【作者】 张志华；

【导师】 侯艳宁；

【作者基本信息】 河北医科大学 ， 药理学， 2009， 博士

【摘要】 结核病是由结核分支杆菌感染引起的疾病,最早以肺结核病多见,近年来肺外结核菌感染及不典型结核病也较常见。结核病在世界范围内仍是发病率和死亡率最高的疾病之一。世界卫生组织(WHO)预计,如果得不到有效的治疗,今后10年内全球至少将有3000万人死于结核病。在我国,仅2008年1-9月就新发结核病77万人,因此目前抗结核治疗形势仍然十分严峻。在结核病的治疗方案中,异烟肼(isoniazid,INH)和利福平(rifampicin,RFP)仍是世界卫生组织推荐的、不可替代的一线用抗结核药。异烟肼通过抑制结核杆菌细胞所特有的分枝菌酸(mycolic acid)的合成,使细菌丧失耐酸性、疏水性和增殖力而死亡;利福平则可以抑制细菌RNA多聚酶,阻碍mRNA合成,从而达到抑菌和杀菌作用。二者对于繁殖期和静止期状态的细菌均有较强的杀菌、抑菌作用,联合应用对杀死细胞内外结核杆菌有协同效果并可减少耐药性,但两药合用时肝脏毒性发生的几率却明显增加,有时甚至发生急性肝脏衰竭危及生命。虽然临床抗结核药物肝脏损伤现状仍十分严重,抗结核药物致肝损伤的机制却尚未阐明,因此对抗结核药物所致肝脏损害的机制进行更深入的研究就显得尤为重要。研究认为异烟肼和利福平合用时,二者在体内的代谢转化过程改变,其特定代谢物水平升高是导致肝毒性的重要因素。肝脏细胞色素P450酶(cytochrome P450,CYPs)是催化异烟肼和利福平在体内代谢的主要酶类。目前已知的CYPs的种类超过1000多种,人肝脏中CYP 3A4的含量最丰富,约占其CYPs总量的40%,并参与50%以上常用药物种类的代谢。CYP 2E1在肝脏中的含量只占其中CYPs总量的7%左右,也只代谢2%左右种类的常用药物。但这两种酶均在异烟肼和利福平合用后其代谢产物乙酰肼和/或肼生成增加、肝毒性产生过程中发挥关键作用。本课题组在前期工作中利用小鼠在体和原代培养的小鼠肝细胞体外实验研究了异烟肼和利福平单/合用及其代谢物对肝细胞的毒性作用,探讨了利福平与异烟肼合用肝毒性增加的发生机制,并对阿魏酸钠、黄苓苷、水飞蓟宾等具有保肝作用的中药成分对抗抗结核药物致肝损害的保护作用进行了研究。本课题首先观察结核病患者血浆异烟肼及其代谢物水平的变化,并以人肝细胞株QSG-7701为对象,研究异烟肼及其代谢物及异烟肼和利福平合用对肝细胞的损伤和毒性作用,探讨细胞色素酶P450同工酶CYP 3A4、CYP 2E1在异烟肼、利福平所致肝毒性中的作用;筛选抗结核药物异烟肼、利福平所致肝毒害的特异性保护药物并探讨其作用机制。旨在阐明异烟肼和利福平合用导致肝毒性增加的机制,探索有效抑制其毒性的药物,为制定抗结核用药的新策略奠定基础。方法1受试者入选与分组给药遵照《人体生物学医学研究国际道德指南》规定的原则选择受试者,并填写《患者知情同意书》。将24名入选的肺结核患者,随机分为异烟肼组、利福平组及异烟肼和利福平合用组。异烟肼组晨起空腹口服0.3 g异烟肼一次,早晚空腹口服0.2 g左氧氟沙星各一次,早中晚空腹口服0.1 g葡萄糖醛酸内酯各一次;利福平组晨起空腹口服0.45 g利福平一次,早晚空腹口服0.2 g左氧氟沙星各一次,早中晚空腹口服0.1 g葡萄糖醛酸内酯各一次;异烟肼和利福平合用组除服用与异烟肼组相同的药物外,晨起空腹口服0.45 g利福平一次,连续服药两周。2血样采集与处理方法患者连续服药两周后,即第15天晨起服药前及服药后2 h、4 h、12 h采用抗凝管收集肘静脉血5 mL,3500 rpm离心10 min,取血浆分装两份,于-70℃低温冰箱保存,备异烟肼及其代谢物浓度检测;分别于开始抗结核前和用药两周后晨起空腹抽取肘静脉血5 mL,检测谷丙转氨酶、谷草转氨酶和总胆红素水平。3 QSG-7701肝细胞培养方法利用QSG-7701肝细胞株研究异烟肼和利福平对离体肝细胞的毒性。将冻存于液氮中的QSG-7701肝细胞于37℃水浴中迅速解冻,离心弃上清后,沉淀加入含10%胎牛血清的高糖DMEM培养基,用吸管吹打,使成单细胞悬液,用台盼蓝染色后,显微镜下用计数板计数,并接种于70 mm培养皿中培养,接种密度为1×105 cells/mL,体积5 mL,于CO2培养箱中培养24 h后换全液,以后隔天换液,待细胞长满后吸出培养液,加入1 mmol/L的EDTANa2清洗,吸弃EDTANa2溶液后,用0.25%的胰酶消化液消化,加入培养液并用吸管吹打使成单细胞悬液,进行传代。用于加药处理的细胞,接种于培养板。4 MTT法检测细胞存活率向培养板培养的细胞培养液中加入MTT溶液使其终浓度为0.05%,继续培养4 h后吸弃培养液,每孔加入DMSO 150μL,振荡使甲瓒结晶溶解,以空白孔调零,用读板机或分光光度计读取570 nm波长处的吸光度值,以空白对照组吸光度的均值为100%,用各样本吸光度值/空白对照组吸光度的均值表示该样本细胞的存活率。5细胞培养液及细胞裂解液乳酸脱氢酶活性的测定药物处理完毕后,分别收集各组细胞培养上清液,-80℃冻存备测。将细胞用生理盐水冲洗一遍后,每孔加入生理盐水1 mL,用细胞刮刀将细胞刮下,将细胞悬液2000 rpm离心5 min,倾弃上清后,沉淀加入0.5 mL细胞裂解液,4℃下裂解30 min,12000 rpm离心20 min,收集上清-80℃冻存备测。用乳酸脱氢酶测试盒,比色法测定440 nm样品的吸光度值,并计算乳酸脱氢酶的活力。用细胞培养液中酶活力与细胞裂解液中酶活力的比值代表药物作用对细胞损伤及乳酸脱氢酶活力的影响。6 HPLC-MS法测定血浆中异烟肼及其代谢物乙酰肼和肼的浓度取血浆样品0.1 mL,依次加入内标溶液20μL,甲醇0.2 mL,涡旋混合3 min,4000 r·min-1离心10 min,取上清液0.2 mL,加入衍生化溶液10μL,室温下暗处反应24 h,取20μL用于HPLC-MS法测定。采用高效液相色谱质谱联用法测定人血浆中异烟肼、乙酰肼和肼的浓度。所采用的色谱方法为:Agilent C18分析柱(5μm,2.1mm×150 mm ID),Agilent SB C18保护柱(5μm),柱温40℃,流动相为甲醇-水(含0.1%冰醋酸),流速为150μL/min。