斑马鱼模型筛选何首乌肝毒性的物质基础
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摘要
目的:用斑马鱼模型探索何首乌肝毒性物质基础,为何首乌肝毒性作用机制研究提供一定的理论依据。方法:将对实验前期所筛选出来的大黄素、大黄酸、芦荟大黄素、大黄素-1-O-葡萄糖苷、大黄素甲醚-8-O-葡萄糖苷、芦荟大黄素-8-O-葡萄糖苷(质量浓度分别为0. 000 73,0. 002 22,0. 015 05,0. 002 36,0. 198 95,0. 072 73 g·L~(-1))这6种何首乌中的化学成分,继续使用肝脏荧光转基因斑马鱼为动物模型,对受精后72 h的幼鱼连续给药3 d。分别测定给药后1,2,3 d的斑马鱼体内丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),谷胱甘肽(GSH)的活力和总胆红素(TBIL)的含量,分别对其进行切片分析,观察斑马鱼肝脏组织病理学改变。结果:大黄素、大黄酸、大黄素-1-O-葡萄糖苷、大黄素甲醚-8-O-葡萄糖苷对斑马鱼体内ALT,AST,GSH的活力和TBIL的含量无明显影响,其斑马鱼肝组织显示正常;芦荟大黄素能显著降低斑马鱼体内ALT,AST,GSH的活力(P <0. 01),显著升高TBIL含量(P <0. 01);芦荟大黄素-8-O-葡萄糖苷能显著降低斑马鱼体内ALT,GSH的活力(P <0. 01),明显升高TBIL含量(P <0. 05,P <0. 01),对斑马鱼体内AST的活力无明显影响,同时此二者的斑马鱼肝组织均出现大片片状坏死,肝组织细胞发生明显形态改变,肝细胞排列不规则等。结论:何首乌中的芦荟大黄素和芦荟大黄素-8-O-葡萄糖苷可对斑马鱼肝脏产生毒性作用,提示芦荟大黄素和芦荟大黄素-8-O-葡萄糖苷可能是何首乌的肝毒性物质基础。
Objective: To explore the hepatotoxic material basis of Polygoni Multiflori Radix with zebrafish model,in order to provide a theoretical basis for the study on the mechanism of Polygoni Multiflori Radix hepatotoxicity. Method: Emodin,rhein,aloe emodin,emodin-1-O-glucoside,physcion-8-O-glucoside and aloe emodin-8-O-glucoside for three days( at the concentrations of 0. 000 73,0. 002 22,0. 015 05,0. 002 36,0. 198 95,0. 072 73 g·L~(-1)) selected from the early stage of the experiment were continuously administered to zebrafish fertilized for 72 hours to establish the liver fluorescence transgenic larvae animal model. The activities of alanine aminotransferase( ALT),aspartate aminotransferase( AST),glutathione( GSH) and total bilirubin( TBIL) in zebrafish at 1,2,3 day after administration were measured respectively,and the pathological changes of zebrafish liver tissue were analyzed. Result: Emodin,rhein,emodin-1-O-glucoside and physcion-8-O-glucoside had no significant effect on the activities of ALT,AST,GSH and content of TBIL( P < 0. 01) in zebrafish,and the liver tissue of zebrafish was normal; aloe-emodin could significantly reduce the activities of ALT,AST,GSH( P < 0. 01),whereas increase the content of TBIL in zebrafish; aloe-emodin-8-O-glucoside could significantly reduce the activities of ALT,GSH,whereas increased the content of TBIL( P < 0. 05,P < 0. 01) in zebrafish,with no significant effect on the activity of AST,then both groups showed large flaky necrosis in zebrafish liver tissue,obvious morphologic changes in liver tissue cells,and irregular arrangement of hepatocytes. Conclusion: Aloe-emodin and aloe-emodin-8-O-glucoside in Polygoni Multiflori Radix have a toxic effect on zebrafish liver,suggesting that aloe-emodin and aloeemodin-8-O-glucoside might be the hepatotoxic material basis of Polygoni Multiflori Radix.
