何首乌不同萃取部位对斑马鱼幼鱼的肝脏毒性观察
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摘要
目的观察何首乌不同萃取部位(氯仿部位、乙酸乙酯部位、剩余水相部位)对斑马鱼幼鱼的肝脏毒性,并探讨其作用机制。方法称取1 000 g生何首乌,制备何首乌氯仿部位、乙酸乙酯部位及剩余水相部位固体提取物,采用24 h急性毒性实验测算何首乌不同萃取部位对斑马鱼幼鱼的10%致死剂量,据此确定后续实验中何首乌不同萃取部位的给药剂量,其中氯仿部位的低、中、高给药剂量分别为3、6、9μg/m L,乙酸乙酯部位的低、中、高给药剂量分别为10、20、30μg/m L,剩余水相部位的低、中、高给药剂量分别为20、40、60μg/m L。将斑马鱼幼鱼分为a、b、c、d组,a组加入高剂量氯仿部位提取物,b组加入高剂量乙酸乙酯部位提取物,c组加入高剂量剩余水相部位提取物,d组加入等量培养水;各组培养24 h后,显微镜下观察各组斑马鱼幼鱼肝脏病理学变化,测算肝体比、卵黄囊占比。将斑马鱼幼鱼分为A1、A2、A3、A4、B1、B2、B3、B4、C1、C2、C3、C4组,其中A1、A2、A3组分别加入低、中、高剂量氯仿部位提取物,B1、B2、B3组分别加入低、中、高剂量乙酸乙酯部位提取物,C1、C2、C3组分别加入低、中、高剂量剩余水相部位提取物,A4、B4、C4组加入等量培养水;各组培养24 h后,采用赖氏法检测各组斑马鱼幼鱼ALT、AST,羟胺法检测各组斑马鱼幼鱼T-SOD。结果 c、d组斑马鱼幼鱼肝脏透明,轮廓明显,形态完整,a、b组斑马鱼幼鱼肝脏变为暗灰色,透明度降低,肝脏变大; a、b、c、d组斑马鱼幼鱼肝体比分别为9. 631%±0. 011%、10. 061%±0. 014%、8. 235%±0. 010%、7. 785%±0. 013%,a、b组与d组相比,P均<0. 05; a、b、c、d组斑马鱼幼鱼卵黄囊占比分别为3. 679%±1. 131%、3. 648%±1. 388%、3. 862%±1. 345%、3. 893%±0. 601%,组间相比,P均> 0. 05;与d组相比,a、b组肝细胞数量减少,并出现空泡变性及排列不规则现象,显示出肝损伤。A1、A2、A3组ALT水平均显著高于A4组(P均<0. 05),B1、B2、B3组ALT水平均显著高于B4组(P均<0. 05),C2、C3组ALT水平均显著高于C4组(P均<0. 05); A3组AST水平显著高于A4组(P <0. 05),B3组AST水平显著高于B4组(P <0. 05); A3组T-SOD水平显著高于A4组(P <0. 05),B1、B2、B3组T-SOD水平均显著高于B4组(P均<0. 05)。结论何首乌氯仿部位和乙酸乙酯部位对斑马鱼幼鱼具有肝脏毒性,其作用机制可能是何首乌中的毒性成分通过破坏斑马鱼幼鱼肝细胞的氧化应激平衡,进而导致肝细胞损伤。
Objective To observe the hepatotoxicity of different extraction sites( chloroform,ethyl acetate and residual aqueous phase) of polygonum multiflorum on zebrafish larvae and to explore its mechanism. Methods We weighed 1000 g polygonum multiflorum,and prepared solid extracts of chloroform,ethyl acetate and residual aqueous phase of polygonum multiflorum,and measured the 10% lethal dose of different extracts of polygonum multiflorum on zebrafish larvae by24 h acute toxicity test. The dosages of different extraction sites of polygonum multiflorum in the subsequent experiments were determined. The low-dose,medium-dose and high-dose chloroform were 3,6,and 9 μg/m L,respectively. The lowdose,medium-dose and high-dose ethyl acetate were 10,20,and 30 μg/m L,respectively. The low,medium and high doses of the remaining aqueous phase were 20,40,and 60 μg/mL,respectively. The zebrafish larvae were divided into groups a,b,c and d. The high-dose chloroform extract was added to the group a,ethyl acetate extract was added to the group b,residual water extract was added to the group c,and the same amount of culture water was added to the group d.After 24-hour incubation,the pathological changes of liver of each group were observed under the microscope,and the liver-body ratio and yolk sac ratio were calculated. The zebrafish larvae were divided into the groups A1,A2,A3,A4,B1,B2,B3,B4,C1,C2,C3,and C4. The low-dose,medium-dose and high-dose chloroform extracts were added to the groups A1,A2 and A3; the low-dose,medium-dose and high-dose ethyl acetate extracts were added to the groups B1,B2 and B3; and the low-dose,medium-dose and high-dose residual water extracts were added to the groups C1,C2 and C3,respectively,and the same amount of culture water were added to the groups A4,B4,and C4,respectively.The ALT and AST of zebrafish larvae in each group were detected by Reiss method,and T-SOD of zebrafish larvae in each group was detected by hydroxylamine method. Results The liver of zebrafish larvae in the groups a,b,c and d was transparent,clear and intact. The liver of zebrafish larvae in the groups a,b,c and d became dark grey,with decreased transparency and enlarged liver. The liver-body ratios of zebrafish larvae in the groups a,b,c and d were9. 631% ± 0. 011%,10. 061% ± 0. 014%,8. 235% ± 0. 010%,and 7. 