【作者】 汤喜兰；

【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， 2008， 硕士

【摘要】 自1993年,比利时Vanherweghem报道服用含马兜铃酸（aristolochic acid,AA）成分的减肥药导致肾脏损害和肿瘤后,含马兜铃酸类中药引起马兜铃酸肾毒性问题便逐渐成为国内、外医药界普遍关注的研究热点。为避免此类药物不良反应的再次发生,美国FDA已从2001年起禁止含马兜铃酸的产品进入美国市场,同时禁止使用可能与含马兜铃酸类中药混淆的品种。国际上还有很多国家禁止进口含马兜铃酸的中药材。国内于2003、2004年也先后发文取消了关木通、青木香、广防已的药用标准,并限制含有马兜铃酸成分的中药及其相关制剂的使用。然而我国仍然还有多种含马兜铃酸成分的中药继续在临床使用,如马兜铃、天仙藤、朱砂莲等。虽然这些品种含的马兜铃酸含量较低,但是由于目前对于马兜铃酸的安全剂量仍然不清楚,因此,深入探讨马兜铃酸毒性机制,搞清楚马兜铃酸毒性的影响因素是非常重要的。临床服用含马兜铃酸中药的人群中,只有约5%出现肾脏毒性。这一现象提示,不同的人对马兜铃酸的敏感性可能存在差异。由于人类的重要药物代谢酶—肝脏细胞色素P450酶存在着基因多态性,不同个体的某些P450同工酶的含量和活性存在很大的差异,因而代谢药物的能力有所不同。马兜铃酸肾脏毒性在不同个体间的差异是否与肝脏代谢酶差异有关,值得深入探讨。此外,有很多药物可影响某些P450同工酶活性,而临床上普遍存在着多种药物联合用药的情况,尤其是化学药与中药合用的情况越来越多。这些具有P450同工酶诱导或抑制作用的药物在与含马兜铃酸中药合用时,是否会影响马兜铃酸的毒性,也值得深入研究。目的采用体外细胞培养系统和整体动物试验系统,研究肝脏微粒体细胞色素P450同工酶对马兜铃酸毒性的影响,以筛选和确定影响马兜铃酸毒性的主要细胞色素P450同工酶,为临床安全用药以及寻找预防马兜铃毒性的方法提供参考。方法第1章:影响马兜铃酸肾小管细胞毒性的细胞色素P450同工酶（CYP450）的体外筛选本实验选择了几种典型的细胞色素P450同工酶抑制剂,主要包括:二乙基二硫代氨基甲酸钠（sodium diethyldithiocarbamate,CYP4502A6和2E1的抑制剂）,奎尼丁（quinidine,CYP4502D6的抑制剂）,α-硫辛酸（α-lipoic acid,NADPH.P450还原酶的抑制剂）,α-萘黄酮（α-naphthoflavone,CYP4501A1和1A2的抑制剂）,磺胺苯吡唑（sulfaphenazole,CYP4502C的抑制剂）,酮康唑（ketoconazole,CYP3A4的抑制剂）。以体外培养的人类肾小管上皮细胞（HK-2）作为检测AA致肾小管毒性的体外模型,用肝脏微粒体混合酶系（S9）加入到HK-2细胞培养中模拟体内代谢环境,将CYP450同工酶的典型抑制剂,分别加入培养体系中,造成不同P450同工酶活性抑制状况,通过细胞增殖抑制率（MTT）试验和LDH释放试验来检测AA单独用药或AA与抑制剂联合用药情况下的细胞毒性。本实验采用5×4×2的析因实验设计,初步筛选影响AA毒性的CYP450亚型。第2章:CYP450 1A2、CYP450 3A4重组酶对马兜铃酸肾小管细胞毒性的影响以前述初步筛选结果为基础,继续在HK-2细胞模型上,进一步验证纯化的细胞色素P450同工酶（CYP1A2,CYP3A4重组酶）对AA肾小管细胞毒性的影响。本实验采用4×4析因实验设计进行体外联合用药,其中AA设3个浓度。CYP1A2或CYP3A4重组蛋白设3个浓度。第3章:不同的细胞色素P450（CYP450）酶诱导剂（苯巴比妥钠、利福平、奥美拉唑）与马兜铃酸联合用药对马兜铃酸致小鼠毒性的影响在体外试验结果的基础上,采用小鼠体内试验,进一步验证并确定影响马兜铃酸毒性的主要细胞色素P450同工酶。本实验选择了几种典型的细胞色素P450同工酶诱导剂—苯巴比妥钠（PB,CYP总酶诱导剂）、利福平（RFP,CYP3A诱导剂）、奥美拉唑（OM,CYP1A诱导剂）。将小鼠随机分为20个试验组,包括:对照组,AA单独用药的4个剂量组(60 mg·kg^-1、48 mg·kg^-1、38.4 mg·kg^-1和30.72mg·kg^-1),PB单独用药组,PB+AA4个剂量组,RFP组,RFP+AA4个剂量组,OM组,OM+AA4个剂量组。实验前将动物禁食16 h,自由饮水:预先3天对照组和马兜铃酸组给予0.5%羧甲基纤维素钠10 mL·kg^-1,诱导组分别给予诱导剂PB 40 mL·kg^-1,RFP 40 mL·kg^-1,OM 200 mL·kg^-1,第3天禁食;第4天先给予诱导剂,3 h后给予马兜铃酸,给完药后,给食给水。连续观察14天,观察动物一般情况,每2天称1次体重,观察给药组动物的体重变化情况;死亡动物解剖检查,查看各脏器情况,并且记录死亡情况计算LD₅₀;观察期结束后,动物禁食16 h后,称禁食后体重,取血测定CRE、UREA,同时取出小鼠肝、胃、肾,计算脏器指数。结果第1章:影响马兜铃酸肾小管细胞毒性的细胞色素P450同工酶（CYP450）的体外筛选实验结果表明:肝脏微粒体总酶（S9）加入到HK-2培养体系中后,对AA所致的肾小管细胞毒性具有一定程度的降低作用。与AA单独用药组相比,AA+S9组的细胞增殖抑制率降低,LDH释放率变化百分数减少,二者有统计学差异（P＜0.05）。MTT检测和LDH释放试验发现,二乙基二硫代氨基甲酸钠、奎尼丁、α-硫辛酸和磺胺苯吡唑所形成的抑制效应对AA的肾小管细胞毒性可能影响不明显。而酮康唑在无明显细胞毒影响的情况下,与AA+S9组相比,AA+酮康唑+S9组可使AA抑制HK-2细胞增殖的作用明显增强（P＜0.05）,抑制的相对倍数增大,表明酮康唑可能通过抑制CYP4503A4酶活性而对AA的细胞毒性具有增强作用。同时本研究结果也显示,在α-萘黄酮略有些细胞毒影响的情况下,与AA+S9组相比,AA+α-萘黄酮(12.5μg·mL^-1)+S9组LDH释放率增高,释放变化的相对倍数增大,二者有显著性差异（P＜0.05）。表明α-萘黄酮可能通过抑制CYP4501A酶活性而影响AA的细胞毒性作用。从而初步筛选得出CYP4503A,CYP4501A是影响AA的代谢活化的主要同工酶。第2章:马兜铃酸与CYP4501A2、CYP4503A4重组蛋白联合对AA肾小管细胞毒性的影响实验结果提示:与同等浓度的单独AA组相比较,AA与重组蛋白CYP3A4联合用药后LDH释放率变化百分数均降低。与同等浓度的单独AA组相比较,AA+CYP1A2(97.5 pmol·mL^-1)联合用药时LDH释放率的变化百分数增高,释放相对倍数＞1,而AA+CYP1A2(25 pmol·mL^-1)联合用药组LDH释放率的变化百分数降低,释放相对倍数＜1。