质谱检测采用电喷雾(ESI)离子化源,毛细管电压4.0 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;正离子方式检测,碎片电压为分别为190V、130V、150V;选择离子监测(SIM)模式,用于定量分析的离子分别为m/z 256.1(异烟肼衍生物)、m/z 193.2(乙酰肼衍生物)、m/z 151.1(肼衍生物)。7 HPLC-MS法测定细胞孵育液中咪达唑仑和对硝基儿茶酚的浓度色谱条件:色谱柱:Gemini C18分析柱(2.0×50 mm,5μm),Phenomenex公司;C18保护柱(5μm,3.0×4.0 mm),Phenomenex公司;流动相:乙腈-水(含0.2%冰醋酸)=60:40;流速为0.2 mL·min-1;柱温为30℃;进样量:10μL。质谱条件:电喷雾(ESI)离子化源,选择离子监测(SIM)模式。对硝基儿茶酚检测条件:毛细管电压2 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;负离子方式检测,碎片电压为150 V,m/z [154.0]-(对硝基儿茶酚)。咪达唑仑:毛细管电压3 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;正离子方式检测,碎片电压为50 V,m/z [326.0]+(咪达唑仑)。8分组及加药处理8.1异烟肼及其代谢物和利福平对细胞存活率的影响细胞接种于96孔板,接种密度为每孔1×104 cells,将细胞分为空白对照组、利福平组、异烟肼组、乙酰肼组、肼组和利福平+异烟肼组,每组6孔。于接种后培养24 h时分别换含有空白溶剂、利福平(100μg/mL,200μg/mL,300μg/mL)、异烟肼(250μg/mL,500μg/mL,2500μg/mL)、乙酰肼(125μg/mL,250μg/mL,1250μg/mL)、肼(0.25μg/mL,2.5μg/mL,25μg/mL)、利福平+异烟肼(100+50μg/mL,200+100μg/mL,300+150μg/mL)的含药培养液,培养箱中培养48 h后采用MTT法测定细胞存活率。8.2异烟肼及其代谢物和利福平对细胞乳酸脱氢酶释放的影响将细胞接种于6孔培养板,接种后24 h换含药培养液培养48 h,分组及加药方法同“8.1”项下操作,收集培养液和细胞用于乳酸脱氢酶活性的测定。8.3异烟肼和利福平对细胞CYP 2E1和3A4活性的的影响将细胞分为8组,每组6孔,分别加入含有溶剂、异烟肼100μg/mL、利福平200μg/mL、异烟肼100μg/mL和利福平100μg/mL的培养液,各两组。继续培养48 h后,吸弃含药培养液,分别加入含有CYP 2E1作用底物对硝基酚(15μg/mL)或CYP 3A4作用底物咪达唑仑(1μg/mL)的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h,吸出培养液,用HPLC-MS法测定对硝基酚代谢产物对硝基儿茶酚和咪达唑仑的水平,用以代表各组细胞CYP 2E1和3A4的活性。8.4 CYP 2E1和3A4抑制剂对异烟肼和利福平合用增加细胞乳酸脱氢酶释放的影响将培养板中的细胞分为14组,分别为空白对照组、异烟肼和利福平合用组、柑桔素组、红霉素组、阿魏酸钠组和三七皂苷组,每组6孔。各组分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL)的培养液继续培养48 h。柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加入CYP 3A4抑制剂柑桔素(1μg/mL、5μg/mL、25μg/mL)和红霉素(1μg/mL、5μg/mL、25μg/mL);阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时分别加入CYP 2E1抑制剂阿魏酸钠(10μg/mL、50μg/mL、250μg/mL)和三七皂苷(4μg/mL、20μg/mL、100μg/mL)共同培养。收集细胞培养上清液和细胞,检测乳酸脱氢酶的活性8.5 CYP 2E1和3A4抑制剂对异烟肼和利福平合用降低细胞存活率的影响将接种于24孔培养板的细胞分为8组,每组6孔,于接种后24 h吸弃培养液,分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL)的培养液继续培养48 h。柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加入CYP 3A4抑制剂柑桔素(5μg/mL)和红霉素(5μg/mL);阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时分别加入CYP 2E1抑制剂阿魏酸钠(50μg/mL)和三七皂苷(20μg/mL)共同培养。采用MTT法测定培养后细胞的存活率的变化。8.6酶抑制剂对细胞内CYP450 2E1和3A4酶活性的影响将接种于24孔培养板的细胞分为8组,每组6孔,各组分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL);柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加含CYP 3A4抑制剂柑桔素(5μg/mL)和红霉素(5μg/mL)的培养液继续培养48 h,吸弃含药培养液,加入含有CYP 3A4作用底物咪达唑仑1μg/mL的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h;阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时加入含CYP 2E1抑制剂阿魏酸钠(50μg/mL)和三七皂苷(20μg/mL)的培养液继续培养48 h,吸弃含药培养液,加入含有CYP 2E1作用底物对硝基酚15μg/mL的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h。