芦荟大黄素对小鼠肾毒性的作用机制
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摘要
目的:研究长期给予不同剂量的芦荟大黄素导致小鼠肾毒性,并探讨其毒性机制。方法:将30只昆明种小鼠随机分为雌雄空白组、雌雄芦荟大黄素低、高剂量组(0.8,1.6 g·kg~(-1))。芦荟大黄素各剂量组连续灌胃给药11周,每日早晚各1次。采用生化试剂盒检测小鼠血清尿素氮(urea nitrogen,BUN),肌酐(creatinine,SCr),超氧化物歧化酶(superoxide dismustase,SOD)和丙二醛(malondialdehyde,MDA)含量,肾脏总谷胱甘肽(total glutathione,T-GSH)/氧化性谷胱甘肽试剂盒(oxidized glutathione,GSSG)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)水平;酶联免疫吸附试验(ELISA)检测血清中肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)和白细胞介素-6(interleukins-6,IL-6)水平;苏木素-伊红(HE)染色检测肾脏病理变化;免疫组化检测肾脏半胱氨酸蛋白酶-3(cysteine aspartic acid specific protease-3,Caspase-3)和转化生长因子-β_1(transforming growth factor-β_1,TGF-β_1)蛋白表达。结果:与同性别空白组比较,芦荟大黄素高剂量组雌、雄小鼠血清中BUN含量升高(P<0.05,P<0.01),芦荟大黄素高剂量组雄性小鼠血清中SCr含量升高(P<0.05),芦荟大黄素低剂量组肾小管轻度损伤,高剂量肾小管和肾小球中度损伤;与空白组比较,芦荟大黄素高剂量组雌、雄小鼠血清MDA含量升高(P<0.05),SOD活性降低(P<0.05),芦荟大黄素高剂量组雌雄小鼠肾脏中GSH/GSSG含量降低(P<0.05),雄性小鼠Caspase-3蛋白表达增加(P<0.05);与空白组比较,芦荟大黄素高剂量组雄性小鼠TNF-α和IL-6含量升高(P<0.05),芦荟大黄素低、高剂量组雌雄小鼠肾脏TGF-β_1蛋白表达均增加(P<0.05)。结论:芦荟大黄素1.6 g·kg~(-1)给药11周,对小鼠肾脏有毒性作用,其机制与机体氧化应激、细胞凋亡和TGF-β_1蛋白表达有关。
Objective:To study nephrotoxicity induced by long-term administration of different doses of aloe-emodin in mice,and explore its mechanism.Method:A total of 30 male and female Kunming mice were randomly divided into normal control group,and low-dose aloe-emodin group,high-dose aloe-emodin group(0.8,1.6 g·kg~(-1)).Every dose of group was administered intragastrically for 11 weeks,twice daily.effect of serum urea nitrogen(BUN),creatinine(SCr),superoxide dismutase(SOD),malondialdehyde(MDA),Glutathione(GSH/GSSG)and Glutathione Peroxidase(GSH-Px)levels were detected by biochemical kits according to manufacturer's instruction.Enzyme-linked immune assay was used to determine serum tumor necrosis factor(TNF)-αand interleukins(IL)-6 levels.Hematoxylin eosin(HE)staining was used to detect renal pathological changes in kidney tissues,and cysteine aspartic acid specific protease(Caspase)-3 and transforming growth factor(TGF)-β_1proteins were determined by immunohistochemistry.Result:According to results,compared with normal control group,the levels of BUN and SCr in serum with high-dose aloe-emodin were increased.The renal tubules in low-dose group were mildly injured,while renal tubules and glomeruli of high-dose group were moderately damaged.Compared with normal control group,the level of SOD was significant decreased(P<0.05),MDA was increased(P<0.05),the levels of GSH/GSSG in kidneys of high-dose groups were decreased(P<0.05).In high-dose group,the expression of Caspase-3 protein was increased in kidneys,especially in males(P<0.05).Compared with normal control group,the levels of TNF-αand IL-6 were increased,the expression of TGF-β_1protein in kidneys was increased in low-dose and high-dose groups(P<0.05).Conclusion:results show that 1.6 g·kg~(-1)aloe-emodin was administered intragastrically for 11 weeks,which had toxic effects on kidney in mice.The mechanism may be related to oxidative stress,apoptosis and TGF-β_1protein expression.