785% ± 0. 013%,respectively. The yolk sac percentage of zebrafish larvae in the groups a,b,c and d were 3. 679% ± 1. 131%,3. 648% ± 1. 388%,3. 862% ±1. 345% and 3. 893% ± 0. 601%,respectively,with no statistically significant difference( all P > 0. 05). Compared with the group d,the number of hepatocytes in the groups a and b decreased,and there were vesicular degeneration and irregular arrangement,indicating the liver injury. The ALT levels of the groups A1,A2,and A3 were significantly higher than those of the group A4( P < 0. 05),the groups B1,B2,and B3 were significantly higher than group B4( P <0. 05),and the groups C2 and C3 were significantly higher than the group C4 group( P < 0. 05). The AST levels of the group A3 was significantly higher than that of the group A4( P < 0. 05),and the group B3 was significantly higher than the group B4( P < 0. 05). T-SOD level in the group A3 was significantly higher than that in the group A4( P < 0. 05),and those in the groups B1,B2 and B3 were significantly higher than that in the group B4( all P < 0. 05). Conclusions The chloroform and ethyl acetate fractions of polygonum multiflorum have hepatotoxicity to zebrafish larvae. The mechanism may be that the toxic components of polygonum multiflorum destroy the balance of oxidative stress in zebrafish,and then lead to hepatocyte damage.
Objective To observe the hepatotoxicity of different extraction sites( chloroform,ethyl acetate and residual aqueous phase) of polygonum multiflorum on zebrafish larvae and to explore its mechanism. Methods We weighed 1000 g polygonum multiflorum,and prepared solid extracts of chloroform,ethyl acetate and residual aqueous phase of polygonum multiflorum,and measured the 10% lethal dose of different extracts of polygonum multiflorum on zebrafish larvae by24 h acute toxicity test. The dosages of different extraction sites of polygonum multiflorum in the subsequent experiments were determined. The low-dose,medium-dose and high-dose chloroform were 3,6,and 9 μg/m L,respectively. The lowdose,medium-dose and high-dose ethyl acetate were 10,20,and 30 μg/m L,respectively. The low,medium and high doses of the remaining aqueous phase were 20,40,and 60 μg/mL,respectively. The zebrafish larvae were divided into groups a,b,c and d. The high-dose chloroform extract was added to the group a,ethyl acetate extract was added to the group b,residual water extract was added to the group c,and the same amount of culture water was added to the group d.After 24-hour incubation,the pathological changes of liver of each group were observed under the microscope,and the liver-body ratio and yolk sac ratio were calculated. The zebrafish larvae were divided into the groups A1,A2,A3,A4,B1,B2,B3,B4,C1,C2,C3,and C4. The low-dose,medium-dose and high-dose chloroform extracts were added to the groups A1,A2 and A3; the low-dose,medium-dose and high-dose ethyl acetate extracts were added to the groups B1,B2 and B3; and the low-dose,medium-dose and high-dose residual water extracts were added to the groups C1,C2 and C3,respectively,and the same amount of culture water were added to the groups A4,B4,and C4,respectively.The ALT and AST of zebrafish larvae in each group were detected by Reiss method,and T-SOD of zebrafish larvae in each group was detected by hydroxylamine method. Results The liver of zebrafish larvae in the groups a,b,c and d was transparent,clear and intact. The liver of zebrafish larvae in the groups a,b,c and d became dark grey,with decreased transparency and enlarged liver. The liver-body ratios of zebrafish larvae in the groups a,b,c and d were9. 