实验结果表明:CYP3A4和CYP1A2是影响AA致HK-2细胞毒性的主要的CYP450亚型,CYP3A4能降低马兜铃酸对HK-2细胞的毒性,而CYP1A2活性较高时可能增加AA马兜铃酸对HK-2细胞的毒性,活性较低时可能降低AA马兜铃酸对HK-2细胞的毒性。第3章:不同的细胞色素P450（CYP450）酶诱导剂（苯巴比妥钠、利福平、奥美拉唑）与马兜铃酸联合用药对马兜铃酸致小鼠毒性的影响采用Bliss法计算LD₅₀,AA单独组的LD₅₀为55.01 mg·kg^-1。经PB诱导的小鼠,再服用AA后其毒性明显降低,可使AA导致的小鼠死亡率降低,死亡时间延迟,表明体内肝脏CYP450总酶的诱导可明显减轻毒性。经RFP诱导后,给小鼠灌胃给予AA,AA的LD₅₀为33.82 mg·kg^-1,比AA单独用药的LD₅₀值(55.01 mg·kg^-1)降低了约38.5%。结果提示CYP450 3A4的诱导可增强AA毒性。经OM诱导后,给小鼠灌胃给予AA,AA的LD₅₀为56.22 mg·kg^-1,与AA单独用药组的LD₅₀值(55.01 mg·kg^-1)相差不明显,表明CYP450 1A的诱导对AA毒性无明显影响。结论1.肝脏细胞色素P450（肝脏微粒体）总酶活性增高可以在一定程度上降低马兜铃酸毒性2.不同的细胞色素P450酶亚型对马兜铃酸肾脏毒性的影响有所不同,其中细胞色素P4503A4活性增高可能增强AA的毒性。本研究结果提示:某些具有肝P450酶诱导或抑制作用的药物与含马兜铃酸中药联合用药时可能影响马兜铃酸毒性。故临床联合用药应注意联合用药的安全性。此外,不同个体体内肝脏P450酶活性的差异可能造成对AA敏感性的不同,从而在服用含AA药物后表现出毒性差异。更多还原

【Abstract】 Since the report from Belgium’s Vanherweghem that taking the weight loss drug containing aristolochic acid composition led to kidney damage and cancer in 1993,aristolochic acid renal toxicity problems caused by chinese medicine containing aristolochic acid has become hotspot in the domestic and foreign medical research fields.To avoid such adverse drug reactions from happening again,the FDA has already banned products containing aristolochic acid to enter the american market and also has prohibited the use of medicine containing aristolochic acid confused species since 2001.Many countries in the international also has banned on imports of chinese herbal medicines containing aristolochic acid.The commissioner of china has issued a document to abolish the medicinal standards of aristolochia manshuriensis,Green costas,widely Menispermaceae and to restrict the use of chinese medicine and related agents containing aristolochic acid component in 2003,2004.However,in our country, there are still a variety of chinese medicines containing aristolochic acid using in clinical,such as aristolochic,Tianxian rattan,cinnabar-lian and so on.Although the content of aristolochic acid in these varieties is low,the security dose of aristolochic acid is still unclear at present,therefore,it is very important to study the toxicity mechanism of aristolochic acid in-depth and to understand the factors which impacts the toxicity of aristolochic acid.In the so-called "herbal nephropathy" incident,the incidence rate of aristolochic acid nephropathy was 5%.This showed that different groups of people had different sensitivity to aristolochic acid.Because of the genetic polymorphism for the important drug metabolizing enzymes in human（the liver cytochrome P450 enzyme）,some CYP450 isozymes of different individuals has a lot of difference,thus the ability of drug metabolism for different individual is different.Whether the differences in aristolochic acid kidney toxicity among different individuals relate to the differences in CYP450 isozymes or not,which will be worth to study in-depth.Furthermore,there are many drugs that can affect the activity of some CYP450 isozymes.Moreover,the combinations of multi-drugs are widespread in clinical,in