Objective: To explore the hepatotoxic material basis of Polygoni Multiflori Radix with zebrafish model,in order to provide a theoretical basis for the study on the mechanism of Polygoni Multiflori Radix hepatotoxicity. Method: Emodin,rhein,aloe emodin,emodin-1-O-glucoside,physcion-8-O-glucoside and aloe emodin-8-O-glucoside for three days( at the concentrations of 0. 000 73,0. 002 22,0. 015 05,0. 002 36,0. 198 95,0. 072 73 g·L~(-1)) selected from the early stage of the experiment were continuously administered to zebrafish fertilized for 72 hours to establish the liver fluorescence transgenic larvae animal model. The activities of alanine aminotransferase( ALT),aspartate aminotransferase( AST),glutathione( GSH) and total bilirubin( TBIL) in zebrafish at 1,2,3 day after administration were measured respectively,and the pathological changes of zebrafish liver tissue were analyzed. Result: Emodin,rhein,emodin-1-O-glucoside and physcion-8-O-glucoside had no significant effect on the activities of ALT,AST,GSH and content of TBIL( P < 0. 01) in zebrafish,and the liver tissue of zebrafish was normal; aloe-emodin could significantly reduce the activities of ALT,AST,GSH( P < 0. 01),whereas increase the content of TBIL in zebrafish; aloe-emodin-8-O-glucoside could significantly reduce the activities of ALT,GSH,whereas increased the content of TBIL( P < 0. 05,P < 0. 01) in zebrafish,with no significant effect on the activity of AST,then both groups showed large flaky necrosis in zebrafish liver tissue,obvious morphologic changes in liver tissue cells,and irregular arrangement of hepatocytes. Conclusion: Aloe-emodin and aloe-emodin-8-O-glucoside in Polygoni Multiflori Radix have a toxic effect on zebrafish liver,suggesting that aloe-emodin and aloeemodin-8-O-glucoside might be the hepatotoxic material basis of Polygoni Multiflori Radix.
引文
[1]袁炜.何首乌的化学成分研究[D].北京:北京中医药大学,2017.
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[4]DONG H H,Slain D,CHENG J C,et al.Eighteen cases of liver injury following ingestion of Polygonum multiflorum[J].Complement Ther Med,2014,22(1):70-74.
[5]赖潇潇,吴俊标,陈设,赖平,汪小惠,王颖彦,罗懿妮,林华.基于回顾性研究的何首乌风险因素分析与安全应用建议[J].中国中药杂志,2018,43(15):3205-3210.
[6]Min H J,Jung K A,Kim H J,et al.951 twelve cases of toxic hepatitis related to the root of Polygonum multiflorum Thunb[J].J Hepatol,2008,48(Suppl2):S356-S356.
[7]全云云,周忆梦,刘美辰,等.斑马鱼模型评价何首乌中18种成分的肝脏毒性[J].天然产物研究与开发,2018,30(05),744-752.
[8]Westerfield M.The Zebrafish Book.A Guide for the Laboratory Use of Zebrafish Danio(Brachydanio)rerio[M].Eugene:Institute of Neuroscience,University of Oregon,2000:1-342.
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[11]Chu J,Sadler K C.New school in liver development:lessons from zebrafish[J].Hepatology,2009,50(5):1656-1663.
[12]王立媛.正常人群血清总蛋白、总胆红素、血红蛋白测定值升高及原因探讨[J].临床和实验医学杂志,2003,2(2):128-129.
[13]Armstrong J S,Steinauer K K,Hornung B,et al.Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line[J].Cell Death Differ,2002,9(3):252-263.
[14]Mazzanti G,Battinelli L,Daniele C,et al.New case of acute hepatitis following the consumption of Shou Wu Pian,a Chinese herbal product derived from Polygonum multiflorum[J].Ann Intern Med,2004,140(7):W30.
[15]Panis B,Wong D R,Hooymans P M,et al.Recurrent toxic hepatitis in a Caucasian girl related to the use of Shou-Wu-Pian,a Chinese herbal preparation[J].JPediatr Gastroenterol Nutr,2005,41(2):256-258.
[16]Cárdenas A,Restrepo J C,Sierra F,et al.Acute hepatitis due to shen-min:a herbal product derived from Polygonum multiflorum[J].J Clin Gastroenterol,2006,40(7):629-632.