631% ± 0. 011%,10. 061% ± 0. 014%,8. 235% ± 0. 010%,and 7. 785% ± 0. 013%,respectively. The yolk sac percentage of zebrafish larvae in the groups a,b,c and d were 3. 679% ± 1. 131%,3. 648% ± 1. 388%,3. 862% ±1. 345% and 3. 893% ± 0. 601%,respectively,with no statistically significant difference( all P > 0. 05). Compared with the group d,the number of hepatocytes in the groups a and b decreased,and there were vesicular degeneration and irregular arrangement,indicating the liver injury. The ALT levels of the groups A1,A2,and A3 were significantly higher than those of the group A4( P < 0. 05),the groups B1,B2,and B3 were significantly higher than group B4( P <0. 05),and the groups C2 and C3 were significantly higher than the group C4 group( P < 0. 05). The AST levels of the group A3 was significantly higher than that of the group A4( P < 0. 05),and the group B3 was significantly higher than the group B4( P < 0. 05). T-SOD level in the group A3 was significantly higher than that in the group A4( P < 0. 05),and those in the groups B1,B2 and B3 were significantly higher than that in the group B4( all P < 0. 05). Conclusions The chloroform and ethyl acetate fractions of polygonum multiflorum have hepatotoxicity to zebrafish larvae. The mechanism may be that the toxic components of polygonum multiflorum destroy the balance of oxidative stress in zebrafish,and then lead to hepatocyte damage.
引文
[1]国家药典委员会.中华人民共和国药典[M].北京:中国医药科技出版社,2015:175-176.
[2]Lin L,Lin H,Zhang M,et al. A novel method to analyze hepatotoxic components in Polygonum multiflorum using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry[J]. J Hazard Mater,2015,299:249-259.
[3]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(5):744-752.
[4]全正扬,孙震晓.基于斑马鱼模型的何首乌水提物及其主要成分的肝毒性研究[J].中国药物警戒,2018,15(1):1-5.
[5]胡锡琴,李娅琳,王磊.何首乌中鞣质对大鼠肝脏生化指标的影响[J].药物评价研究,2010,33(1):63-65.
[6]Meng Y,Li C,Li R,et al. Cis-stilbene glucoside in Polygonum multiflorum induces immunological idiosyncratic hepatotoxicity in LPS-treated rats by suppressing PPAR-γ[J]. Acta Pharmacol Sin,2017,38(10):1340-1352.
[7]Li C,Niu M,Bai Z,et al. Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb,Polygonum multiflorum[J]. Front Medprc,2017,11(2):253-265.
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[10]Barbazuk WB,Korf I,Kadavi C,et al. The Syntenic relationship of the zebrafish and human genomes[J]. Genome Res,2000,10(9):1351.
[11]Santos-Ledo,Adrián,Jenny A,et al. Comparative gene expression analysis of the fmnl family of formins during zebrafish development and implications for tissue specific functions[J]. Gene Expr Patterns,2013,13(1-2):30-37.
[12]Jardine D,Litvak MK. Direct yolk sac volume manipulation of zebrafish embryos and the relationship between offspring size and yolk sac volume[J]. J Fish Biol,2003,63(2):10.
[13]Li DK,Chen J,Ge ZZ,et al. Hepatotoxicity in rats induced by aqueous extract of polygoni multiflori radix,root of polygonum multiflorum related to the activity inhibition of CYP1A2 or CYP2E1[J].Evid-Based Compl Alt,2017,2017(2):1-11.
[14]黄春连,范雪梅,黎倩,等.制何首乌对大鼠肝脏P450酶5种亚型mRNA表达的影响[J].中国中药杂志,2017,42(2):352-356.
[15]Conrado DJ,Rogers HL,Zineh I,et al. Consistency of drug-drug and gene-drug interaction information in US FDA-approved drug labels[J]. Pharmacogenomics,2013,14(2):215-223.
[16]Li CY,Tu C,Gao D,et al. Metabolomic study on idiosyncratic liver injury induced by different extracts of polygonum multiflorumin rats integrated with pattern recognition and enriched pathways analysis[J]. Front Pharmacol,2016,7(30):483.