particular,the combination of chemical medicine with chinese medicine are used more and more.Whether the kidney toxicity of aristolochic acid will change when chinese medicine containing aristolochic acid combined with those drugs which can inhibit or induce CYP450 isozymes.It is worthy of study in depth.This study funded by the Beijing Municipal Science（issue No.:7052058）.ObjectiveBy using cell culture technology in vitro and and animals as experimental methods,to investigate the effects of cytochrome P450 isozymes on aristolochic acid renal toxicity and to screen the major CYP450 isozymes that impact AA renal toxicity,which will provide reference for the safety of clinical drug-using and to find ways to prevent aristolochic renal toxicity.MethodsChapter 1:CYP450 isoforms that participate in the drug metabolism of aristolochic acid were screened by CYP450 enzymes inhibitory effect in vitroWe chose several of classic inhibitors of CYP450 isoenzymes,including sodium diethyldithiocarbamate（CYP4502A6 and 2E1 inhibitors）,quinidine（CYP4502D6 inhibitor）,α-lipoic acid（NADPH:P450 reductase inhibitor）,α-naphthoflavone（CYP4501A1 and 1A2 inhibitors）,α-lipoic acid（CYP4502C inhibitor）,ketoconazole（CYP3A4 inhibitor）.Aristolochic acid renal toxicity was tested by using human renal epithelial cells （HK-2） as model in vitro.The metabolism environment in vivo simulated by adding the liver microsomal mixed enzymes（S9） into HK-2 cell culture system.The inhibitors were added into HK-2 cell culture system respectively to form different activity inhibition situation for CYP450 isoenzymes.The cytotoxicity of AA alone and the combination of AA with inhibitors were determined by inhibition of cell proliferation（MTT） test and LDH release test.In this experiment,we used 5×4×2 factorial experimental design to initially screen CYP450 subtypes that impacted AA renal toxicity.Chapter 2:The effects of combination of aristolochic acid with recombinant protein CYP4501A2/CYP4503A4 on AA renal cytotoxicityAccording to the preliminary screening results in chapter 1,we will further verify the effects of the purification CYP450 isozymes（CYP1A2 and CYP3A4 enzymes） on aristolochic acid renal cytotoxicity.In this experiment,we used 4×4 factorial experimental design with AA three concentrations,CYP1A2/CYP3A4 three concentrations and detected that through LDH release test.Chapter 3:The combination of aristolochic acid with different cytochrome P450（CYP450） inducers（phenobarbital,rifampicin,omeprazole） effects AA acute toxicity.Based on the experimental results in vitro,mice experiment in vivo were carried out to verify and identify the major CYP450 isozymes that effects aristolochic acid renal toxicity.we selected three CYP450 isozymes inducers—phenobarbital（PB）（CYP450 inducer）,rifampicin（RFP）（CYP3A inducer）,omeprazole（OM）（CYP1A inducer）.206 mice were randomly divided into 20 groups,namely the control group,that groups of four AA concentration,including