孵育后吸出培养液,用HPLC-MS法测定咪达唑仑和对硝基儿茶酚的浓度。9数据处理与统计分析统计分析采用SPSS 11.5 for windows软件,数据以均数±标准差(mean±SD)表示,组间差异采用成组设计的t检验;多组间比较采用方差分析(ANOVA)继于Dunnett t test;量效关系采用二元变量相关分析,计算Pearson相关系数,统计结果以P<0.05为具有显著性差异。结果1肺结核病患者血浆异烟肼及其代谢物乙酰肼和肼浓度的变化经过对肺结核病患者服药后血浆中药物浓度的测定发现,与单用异烟肼组比较,异烟肼和利福平合用组肺结核病患者血浆中肼的浓度在服药后2 h即显著升高(P<0.05),在服药后4 h和12 h时仍显著高于单用异烟肼组(均为P<0.01)。与单用异烟肼组比较,异烟肼和利福平合用组患者血浆中异烟肼和乙酰肼的浓度未见显著性变化(P>0.05)。各组患者服药前后谷丙转氨酶、谷草转氨酶和总胆红素水平均未见异常(P>0.05)。2异烟肼、利福平及异烟肼代谢物乙酰肼和肼对细胞存活率的影响进一步对异烟肼、利福平及异烟肼代谢物乙酰肼和肼对离体肝细胞的毒性作用进行研究发现,与对照组比较,500μg/mL和2500μg/mL的异烟肼处理48 h,肝细胞的存活率均显著降低,其存活率分别为64.3% (P<0.01)和9.9% (P<0.01),异烟肼浓度与细胞存活率的Pearson相关系数为r = -0.959 (P<0.01);300μg/mL的利福平处理肝组细胞的存活率显著降低(56.2%, P<0.01);中、高剂量利福平和异烟肼合用组肝细胞存活率依次显著降低(分别为73.7%, P<0.05和33.3%, P<0.01),异烟肼和利福平合用浓度与细胞存活率的Pearson相关系数为r = -0.892 (P<0.01);2.5μg/mL和25μg/mL肼处理组肝细胞的存活率显著降低(分别为53.5%, P<0.05和46.0%, P<0.01)。结果表明,异烟肼和利福平合用的肝细胞毒性作用大于其单用,异烟肼代谢物肼对肝细胞的毒性作用最强。3异烟肼、利福平及其异烟肼代谢物对细胞乳酸脱氢酶活性的影响检测药物处理后肝细胞外/内乳酸脱氢酶活性的比值,反映细胞的损伤程度。与对照组比较,三种浓度的异烟肼处理后,细胞外/细胞内LDH的比值分别升高83.8%(P<0.01)、112.0%(P<0.01)和124.9%(P<0.01);300μg/mL的利福平处理使细胞外/细胞内LDH的比值显著升高50.3% (P<0.05);中、高浓度的利福平和异烟肼合用剂量依赖地使细胞外/细胞内LDH比值显著升高,升高幅度分别为76.2%(P<0.05)、210.8%(P<0.01)和1010.3% (P<0.01);异烟肼和利福平合用浓度与LDH比值的Pearson相关系数为r = 0.907 (P<0.01);三种浓度的肼处理后,细胞外/细胞内LDH比值分别升高73.3%(P<0.05)、102.1%(P<0.01)和115.5%(P<0.01);250μg/mL和1250μg/mL的乙酰肼处理后,细胞外/细胞内LDH比值分别升高121.1%(P<0.01)和153.1%(P<0.01);乙酰肼浓度与LDH比值的Pearson相关系数为r = 0.674 (P<0.01)。结果表明,异烟肼和利福平合用的对肝细胞的损伤作用大于其单用,异烟肼代谢物乙酰肼和肼均有损伤作用,其中肼对肝细胞的损伤作用最强。4异烟肼和利福平对肝细胞CYP 2E1和CYP 3A4活性的影响本实验关于异烟肼和利福平对肝细胞CYPs活性影响的结果显示,与对照组比较,单用异烟肼和异烟肼与利福平合用处理48 h,肝细胞CYP 2E1酶活性均显著升高(均为P<0.05);而单用利福平处理48 h,肝细胞CYP 2E1酶活性则未见明显变化(P>0.05);单用异烟肼和单用利福平处理48 h,分别使肝细胞CYP 3A4酶活性显著升高(P<0.05和P<0.01);异烟肼和利福平合用处理48 h,肝细胞CYP 3A4酶活性进一步升高(P<0.01)。表明异烟肼和/或利福平单用和合用均可上调细胞CYP 2E1和CYP 3A4的活性,二者合用更可进一步上调CYP 3A4的活性。5 CYP450 3A4抑制剂对异烟肼和利福平致肝细胞毒性作用的影响采用柑桔素或红霉素与异烟肼和利福平共同处理肝细胞发现,与异烟肼和利福平合用组比较,给予异烟肼和利福平处理的同时,加入柑桔素或红霉素共同培养,肝细胞中CYP 3A4的酶活性均显著低于异烟肼和利福平合用组(均为P<0.01);肝细胞的存活率显著升高(分别为95.2%和107.8%,均为P<0.01);中、高剂量的柑桔素和低、中、高剂量的红霉素均显著降低了细胞外和细胞内乳酸脱氢酶的比值(均为P<0.01),其中柑桔素浓度与乳酸脱氢酶比值的Pearson相关系数为r = -0.712 (P<0.01)。表明柑桔素和红霉素可以通过抑制异烟肼和利福平对CYP 3A4的诱导作用,减轻其对肝细胞的损伤。6 CYP450 2E1抑制剂对异烟肼和利福平致肝细胞毒性作用的影响采用阿魏酸钠和三七皂苷与异烟肼和利福平共同处理肝细胞发现,与异烟肼和利福平合用组比较,给予异烟肼和利福平处理的同时,加入阿魏酸钠或三七皂苷共同培养,肝细胞中CYP 2E1的酶活性均显著降低(均为P<0.01);阿魏酸钠组肝细胞的存活率显著升高(77.1%,P<0.05);中、高剂量的三七皂苷和低、中、高剂量的阿魏酸钠均显著降低了细胞外/内乳酸脱氢酶的比值(均为P<0.01),其中三七皂苷浓度与乳酸脱氢酶比值的Pearson相关系数为r = -0.659 (P<0.01)。表明阿魏酸钠和三七皂苷可以通过抑制异烟肼和利福平对CYP 2E1的诱导作用,减轻其对肝细胞的损伤。结论1.肺结核病患者使用异烟肼和利福平联合治疗时,血浆中异烟肼代谢物肼的浓度较单用异烟肼时显著升高,这可能是两药合用导致肝毒性增加的机制之一。2.采用体外肝细胞培养的方法进一步证明,不同剂量的异烟肼、利福平、肼及异烟肼和利福平合用能明显造成肝细胞损伤、显著降低肝细胞的存活率,其中异烟肼和利福平合用的肝细胞毒性强于其单用,异烟肼代谢物肼对肝细胞的毒性作用远大于其母体异烟肼,是异烟肼产生肝毒性的重要代谢产物。3.异烟肼和利福平单独或合用可不同程度地使肝细胞CYP 3A4和CYP 2E1的活性增加,推测此为异烟肼和其与利福平合用时异烟肼代谢产物水平升高,肝细胞存活率降低、损伤增加,乳酸脱氢酶向细胞外释放增加的机制之一。4.柑桔素和红霉素可以显著降低肝细胞内CYP 3A4的活性,阿魏酸钠和三七皂苷可以显著降低肝细胞内CYP 2E1的活性。不同浓度的柑桔素、红霉素、阿魏酸钠和三七皂苷均可抑制异烟肼和利福平合用致肝细胞存活率降低和/或乳酸脱氢酶释放增加的作用,具有对其肝细胞毒性的保护作用,对相应酶活性的调节可能是这些药物发挥作用的途径之一。更多还原