[17]余芬芳,唐天乐,白娟娟,等.不同再生水消毒方式对斑马鱼胚胎毒性的影响及其危害分级[J].生态毒理学报,2015,10(2):313-319.
[18]柳敏娜,刘天龙,刘利敏,等.α-乳香酸灌胃对小鼠肾间质纤维化的防治作用及其机制探讨[J].山东医药,2018,58(7):18-22.
[19]田文静,白伟,赵春禄,等.纳米ZnO对斑马鱼胚胎抗氧化酶系统的影响[J].中国环境科学,2010,30(5):705-709.
[20]Zhang C,Zhou T,Wang J,et al. Acute and chronic toxic effects of fluoxastrobin on zebrafish(Danio rerio)[J]. Sci Total Environ,2018,610-611:769-775.
[2]Lin L,Lin H,Zhang M,et al. A novel method to analyze hepatotoxic components in Polygonum multiflorum using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry[J]. J Hazard Mater,2015,299:249-259.
[3]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(5):744-752.
[4]全正扬,孙震晓.基于斑马鱼模型的何首乌水提物及其主要成分的肝毒性研究[J].中国药物警戒,2018,15(1):1-5.
[5]胡锡琴,李娅琳,王磊.何首乌中鞣质对大鼠肝脏生化指标的影响[J].药物评价研究,2010,33(1):63-65.
[6]Meng Y,Li C,Li R,et al. Cis-stilbene glucoside in Polygonum multiflorum induces immunological idiosyncratic hepatotoxicity in LPS-treated rats by suppressing PPAR-γ[J]. Acta Pharmacol Sin,2017,38(10):1340-1352.
[7]Li C,Niu M,Bai Z,et al. Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb,Polygonum multiflorum[J]. Front Medprc,2017,11(2):253-265.
[8]Westerfield M. The zebrafish book[M]. Eugene:University of Oregon Press,1995:7-196.
[9]赵崇军,田敬欢,王金凤,等.斑马鱼在中药研究中的应用进展[J].中草药,2015,46(17):2635-2648.
[10]Barbazuk WB,Korf I,Kadavi C,et al. The Syntenic relationship of the zebrafish and human genomes[J]. Genome Res,2000,10(9):1351.
[11]Santos-Ledo,Adrián,Jenny A,et al. Comparative gene expression analysis of the fmnl family of formins during zebrafish development and implications for tissue specific functions[J]. Gene Expr Patterns,2013,13(1-2):30-37.
[12]Jardine D,Litvak MK. Direct yolk sac volume manipulation of zebrafish embryos and the relationship between offspring size and yolk sac volume[J]. J Fish Biol,2003,63(2):10.
[13]Li DK,Chen J,Ge ZZ,et al. Hepatotoxicity in rats induced by aqueous extract of polygoni multiflori radix,root of polygonum multiflorum related to the activity inhibition of CYP1A2 or CYP2E1[J].Evid-Based Compl Alt,2017,2017(2):1-11.
[14]黄春连,范雪梅,黎倩,等.制何首乌对大鼠肝脏P450酶5种亚型mRNA表达的影响[J].中国中药杂志,2017,42(2):352-356.
[15]Conrado DJ,Rogers HL,Zineh I,et al. Consistency of drug-drug and gene-drug interaction information in US FDA-approved drug labels[J]. Pharmacogenomics,2013,14(2):215-223.
[16]Li CY,Tu C,Gao D,et al. Metabolomic study on idiosyncratic liver injury induced by different extracts of polygonum multiflorumin rats integrated with pattern recognition and enriched pathways analysis[J]. Front Pharmacol,2016,7(30):483.
[17]余芬芳,唐天乐,白娟娟,等.不同再生水消毒方式对斑马鱼胚胎毒性的影响及其危害分级[J].生态毒理学报,2015,10(2):313-319.
[18]柳敏娜,刘天龙,刘利敏,等.α-乳香酸灌胃对小鼠肾间质纤维化的防治作用及其机制探讨[J].山东医药,2018,58(7):18-22.
[19]田文静,白伟,赵春禄,等.纳米ZnO对斑马鱼胚胎抗氧化酶系统的影响[J].中国环境科学,2010,30(5):705-709.
[20]Zhang C,Zhou T,Wang J,et al. Acute and chronic toxic effects of fluoxastrobin on zebrafish(Danio rerio)[J]. Sci Total Environ,2018,610-611:769-775.
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