AA1(60 mg·kg^-1),AA2(48 mg·kg^-1), AA3(38.4 mg·kg^-1),AA4(30.72 mg·kg^-1),the PB group,the PB+AA four concentration’s groups,the RFP group,the RFP+AA four concentration’s groups,the OM group,the OM + AA four concentration’s groups.The experiment began after the mice were fasted for 16 hours,free of drinking.For the first three days,the normal control group and the aristolochic acid group were given 0.5%sodium carboxymethyl cellulose 10 mL·kg^-1,the induction groups were given inducers as PB 40 mg·kg^-1,RFP 40 mg·kg^-1,OM 200 mg·kg^-1 respectively.The mice were fasted on the third day.On the fourth day,three hours after giving the inducers,the aristolochic acid were given and the fresh water and food were supplied.During the experiment,general conditions of mice had been observed for 14days.Weighing body weight in mice once every two days to observe drug-treated animals’ weight changes.The changes in the various organs of the dead animals were observed and death records were maken.After a period of observation,the mice were weighed body weight after fasting for 16 hours.The blood was gathered for determination of CRE、UREA and the liver,stomach,kidney were removed for the calculation of organ index.ResultsChapter 1:CYP450 isoforms that participate in the drug metabolism of aristolochic acid were screened by CYP450 enzyme inhibitory effect in vitroThe experimental results showed that:adding liver microsomal enzymes（S9） into the HK-2 culture system,AA cytotoxicity on renal tubular cell had reduced to a certain extent.Compared with AA group,cell proliferation rate and the percentage changes in LDH release rate of AA + S9 group decreased,there is a significant difference between them（P＜0.05）.MTT assay and LDH release found that the inhibiting effect formed by sodium diethyldithiocarbamate、quinidine、α-lipoic acid and sulfaphenazole may be not obvious on the AA cytotoxicity.However,under the circumstances of adding ketoconazole without obvious cytotoxicity into the HK-2 culture system,there was a significant difference between AA+ketoconazole+S9 group and AA+S9 group.Compared with AA+S9 group,AA+ketoconazole+S9 can significantly enhanced the proliferation inhibition of aristolochic acid on HK-2 cell（P＜0.05）,the relative multiples of inhibition increased.The results showed that ketoconazole maybe influence AA cytotoxicity through inhibiting the activity of CYP3A4.At the same time,the result of this study also showed that under the circumstances of addingα-naphthoflavone with low cytotoxic effects into the HK-2 culture system,there was a significant difference between AA+α-naphthoflavone + S9 group and AA+S9 group.Compared with AA+S9 group,AA+α-naphthoflavone +S9 can significantly increased LDH release rate of aristolochic acid on HK-2 cell（P＜0.05）,the relative multiple of LDH release changes increased.Experimental results indicated that:α-naphthoflavone maybe influence AA cytotoxicity through inhibiting the activity