CN106539803A - 18β‑甘草次酸（或盐）作为减轻化疗药顺铂引起的急性肾损伤的辅助治疗药物的应用 - Google Patents
18β‑甘草次酸（或盐）作为减轻化疗药顺铂引起的急性肾损伤的辅助治疗药物的应用 Download PDFInfo
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Abstract
本发明属于药物治疗学领域，具体涉及甘草中的主要成分之一的18β‑甘草次酸（或盐）作为减轻化疗药顺铂引起的急性肾损伤的辅助治疗药物的应用。顺铂作为常用化疗药物，其肾脏毒性制约了其临床应用。本发明中的18β‑甘草次酸可抑制顺铂诱导的急性肾损伤中肌酐、尿素氮的水平，降低KIM‑1损伤蛋白的表达，抑制顺铂引起的肾小管上皮细胞的凋亡，本发明首次发现18β‑甘草次酸对顺铂引起的急性肾损伤具有保护作用，其机制与其抑制肾小管上皮细胞凋亡有关，在中药抗癌的辅助治疗中提供了新的潜在药物。
Description
技术领域
本发明属于药物治疗学领域，公开了18β-甘草次酸(或盐)作为减轻化疗药顺铂引起的急性肾损伤的辅助治疗药物的应用。
背景技术
临床化疗药物的肾毒性一直是肿瘤治疗的瓶颈，如何降低化疗药物的毒副作用，是肿瘤治疗过程中研究的重点之一。顺铂(cis-dichlorodiammino platinumⅡorCisplatin,CP)作为广泛使用的细胞毒性化疗药物，对多种肿瘤具有较好的疗效。顺铂活性与氯离子浓度相关，在细胞内液的低氯环境中，顺铂结构中氯离子发生水合解离，被水取代，生成带正电荷的水合配离子，与DNA发生加成反应，破坏细胞DNA的复制、转录等功能，阻滞细胞周期，导致细胞凋亡。临床观察发现，应用顺铂治疗的住院病人中肾损害发生率达25％～35％，其引起的急性肾损伤严重限制了其临床应用。顺铂进入体内大部分直接经肾脏排泄，还可以被肾小管分泌，表明肾脏细胞对顺铂有较强的聚集作用，尤其是肾小管细胞，因其浓缩和重吸收作用更易受到药物毒性效应的影响，在肾小管上皮细胞中的顺铂浓度比血液中高5倍左右，顺铂在肾脏组织中高浓度蓄积是其肾毒性作用的基础。顺铂导致的肾损伤涉及多种机制，包括炎症介质释放、氧化应激产生、凋亡及坏死、自噬等多种途径。研究发现，顺铂在体内产生的自由基可攻击心磷脂，促进细胞色素C释放，从而激活caspase-3。此外，顺铂还能够激活促凋亡蛋白Bax、Bak，这些促凋亡蛋白能够使线粒体膜通透性增加，引起细胞色素C的释放和caspases的激活，引起肾小管上皮细胞凋亡。因此阐明顺铂引起的急性肾损伤形成的相关分子机制，寻找药物可能的作用靶点，以预防和减轻顺铂引起的急性肾损伤，对于降低临床化疗药物的肾毒性，增加其临床应用具有重要的意义。
甘草为豆科植物，性平味甘，具有补脾益气，和中缓急，调和诸药的功效，广泛地被用于保肝、降血脂、抗癌及增强细胞免疫调节等方面。甘草中有100多种化学成分，甘草酸(Glycyrrhizic acid,GA)为其主要活性成分，药理作用广泛，具有抗炎、抗肿瘤等作用。甘草次酸是口服甘草酸的最主要的活性代谢产物，是甘草酸在体内发挥活性的主要形式之一。本发明拟通过整体、细胞、蛋白水平研究，深入观察探讨18β-甘草次酸(18β-GA)对CP诱导的急性肾损伤保护作用及可能的作用机制，为预防和减轻顺铂引起的急性肾损伤发现新的药物作用靶点提供实验依据。
发明内容
本发明公开了18β-甘草次酸(或盐)作为减轻化疗药顺铂引起的急性肾损伤的辅助治疗药物的应用。
本发明的目的是为寻找预防和减轻顺铂引起的肾脏损伤新的药物，为实现上述目的，探讨了甘草的活性成分甘草次酸对顺铂引起的急性肾损伤的保护作用。药效学结果显示在整体动物和细胞水平上发现甘草次酸对顺铂诱导的急性肾损伤模型具有较强的保护作用，探究其机制，在整体动物和细胞水平上均证实甘草次酸可通过抑制肾小管上皮细胞凋亡对顺铂引起的急性肾损伤发挥保护作用。
本发明的研究方案如下：
(1)在整体动物水平考察甘草次酸对急性肾损伤的保护作用，通过动物大体观、肾脏的病理、血清的肌酐、尿素氮水平，损伤蛋白KIM-1的表达变化，证明甘草次酸对急性肾损伤具有保护作用。