【Abstract】 Tuberculosis (TB) is one of the major causes of death from infectious disease. Recommended standard treatment by the World Health Organization (WHO) for adult respiratory TB is a regimen of isoniazid, rifampicin, and pyrazinamide for 2 months, followed by 4 months of isoniazid and rifampicin. The most frequent and most serious adverse effect of anti-tuberculosis treatment is hepatotoxicity. Evidence accumulated during the last years documented that cytochrome enzymes that mediate the metabolism of isoniazid play an important role in the development of hepatotoxicity. Moreover, the cytochrome enzymes CYP 3A4 and 2E1 are thought to mediate the hepatotoxicity. However, mechanisms that mediate anti-tuberculosis treatment induced hepatotoxicity are still not clear.Studies by others and our previous studies in mice suggested that hydrazine, the metabolite of isoniazed, was the main hepatotoxic compound. And drugs administered, such as sodium ferulate, baicalin and silibinin are protective in isoniazid and rifampicin exacerbated heptotoxicity.The present study was carried out to research the levels of isoniazid and its metabolites in the plasma of lung tuberculosis patients administered with isoniazid with or without rifampicin for two weeks. And the hepatocyte toxicity of isoniazid, its metabolites and rifampicin was investigated in cultured hepatocytes by using cell viability assays and lactate dehydrogenase release, a marker of cell death. Furthermore, the role of CYP 3A4, 2E1 and their inhibitors in the occurrence and preservation of isoniazid-rifampicin induced hepatoxicity was demonstrated.Methods1. Patients and drugs administrationTwenty-four lung tuberculosis patients were designated to three groups randomly. In the isoniazid group, patients were administered with isoniazid, levofloxacin and glucurolactone. In the rifampicin group, patients were administered with rifampicin, levofloxacin and glucurolactone. In the isoniazid and rifampicin group, patients were administered with isoniazid, rifampicin and glucurolactone.2. Plasma sample collection and disposalPlasma samples were collected before and 2 hours, 4 hours and 12 hours after the last administration of isoniazid and/or rifampicin following a two week duration of therapy and the samples were then stored at -70℃until HPLC-MS analysis. Alanine aminotransferase, aspartate