of CYP1A2.So we got preliminary screening results:CYP4503A, CYP4501A were the major CYP450 isozymes that affected the metabolic activation of AA.Chapter 2:The effects of the combination of aristolochic acid with recombinant protein CYP4501A2/CYP4503A4 on AA renal cytotoxicityThe experimental results showed that:compared with the same concentration of AA groups,the LDH release rate percentage changes of the groups that AA combined with recombinant protein CYP3A4 reduced.Compared with the same concentration of AA groups,the LDH release rate percentage changes of the groups that AA combined with recombinant protein CYP1A2(97.5 pmol·mL^-1) increased,with release relative multiples＞1,but the LDH release rate percentage changes of the groups that AA combined with recombinant protein CYP1A2(25 pmol·mL^-1)reduced,with release relative multiples＜1.The experimental results showed that:CYP3A4 and CYP1A2 were the major CYP450 isoforms that affected HK-2 cell toxicity caused by AA.CYP3A4 could reduce HK-2 cell toxicity;CYP1A2 with high activity maybe increase HK-2 cells toxicity and CYP1A2 with lower activity maybe reduce HK-2 cells toxicity.Chapter 3:The effects of the combination of aristolochic acid with cytochrome P450（CYP450） inducers（Phenobarbital,rifampicin,omeprazole） on AA acute toxicity.The determination of LD₅₀ was calculated by bliss method,the LD₅₀ of the AA alone group was 55.01 mg·kg^-1.The toxicity of the PB-induced mice significantly decreased after its taking AA.the induction of PB reduced the mortality rate in mice and delayed the death of the mice.The results suggested that the induction for CYP450 enzymes in the liver caused by PB can significantly reduce the toxicity.Compared with the LD₅₀ of AA alone group,the LD₅₀ of RFP + AA group was 33.82 mg·kg^-1,which decreased by about 38.5 percents.The results showed that the induction for CYP3A4 enzyme caused by RFP significantly enhanced the AA kidney toxicity.The LD₅₀ of AA + OM group was 56.22 mg·kg^-1.compared with the LD₅₀ of AA alone group,there was not obvious difference.The results showed that the induction for CYP1A enzyme caused by OM had no obvious effect on AA kidney toxicity.Conclusion1.the increasing activity of the total cytochrome P450 can reduce the toxicity of aristolochic acid to a certain extent.2.different cytochrome P450 enzymes have different effects on aristolochic acid renal toxicity,and the increasing activity of cytochrome P4503A4 may increase aristolochic acid toxicity.The results of this study suggested that the combination of chinese medicine containing aristolochic acid with drugs that can induce or inhibit some CYP450 enzymes maybe impact aristolochic acid toxicity.Therefore,the safety of drugs combined should be noted.Furthermore,CYP450 enzymes activity from different individuals are different,which may cause the AA sensitivity different and show differences in toxicity after taking drugs containing aristolochic acid.更多还原