(2)在整体动物水平，运用TUNEL染色及组织中Caspase3的活性变化证明甘草次酸可通过抑制肾小管上皮细胞的凋亡对顺铂引起的急性肾损伤发挥保护作用。
(3)运用药代动力学方法，考察小鼠口服灌胃给药后，甘草次酸在肾脏组织中的浓度变化，为甘草次酸的肾脏药理学研究提供物质基础。
本发明拟通过整体、细胞、蛋白水平研究，深入观察探讨18β-GA对CP诱导的急性肾损伤保护作用及可能的作用机制，为预防和减轻顺铂引起的急性肾损伤发现新的药物作用靶点提供实验依据。
附图说明
图1为18β-GA对CP(20mg/kg,3d)诱导的AKI小鼠一般状况与肾脏大体的影响图；
图2为18β-GA对CP(20mg/kg,3d)诱导AKI小鼠肾脏病理的影响图；
图3为18β-GA对CP(20mg/kg,3d)诱导AKI小鼠血清Cr、BUN的影响；
A.18β-GA对CP(20mg/kg,3d)诱导AKI小鼠血清Cr的影响；
B.18β-GA对CP(20mg/kg,3d)诱导AKI小鼠血清BUN的影响；
**p<0.01vs vehicle；#p<0.05,##p<0.01vs model；
图4为18β-GA对CP(20mg/kg,3d)诱导AKI小鼠肾脏组织中KIM-1表达的影响；
A.KIM-1免疫荧光变化(x40)；B.KIM-1蛋白变化，**p<0.01vs vehicle,##
p<0.01vs model；
图5为18β-GA(50mg/kg)抑制CP(20mg/kg,3d)诱导的AKI小鼠肾脏组织凋亡(x40)；
图 6为18β-甘草次酸在肾脏匀浆液中代表性色谱图。
A.18β-GA B.空白肾组织匀浆
C.空白肾组织匀浆+25μg/ml 18β-GA D.小鼠给药 1h后肾组织匀浆1：18β-GA。
具体实施方式：
实施例1：18β-GA对CP诱导的急性肾损伤(AKI)小鼠一般状况与肾脏的影响主要步骤如下：
60只雄性C57BL/6J小鼠，体重18±2克，随机分为如下6组(n＝10)：对照组(Vehicle)、对照+18β-GA(100mg/kg)组、CP组、CP+18β-GA(25mg/kg)组、CP+18β-GA(50mg/kg)组和CP+18β-GA(100mg/kg)组。第1天对照组及对照+18β-GA(100mg/kg)组经腹腔注射0.9％氯化钠0.5ml，CP组、CP+18β-GA(25mg/kg)组、CP+18β-GA(50mg/kg)组和CP+18β-GA(100mg/kg)组腹腔注射CP(20mg/kg)，与此同时CP+18β-GA(25mg/kg)组、CP+18β-GA(50mg/kg)组和CP+18β-GA(100mg/kg)连续灌胃给药3天，第四天腹腔注射10％的水合氯醛，麻醉后处死动物，称重并收集血液，用于生化指标的检测；收集肾组织冻存或固定，用于石蜡切片和冰冻切片的制备和蛋白及RNA的制备。
结果发现：与CP诱导AKI小鼠相比，应用18β-GA(25、50、100mg/kg)，可不同程度改善小鼠一般状态。
大体观，与CP诱导AKI小鼠相比，18β-GA(25、50、100mg/kg)可缓解CP诱导的AKI小鼠肾脏紧张度，颜色亦逐渐加深(见图1)。
实施例2：18β-GA对CP诱导的小鼠肾脏病理的影响
主要操作步骤：将不同组别的小鼠肾脏组织固定于福尔马林溶液中24h～48h，样品经过酒精脱水和二甲苯透明后，用石蜡包埋。待包埋好的组织块变硬，用切片机进行切片。染色前，在二甲苯中脱去石蜡切片中的石蜡，经过由高浓度到低浓度的乙醇脱水后，最后用清水(蒸馏水)冲洗即可开始染色。于苏木素染色液中染色5min～15min，用流水稍洗去多余的染色液。在1％盐酸乙醇中作用1～3s，稍水洗后加入蓝液(0.5％淡氨水)返蓝10～30s后，用0.5％伊红染液进行染色1～3min左右。蒸馏水稍洗后，再在由低浓度到高浓度乙醇溶液中进行脱水处理，二甲苯中透明、中性树脂胶封片后在显微镜下观察，分析结果后选取需要的位置拍照。
结果：H&E染色检测小鼠肾脏病理发现，与CP诱导AKI小鼠相比，18β-GA(25、50、100mg/kg)可减轻肾血管充血，降低肾小管上皮细胞水肿(图2)。