Objective:To study nephrotoxicity induced by long-term administration of different doses of aloe-emodin in mice,and explore its mechanism.Method:A total of 30 male and female Kunming mice were randomly divided into normal control group,and low-dose aloe-emodin group,high-dose aloe-emodin group(0.8,1.6 g·kg~(-1)).Every dose of group was administered intragastrically for 11 weeks,twice daily.effect of serum urea nitrogen(BUN),creatinine(SCr),superoxide dismutase(SOD),malondialdehyde(MDA),Glutathione(GSH/GSSG)and Glutathione Peroxidase(GSH-Px)levels were detected by biochemical kits according to manufacturer's instruction.Enzyme-linked immune assay was used to determine serum tumor necrosis factor(TNF)-αand interleukins(IL)-6 levels.Hematoxylin eosin(HE)staining was used to detect renal pathological changes in kidney tissues,and cysteine aspartic acid specific protease(Caspase)-3 and transforming growth factor(TGF)-β_1proteins were determined by immunohistochemistry.Result:According to results,compared with normal control group,the levels of BUN and SCr in serum with high-dose aloe-emodin were increased.The renal tubules in low-dose group were mildly injured,while renal tubules and glomeruli of high-dose group were moderately damaged.Compared with normal control group,the level of SOD was significant decreased(P<0.05),MDA was increased(P<0.05),the levels of GSH/GSSG in kidneys of high-dose groups were decreased(P<0.05).In high-dose group,the expression of Caspase-3 protein was increased in kidneys,especially in males(P<0.05).Compared with normal control group,the levels of TNF-αand IL-6 were increased,the expression of TGF-β_1protein in kidneys was increased in low-dose and high-dose groups(P<0.05).Conclusion:results show that 1.6 g·kg~(-1)aloe-emodin was administered intragastrically for 11 weeks,which had toxic effects on kidney in mice.The mechanism may be related to oxidative stress,apoptosis and TGF-β_1protein expression.
引文
[1]李牧,杜智敏.芦荟大黄素的药理作用研究进展[J].中国临床药理学杂志,2015,31(9):765-768.
[2]CHEN R, ZHANG J, HU Y, et al. Potential antineoplastic effects of Aloe-emodin:a comprehensive review[J]. Am J Chin Med,2014,42(2):275-288.
[3]陶明宝,张乐,刘飞,等.含蒽醌类成分中药的安全性研究进展[J].中药药理与临床,2016,32(6):238-243.
[4]王建平.大黄有毒物质基础的研究[J].中国民族民间医药,2011,20(24):63-64.
[5]王青秀.大黄及其主要成份的毒性毒理研究[D].北京:中国人民解放军军事医学科学院,2007.
[6]王伽伯,马永刚,张萍,等.炮制对大黄化学成分和肝肾毒性的影响及其典型相关分析[J].药学学报,2009,44(8):885-890.
[7]任历,曾滨阳,张诗缇,等.大黄总蒽醌对人肾小管上皮细胞毒性作用及相关机制研究[J].中药药理与临床,2015,31(1):79-83.
[8]Program N T. NTP toxicology and carcinogenesis studies of EMODIN(CAS NO. 518-82-1)feed studies in F344/N rats and B6C3F1 mice[J]. Natl Toxicol Program Tech Rep Ser,2001,493(6):1-278.
[9]笪红远,江振洲,王翠芬,等.大黄酸和大黄素在体外对人肾小管上皮细胞的毒性作用研究[J].中草药,2009,40(1):102-105.
[10]王艳辉,赵海平,王伽伯,等.基于“有故无殒”思想的熟大黄对肝脏量-毒/效关系研究[J].中国中药杂志,2014,39(15):2918-2923.
[11]毕礼明,陈英兰,陆曙,等.大黄治疗肾脏病安全性探析[J].医学争鸣,2016,7(6):29-32,36.