aminotransferase and total bilirubin levels in serum were determined before and after therapy.3. Culture of QSG-7701 hepatocellularsThe QSG-7701 cell strain was cultured in DMEM culture media that contained high glucose at a cell density of 1×105 cells/mL. Pancreatin (0.25%) was used for trypsinization and sub-culturing.4. Cell viability assayThe MTT assay was used to evaluate the viability of the hepatocytes cultured in 96-well plates. Briefly, MTT was added to the culture media with a final concentration of 0.05%. After 4 hours of culture, the culture media was removed and 150μL DMSO was added to dissolve the crystals. The absorbance of the solution was read at 570nm on a spectrophotometer and the cell viability was calculated.5. Measurement of lactate dehydrogenase activity in cells and culture mediaThe cells in the culture plate were flushed with sodium solution and scraped off. After centrifugation, the cells were lyzed with lysis buffer that was incubated with the cells for 30 minutes. The resultant supernatant was used for the assay. Lactate dehydrogenase activity in cell lysates and culture media were determined using a lactate dehydrogenase activity kit and absorbance spectrophotometery. The ratio of extracellular and intracellular activity of lactate dehydrogenase was calculated.6. Determination of plasma isoniazid, acetylhydrazine and hydrazine concentrations using HPLC-MS Internal standard and methanol were added to plasma samples that were then vortexed for 3 minutes. After centrifugation of the samples, the supernatants were incubated with derivative agent for 24 hours and then analyzed with HPLC-MS using a 10μL on each HPLC run. An Agilent C18 analyze column (5μm, 2.1mm×150 mm ID) was used with an Agilent SB C18 guard column (5μm). The mobile phase consisted of methanol: water (containing 0.1% glacial acetic acid) and was pumped into the system at a rate of 0.15 mL·min-1. ESI ionization was used in the mass detection in selected ion monitoring. The ions selected for isoniazid, acetylhydrazine and hydrazine- were m/z [256.1]+, m/z [193.2]+ and m/z [151.1]+ , respectively.7. Determination of midazolam and 4-nitrocatichol concentrations in culture media using HPLC-MSA Gemini C18 analyze column (2.0×50 mm, 5μm) was used with a C18 guard column (5μm,3.0×4.0 mm). The mobile phase consists of acetonitrile: water (60:40, v/v, containing 0.2% glacial acetic acid) and was pumped in 0.2 mL·min-1. Ten milliliters of sample was analyzed on the column. ESI ionization was used in mass detection in selected ion monitoring. The ions selected for 4-nitrocatechol and midazolam- were m/z [154.0]- and m/z [326.0]+, respectively.8. Grouping and drug treatment8.1 Effect of isoniazid, its metabolites and rifampicin on cellular viabilityThe cells were plated in 96-well plates at a cell density of 1×104 cells per well. The cells were designated to sixteen groups with six wells in each group. The six treatment groups consisted of the blank vehicle, isoniazid (250μg/mL, 500μg/mL, 2500μg/mL), rifampicin (100μg/mL, 200μg/mL, 300μg/mL), acetylhydrazine (125μg/mL, 250μg/mL, 1250μg/mL), hydrazine (0.25μg/mL, 2.5μg/mL, 25μg/mL) and isoniazid+rifampicin (100+50μg/mL, 200+100μg/mL, 300+150μg/mL). All treatments were added to the culture media and after 48 hours of culture, cell viability was determined using the MTT method.8.2 Effect of isoniazid, its metabolites and rifampicin on lactate dehydrogenase activityCells were plated in to 6-well plates. The groups and treatments were the same as item“8.1”. Cells and culture media were collected for assay of lactate dehydrogenase.8.3 Effect of isoniazid and rifampicin on the activity of CYP 2E1 and 3A4Cells were designated to eight treatment groups with six wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL), rifampicin (200μg/mL), isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. After a 48 hour incubation period the culture media was removed. 4-nitrophenol (15μg/mL) and midazolam (1μg/mL), substrates of CYP 2E1 and 3A4 respectively, were added to the wells and cultured for an additional 2 hours. The concentration of 4-nitrocatechol, a metabolite of 4-nitrophenol, and midazolam were determined using HPLC-MS. The activity of CYP 2E1 and 3A4 were calculated by the increasing level of 4-nitrocatechol and the decreasing level of midazolam.8.4 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin on lactate dehydrogenase releaseCells were plated onto 24-well plates, and were designated to fourteen groups with six-wells in each group. Three of the fourteen groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) treatments that were added to the culture media. The other groups consisted of the naringenin and erythromycin groups where naringenin (1μg/mL, 5μg/mL, 25μg/mL) or erythromycin (1μg/mL, 5μg/mL, 25μg/mL) were added in addition. In the sodium ferulate and sanchinoside groups, sodium ferulate (10μg/mL, 50μg/mL, 250μg/mL) and sanchinoside (4μg/mL, 20μg/mL, 100μg/mL) were added in addition. After 48 hours of culture, lactate dehydrogenase activity in the cells and culture media were determined.8.5 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin decreased cellular viabilityCells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were add to the culture media. In naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. In sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the cell viability was determined using the MTT method.8.6 Effect of enzyme inhibitors on the activity of CYP 2E1 and 3A4Cells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. In the naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 3A4 determined. In the sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 2E1 determined.9 Data and statisticsStatistic was performed with SPSS 11.5 for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA with the Dunnett t test and binary variable correlation analysis. Significance was considered when P<0.05.Results1. Concentrations of isoniazid, acetylhydrazine and hydrazine in the plasma of lung tuberculosis patientsThe concentration of hydrazine increased significantly 2 h, 4 h and 12 h (P<0.05 or P<0.01) in plasma after isoniazid and rifampicin were co-administered to patients with lung tuberculosis compared with treatment with isoniazid alone. There were no significant differences between the concentrations of isoniazid and acetylhydrazine between the groups (P>0.05). There was no significant abnormality in the levels of GPT, GOT and total bilirubin before and after two weeks of drug therapy (P>0.05).2. Viability of isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytesAfter 48 hours of treatment with isoniazid (500μg/mL or 2500μg/mL), the viability of cultured QSG-7701 cells decreased significantly compared with the control group with the value of 64.3% (P<0.01) and 9.9% (P<0.01). The Pearson correlation coefficient was -0.959 (P<0.01) between the concentration of isoniazid and cell viability. The viability of cells decreased to 56.2% (P<0.01), 53.5% (P<0.05) and 46.0% (P<0.01) in the rifampicin (300μg/mL) and hydrazine (2.5μg/mL and 25μg/mL) treated groups. Co-administration of isoniazid and rifampicin dose dependently decreased cell viability and the Pearson correlation coefficient was -0.892 (P<0.01).3. Activity of lactate dehydrogenase released from isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytesIsoniazid, rifampicin, acetylhydrazine, hydrazine, isoniazid and rifampicin co-treatment increased the release of lactate dehydrogenase from cells after 48 hours of culture (P<0.01 or P<0.01). The effect of acetylhydrazine and isoniazid-rifampicin co-treatment was dose dependent and the Pearson correlation coefficient was 0.674 (P<0.01) and 0.907 (P<0.01), respectively.4. Effect of isoniazid and rifampicin on activity of CYP 3A4 and 2E1 in hepatocytesThe activity of CYP 2E1 increased significantly in QSG-7701 cells cultured for 48 