实施例3:18β-GA对CP诱导的小鼠血清Cr与BUN的影响
主要步骤：取不同组别的小鼠血清作为样品，按照肌酐Cr测试盒(肌苷酸氧化酶法，微孔96T微板法)及尿素氮BUN测试盒(尿酶法)(南京建成生物工程研究所)说明书进行操作。
检测血清Cr与BUN发现，与CP诱导AKI小鼠相比18β-GA(50、100mg/kg)可明显降低血清Cr与BUN水平(p<0.01)(图3A，图3B)。上述实验提示18β-GA对CP诱导AKI具有保护作用。
实施例4：18β-GA抑制CP引起的AKI中KIM-1的表达
主要步骤：将不同组别小鼠肾组织切片进行脱蜡和水化；用PBS溶液洗涤2～3次，每次5min左右；以0.3％H2O2溶液室温静置封闭10～20min；再次用PBS溶液洗涤2～3次，每次5min左右；在正常山羊血清封闭液中室温静置封闭30～60min；PBS溶液洗涤2～3次，每次5min左右；加入50μl一抗(KIM-1抗体)，室温静置1h或者4℃过夜；再次用PBS溶液洗涤2～3次去除多余的一抗，每次5min左右；加入生物素化标记的二抗45～50μl，室温孵育1～2h；用PBS溶液洗涤2～3次去除多余的二抗，每次5min左右；将DAB显色剂A，B，C各1滴加入1ml蒸馏水中，显色5min后观察；以流水冲洗，10～15min；以苏木精溶液复染，在盐酸酒精溶液中进行分化处理；流水冲洗10～15min；梯度乙醇脱水，以二甲苯透明和树胶封片。对于阴性对照组，其以正常山羊血清替代上述一抗，其余步骤均一致。
结果：免疫荧光染色发现正常组KIM-1荧光表达较弱，CP诱导的AKI小鼠KIM-1荧光表达增强，主要表达于肾小管，18β-GA给药组KIM-1荧光表达明显降低(图4A)。
Western blot检测发现，Vehicle组肾组织KIM-1蛋白表达量低，CP诱导的AKI小鼠肾组织KIM-1蛋白表达明显升高，18β-GA可明显降低CP诱导的AKI中KIM-1蛋白的表达，与模型组相比，有统计学意义(p<0.05)(图4B)。提示18β-GA可抑制CP引起的KIM-1表达。
实施例5：18β-GA抑制CP诱导的肾组织中细胞凋亡
主要步骤：以不同组小鼠肾组织冰冻切片为样本，按照凯基一步法TUNEL细胞凋亡原位检测试剂盒(绿色FITC标记荧光检测法、细胞组织通用型)说明书操作进行。
结果：应用TUNEL原位凋亡染色法检测18β-GA对CP诱导的AKI肾组织凋亡的影响，结果发现，与CP诱导的AKI小鼠相比，18β-GA可明显降低CP诱导的AKI小鼠肾脏组织细胞凋亡(图5)。
实施例6：18β-GA对CP诱导的急性肾损伤肾组织中caspase3活性的影响。
主要步骤：根据待测样品数准备裂解缓冲液和检测缓冲液，每1ml缓冲液中加入10μl DTT。按小鼠分组，每组取适量组织样本剪碎，按照50mg/100UI冷的裂解缓冲液，用组织匀浆器匀浆至无明显肉眼可见固体(或用液氮研磨)，冰上静置15分钟，小心将上清吸入另一预冷的干净离心管。在4℃，500×g条件下离心5分钟。快速将上清吸入另一预冷的干净离心管，置于冰上或-80℃冰箱保存备用。对上述处理过的上清用Bradford法进行蛋白定量。取l0μl大约含20-50mg蛋白的裂解上清，加入90μl检测缓冲液。再加入l0μl Ac-DEVD-pNA(使用前请混匀)，在37℃下避光反应1-2小时，有黄色颜色产生即可进行检测，如果颜色变化不明显，时间可以延长，甚至孵育过夜。部分样品中激活的caspase-3水平较低，颜色变化不明显，直接上机检测，与对照组样品产品变化即可。测定A405nm或A400nm。根据凋亡诱导的细胞的吸光值与空白对照细胞的吸光值的比值计算相对的Caspase3活性程度。
结果发现：与CP诱导的急性肾损伤模型组小鼠相比，18β-GA(50mg/kg)给药组可明显抑制肾组织中Caspase-3的活性。
实施例7：18β-GA在CP诱导的小鼠肾组织中分布
主要步骤：
(1)确定最佳的色谱条件：
色谱柱：Phenomenex Luna C18column(250mm×4.6mm，5μm)，C18预柱(4×2mm)；
流动相：0.1％甲酸水：乙腈＝(20:80,v/v)；流速：1000μl/min；
