction of drugs. The CYPs activity showed some difference between female and male rats, the activity of the totle CYP enzymes, CYP3A and CYP1A in male was higher than that in female. The GMT alone group had shown different renal toxicity between the male and the female, too. and the toxicity in female was more serious. Some result was not concordance between the female and the male in the present study, we will have the advanced research to infirm these result. In a word, it is important to identify the enzymes involved in the metabolism of the aristolochic acid and to analyze the effects of the enzymes on the renal toxicity of GMT. Which would guide the doctor to prescribe the drugs reasonably and safely.更多还原