[12]李一飞,徐婷婷,姚广涛,等.尿液NGAL,KIM-1,IL-18在商陆所致的大鼠肾损伤中的变化特征及其联合检测的意义[J].中国中药杂志,2012,37(23):3611-3617.
[13]赵平.大黄附子不同配伍比例灌肠治疗慢性肾衰竭[J].中国实验方剂学杂志,2012,18(15):302-304.
[14]高峰,刘承萍,曹骋,等.大鼠体内大承气汤蒽醌成分的药代动力学研究[J].中华中医药杂志,2011,26(5):1018-1021.
[15]柴宝娟,李祥,陈建伟.大黄配伍前后对大鼠肝肾的影响[J].中药药理与临床,2012,28(3):66-69.
[16]王青秀,吴纯启,周莉,等.大黄素诱导HK-2细胞凋亡的机制探讨[J].中国新药杂志,2010,19(22):2034-2038,2044.
[17]黄伟,孙蓉.益母草肾毒性与氧化损伤机制的相关性研究[J].中药药理与临床,2010,26(2):54-56.
[18]刘德明,周春燕,吴嘉思,等.大黄素通过线粒体通路诱导Hep G2细胞凋亡[J].中国实验方剂学杂志,2018,24(3):104-108.
[19]张晓田,宋天保. Caspase-3与细胞凋亡的研究[J].医学综述,2002,8(11):621-623.
[20]WANG C,WU X,CHEN M,et al. Emodin induces apoptosis through Caspase-3-dependent pathway in HK-2cells[J]. Toxicology,2007,231(2/3):120-128.
[21]Panigrahi G K,Verma N,Singh N,et al. Interaction of anthraquinones of Cassia occifentalis seeds with DNA and Glutathione[J]. Toxicol Rep,2018,5:164-172.
[22]郑媛,王淳,李伟,等.复方痛风康对高尿酸血症模型大鼠肾功能的保护作用及影响IL-1β、TNF-α、TGF-β1的研究[J].中国中西医结合肾病杂志,2015,16(7):575-578,659.
[23]阮雄中.“脂质肾毒性学说”的最新研究进展[J].中国科学:生命科学,2018,48(1):47-55.
[24]XIAO Z,LI C W,SHAN J,et al. Interventions to improve chronic cy-closporine A nephrotoxicity through inhibiting renal cell apoptosis:asystematic review[J].Chin Med J(Engl),2013,126(19):3674-3774.
[25]Martin-Martin N,Slattery C,Mc Morrow T,et al. TGF-β1medi-ates sirolimus and cyclosporine A-induced alteration of barrier func-tion in renal epithelial cells via a noncanonical ERK1/2 signalingpathway[J]. Am J Physiol Renal Physiol,2011,301(6):1281-1292.
[26]段晓虹,董竞成,何立群,等.补肾活血方对慢性肾炎肾虚血瘀证患者蛋白尿、尿IL-6、TGF-β1及MCP-1的影响[J].中国中西医结合杂志,2011,31(6):765-768.
[27]陈红梅,王秀兰,恩和苏仁,等.白豆蔻对阿霉素肾病模型大鼠肾脏组织TGF-β1、PAI-1表达的影响研究[J].中药药理与临床,2017,33(3):105-108.
[2]CHEN R, ZHANG J, HU Y, et al. Potential antineoplastic effects of Aloe-emodin:a comprehensive review[J]. Am J Chin Med,2014,42(2):275-288.
[3]陶明宝,张乐,刘飞,等.含蒽醌类成分中药的安全性研究进展[J].中药药理与临床,2016,32(6):238-243.
[4]王建平.大黄有毒物质基础的研究[J].中国民族民间医药,2011,20(24):63-64.
[5]王青秀.大黄及其主要成份的毒性毒理研究[D].北京:中国人民解放军军事医学科学院,2007.
[6]王伽伯,马永刚,张萍,等.炮制对大黄化学成分和肝肾毒性的影响及其典型相关分析[J].药学学报,2009,44(8):885-890.
[7]任历,曾滨阳,张诗缇,等.大黄总蒽醌对人肾小管上皮细胞毒性作用及相关机制研究[J].中药药理与临床,2015,31(1):79-83.