[17]程生辉,张妍妍,李会芳,等.基于黄疸模型大鼠的栀子苷急性肝肾毒性研究[J].中国实验方剂学杂志,2015,21(4):174-178.
[18]程生辉,唐超,李会芳,等.栀子苷对正常大鼠急性肝、肾毒性的时-毒关系分析[J].中国实验方剂学杂志,2016,22(1):162-165.
[19]张海虹,卫璐戈,李会芳.栀子苷对正常和黄疸模型大鼠的亚急性肝肾毒性[J].中国实验方剂学杂志,2018,24(20):140-144.
[2]YI T,Leung K S,LU G H,et al.Identification and determination of the major constituents in traditional Chinese medicinal plant Polygonum multiflorum thunb by HPLC coupled with PAD and ESI/MS[J].Phytochem Anal,2007,18(3):181-187.
[3]LEI X,CHEN J,REN JT,et al.Liver damage associated with Polygonum ultiflorum thunb.:a systematic review of case reports and case series[J].Evid Based Complement Alternat Med,2015(5):459749.
[4]DONG H H,Slain D,CHENG J C,et al.Eighteen cases of liver injury following ingestion of Polygonum multiflorum[J].Complement Ther Med,2014,22(1):70-74.
[5]赖潇潇,吴俊标,陈设,赖平,汪小惠,王颖彦,罗懿妮,林华.基于回顾性研究的何首乌风险因素分析与安全应用建议[J].中国中药杂志,2018,43(15):3205-3210.
[6]Min H J,Jung K A,Kim H J,et al.951 twelve cases of toxic hepatitis related to the root of Polygonum multiflorum Thunb[J].J Hepatol,2008,48(Suppl2):S356-S356.
[7]全云云,周忆梦,刘美辰,等.斑马鱼模型评价何首乌中18种成分的肝脏毒性[J].天然产物研究与开发,2018,30(05),744-752.
[8]Westerfield M.The Zebrafish Book.A Guide for the Laboratory Use of Zebrafish Danio(Brachydanio)rerio[M].Eugene:Institute of Neuroscience,University of Oregon,2000:1-342.
[9]Carlo Alberto R.Transgenesis techniques-Principles and protocols[M].Clifton:Humana Press,2010:1-331.
[10]Her G M,Chiang C C,CHEN W Y,et al.In vivo studies of liver-type fatty acid binding protein(L-FABP)gene expression in liver of transgenic zebrafish(Danio rerio)[J].Febs Letters,2003,538(1/3):125-133.
[11]Chu J,Sadler K C.New school in liver development:lessons from zebrafish[J].Hepatology,2009,50(5):1656-1663.
[12]王立媛.正常人群血清总蛋白、总胆红素、血红蛋白测定值升高及原因探讨[J].临床和实验医学杂志,2003,2(2):128-129.
[13]Armstrong J S,Steinauer K K,Hornung B,et al.Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line[J].Cell Death Differ,2002,9(3):252-263.
[14]Mazzanti G,Battinelli L,Daniele C,et al.New case of acute hepatitis following the consumption of Shou Wu Pian,a Chinese herbal product derived from Polygonum multiflorum[J].Ann Intern Med,2004,140(7):W30.
[15]Panis B,Wong D R,Hooymans P M,et al.Recurrent toxic hepatitis in a Caucasian girl related to the use of Shou-Wu-Pian,a Chinese herbal preparation[J].JPediatr Gastroenterol Nutr,2005,41(2):256-258.
[16]Cárdenas A,Restrepo J C,Sierra F,et al.Acute hepatitis due to shen-min:a herbal product derived from Polygonum multiflorum[J].J Clin Gastroenterol,2006,40(7):629-632.
[17]程生辉,张妍妍,李会芳,等.基于黄疸模型大鼠的栀子苷急性肝肾毒性研究[J].中国实验方剂学杂志,2015,21(4):174-178.
[18]程生辉,唐超,李会芳,等.栀子苷对正常大鼠急性肝、肾毒性的时-毒关系分析[J].中国实验方剂学杂志,2016,22(1):162-165.
[19]张海虹,卫璐戈,李会芳.栀子苷对正常和黄疸模型大鼠的亚急性肝肾毒性[J].中国实验方剂学杂志,2018,24(20):140-144.