Abstract:

Objective To systematically evaluate the developmental toxicity and potential cardiac and hepatic toxicity of the aqueous extract of Asarum heterotropoides based on zebrafish embryo model， to clarify its dose-response relationship and sensitive target organs， and to provide experimental evidence for the safety assessment of Asarum heterotropoides. Methods Wild-type AB strain zebrafish embryos at 1 day post-fertilization （dpf） were selected. After excluding unfertilized and abnormally developed individuals， they were randomly divided into a control group （culture medium only） and groups treated with different concentrations of the aqueous extract of Asarum heterotropoides （100， 200， 300， 500， 1 000， and 2 000 mg·L?1）， with 18 embryos in each group. Exposure lasted until 5 dpf. The hatching rate at 2 dpf and mortality rate at 5 dpf of zebrafish embryos in various groups were recorded. Concentration-effect curves were plotted， and Probit regression analysis was performed to calculate the median lethal concentration （LC₅₀）， as well as 20% lethal concentration （LC₂₀）， 10% lethal concentration（LC₁₀）， and 5% lethal concentration（LC₅）. At 5 dpf， morphological changes in zebrafish embryos in various groups were observed. Heart rate， sinus venosus-bulbus arteriosus （SV-BA） distance， and pericardial area of zebrafish embryos in various groups were measured to evaluate embryonic cardiac structure and function. Oil Red O staining was used to determine the hepatic integrated optical density （IOD） values to assess the levels of lipid accumulation in the liver of zebrafish embryos in various groups. Results Compared with control group， the hatching rates of zebrafish embryos in 300 mg·L^-1 group and higher concentration groups were significantly decreased （P<0.05）， while the mortality rates were significantly increased （P<0.01）， in a concentration-dependent manner. Probit regression analysis results showed that the LC₅₀ of the aqueous extract of Asarum heterotropoides was 641.11 mg·L^-1， and the LC₂₀， LC₁₀， and LC₅ were 317.78， 223.33， and 146.67 mg·L^-1， respectively. Morphological observation revealed that the zebrafish embryos in 200 mg·L^-1 group and higher concentration groups exhibited abnormalities such as delayed yolk sac absorption， pericardial edema， jaw malformation， and shortened body length. Compared with control group， the heart rates of zebrafish embryos in LC₁₀ group and LC₂₀ group were significantly decreased （P<0.01）， while the SV-BA distances and pericardial areas of zebrafish embryos were significantly increased （P<0.01）， indicating impaired cardiac structural development and functional status. The Oil Red O staining results showed increased lipid deposition in the liver of the zebrafish embryos in LC₅ group， LC₁₀ group and LC₂₀ group， and the levels of lipid accumulation in the liver were significantly higher than that in control group （P<0.01）， among which LC₂₀ group showed the highest degree of lipid deposition in the liver. Conclusion The aqueous extract of Asarum heterotropoides showed concentration-dependent developmental toxicity on zebrafish embryos and may induce cardiac structural and functional abnormalities as well as increased hepatic neutral lipid deposition at doses below the LC₅₀. The results suggested that the heart and liver may be its sensitive target organs.

Key words: Asarum heterotropoides, Zebrafish embryo, Developmental toxicity, Cardiotoxicity, Hepatotoxicity