[8]Program N T. NTP toxicology and carcinogenesis studies of EMODIN(CAS NO. 518-82-1)feed studies in F344/N rats and B6C3F1 mice[J]. Natl Toxicol Program Tech Rep Ser,2001,493(6):1-278.
[9]笪红远,江振洲,王翠芬,等.大黄酸和大黄素在体外对人肾小管上皮细胞的毒性作用研究[J].中草药,2009,40(1):102-105.
[10]王艳辉,赵海平,王伽伯,等.基于“有故无殒”思想的熟大黄对肝脏量-毒/效关系研究[J].中国中药杂志,2014,39(15):2918-2923.
[11]毕礼明,陈英兰,陆曙,等.大黄治疗肾脏病安全性探析[J].医学争鸣,2016,7(6):29-32,36.
[12]李一飞,徐婷婷,姚广涛,等.尿液NGAL,KIM-1,IL-18在商陆所致的大鼠肾损伤中的变化特征及其联合检测的意义[J].中国中药杂志,2012,37(23):3611-3617.
[13]赵平.大黄附子不同配伍比例灌肠治疗慢性肾衰竭[J].中国实验方剂学杂志,2012,18(15):302-304.
[14]高峰,刘承萍,曹骋,等.大鼠体内大承气汤蒽醌成分的药代动力学研究[J].中华中医药杂志,2011,26(5):1018-1021.
[15]柴宝娟,李祥,陈建伟.大黄配伍前后对大鼠肝肾的影响[J].中药药理与临床,2012,28(3):66-69.
[16]王青秀,吴纯启,周莉,等.大黄素诱导HK-2细胞凋亡的机制探讨[J].中国新药杂志,2010,19(22):2034-2038,2044.
[17]黄伟,孙蓉.益母草肾毒性与氧化损伤机制的相关性研究[J].中药药理与临床,2010,26(2):54-56.
[18]刘德明,周春燕,吴嘉思,等.大黄素通过线粒体通路诱导Hep G2细胞凋亡[J].中国实验方剂学杂志,2018,24(3):104-108.
[19]张晓田,宋天保. Caspase-3与细胞凋亡的研究[J].医学综述,2002,8(11):621-623.
[20]WANG C,WU X,CHEN M,et al. Emodin induces apoptosis through Caspase-3-dependent pathway in HK-2cells[J]. Toxicology,2007,231(2/3):120-128.
[21]Panigrahi G K,Verma N,Singh N,et al. Interaction of anthraquinones of Cassia occifentalis seeds with DNA and Glutathione[J]. Toxicol Rep,2018,5:164-172.
[22]郑媛,王淳,李伟,等.复方痛风康对高尿酸血症模型大鼠肾功能的保护作用及影响IL-1β、TNF-α、TGF-β1的研究[J].中国中西医结合肾病杂志,2015,16(7):575-578,659.
[23]阮雄中.“脂质肾毒性学说”的最新研究进展[J].中国科学:生命科学,2018,48(1):47-55.
[24]XIAO Z,LI C W,SHAN J,et al. Interventions to improve chronic cy-closporine A nephrotoxicity through inhibiting renal cell apoptosis:asystematic review[J].Chin Med J(Engl),2013,126(19):3674-3774.
[25]Martin-Martin N,Slattery C,Mc Morrow T,et al. TGF-β1medi-ates sirolimus and cyclosporine A-induced alteration of barrier func-tion in renal epithelial cells via a noncanonical ERK1/2 signalingpathway[J]. Am J Physiol Renal Physiol,2011,301(6):1281-1292.
[26]段晓虹,董竞成,何立群,等.补肾活血方对慢性肾炎肾虚血瘀证患者蛋白尿、尿IL-6、TGF-β1及MCP-1的影响[J].中国中西医结合杂志,2011,31(6):765-768.
[27]陈红梅,王秀兰,恩和苏仁,等.白豆蔻对阿霉素肾病模型大鼠肾脏组织TGF-β1、PAI-1表达的影响研究[J].中药药理与临床,2017,33(3):105-108.