hours with isoniazid or isoniazid and rifampicin (P<0.05) compared with the control group. There was no significant change in CYP 2E1 activity in the rifampicin alone treated group (P>0.05). Treatment with isoniazid, rifampicin alone or isoniazid and rifampicin all increased the activity of CYP 3A4 in QSG-7701 cells compared with the control group(P<0.05 or P<0.01).5.Effect of CYP 3A4 inhibitors on the hepatotoxicity of isoniazid and rifampicin Naringenin and erythromycin inhibited the activity of CYP 3A4 enhanced by isoniazid and rifampicin, and attenuated the decrease in cell viability and depressed lactate dehydrogenase release (P<0.01) from cultured QSG-7701 cells. The effect of naringenin on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.712 (P<0.01).6. Effect of CYP 2E1 inhibitors on the hepatotoxicity of isoniazid and rifampicinSodium ferulate and sanchinoside inhibited the activity of CYP 2E1 enhanced by isoniazid and rifampicin, attenuated the decrease in cell viability and depressed the increase in lactate dehydrogenase release (P<0.05 or P<0.01) from cultured QSG-7701 cells. The effect of sanchinoside on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.659 (P<0.01).Conclusions1 The concentration of hydrazine increased significantly in the plasma of lung tuberculosis patients that were co-administered isoniazid and rifampicin compared with patients that were treated with isoniazid alone, and the co-treatment may be the mechanism by which isoniazid and rifampicin induce hepatotoxicity.2 Different co-administered doses of isoniazid, hydraine, rifampicin, isoniazid and rifampicin result in increasing lactate dehydrogenase release and decrease the viability of hepatocytes. The effect of co-administration is more intensive than isoniazid or rifampicin treatment alone. And hydrazine, the metabolite of isoniazid, induced more severe hepatocytes toxicity than its precursor, which suggests a pivotal and considerable role of hydrazine in anti-tuberculosis induced hepatotoxicity.3 The activity of CYP 3A4 and 2E1 could be enhanced by isoniazid and rifampicin alone and their co-treatment, which may be responsible for the excessive production of hydrazine and the hepatocyte toxicity following isoniazid and rifampicin co-therapy.4 The activity of CYP 3A4 could be depressed by naringenin and erythromycin and the activity of CYP 2E1 could be depressed by sodium ferulate and sanchinoside. Moreover, naringenin, erythromycin, sodium ferulate and sanchinoside attenuate isoniazid and rifampicin co-treatment induced hepatocyte viability and decrease lactate dehydrogenase release through their effect on CYPs.更多还原