检测波长：254nm；
柱温：35℃；
进样量：20μl。
(2)肾脏组织样品处理方法
将小鼠肾脏组织用生理盐水匀浆，按照每200mg肾脏组织加入600μl生理盐水，玻璃匀浆器中匀浆后，精密吸取肾脏匀浆液400μl，加入2000μl乙酸乙酯，涡旋3min后，12000g离心15min，提取上层有机相，N2下吹干后，100μl流动相复溶，涡旋3min后，12000g离心5min，取上清液20μl通过HPLC进样分析。
(3)标准曲线的制备
取空白离心管8个，其中1份为空白样本，另外7份分别加入上述甘草次酸系列工作溶液各20μl(浓度分别为3.125、6.25、12.5、25、50、100、200μg/ml)，再加入空白肾脏匀浆液400μl，使肾脏匀浆液中甘草次酸浓度分别为0.156、0.3125、0.625、1.25、2.5、5、10μg/ml，充分混匀后依3.2.3项下处理。以待测物浓度(X)为横坐标，待测物的峰面积(Y，As)为纵坐标，用加权法(W＝1/X2)进行回归运算，求得的直线方程即为标准曲线。
(4)进行肾脏匀浆液专属性、肾脏匀浆液中18β-GA标准曲线和定量下限、方法的精密度实验、提取回收率的测定、18β-GA在小鼠肾脏匀浆液中稳定性实验的方法学考察。
结果：
(1)精密量取6份不同小鼠空白肾脏匀浆混合液400μl，按“肾脏组织样品处理方法”操作；取50μg/ml的18β-GA标准溶液20μl，加入80μl流动相混匀，20μl进样分析，得色谱图(图6A)；取25μg/ml的18β-GA标准溶液20μl，加入小鼠空白肾脏匀浆液400μl，按“肾脏组织样品处理方法”项下操作得色谱图(图6C)；取小鼠灌胃给予18β-GA(50mg/kg)1h后收集的肾脏样品，按“肾脏组织样品处理方法”项下操作得色谱图(图6D)；结果表明18β-GA保留时间为10.2min左右，肾脏匀浆液中内源性物质不干扰18β-GA的测定(图6B)。
(2)采用HPLC观察18β-GA在肾脏组织的分布情况，结果如表1所示，发现正常组与CP诱导AKI模型组未检测出18β-GA，给药组肾组织匀浆中检测到18β-GA，表明18β-GA灌胃给药后，分布于肾脏，提示18β-GA可能对肾脏产生药理作用，为后续的药理实验围绕肾脏进行研究奠定了物质基础。
表1 18β-GA在肾脏组织中的分布
Claims (2)
1.18β-甘草次酸作为减轻化疗药顺铂引起的急性肾损伤的辅助治疗药物的应用。
2.18β-甘草次酸盐作为减轻化疗药顺铂引起的急性肾损伤的辅助治疗药物的应用。
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2016
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| CN110604758A (zh) * | 2019-09-03 | 2019-12-24 | 华中农业大学 | 一种用于缓解化疗药物副作用的中药 |
| CN110604758B (zh) * | 2019-09-03 | 2021-12-07 | 华中农业大学 | 一种用于缓解化疗药物副作用的中药 |
| CN112716960A (zh) * | 2021-02-04 | 2021-04-30 | 福建医科大学附属第一医院 | 甘草次酸在制备治疗肾小管间质炎症药物中的应用 |
| CN113440596A (zh) * | 2021-07-12 | 2021-09-28 | 杭州市第一人民医院 | 一种Epitalon在制备用于减轻顺铂诱导的急性肾损伤药物中的用途 |
| CN114272325A (zh) * | 2022-02-10 | 2022-04-05 | 天津科技大学 | 百合固金丸在作为或制备防治顺铂急性肾损伤药物中的应用 |
| CN117357545A (zh) * | 2023-09-20 | 2024-01-09 | 复旦大学附属中山医院 | 甘草酸苷在制备治疗万古霉素引起肾损伤药物中的应用 |
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