何首乌不同萃取部位对斑马鱼幼鱼的肝脏毒性观察

Observation of liver toxicity of different extraction parts of polygonum multiflorum on zebrafish larvae

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.