基于斑马鱼模型研究补骨脂肝损伤效应及作用机制

目的 采用斑马鱼模型探究补骨脂提取物对肝脏的毒性作用。方法 根据斑马鱼幼鱼在不同浓度的补骨脂水提物和醇提物中的存活数和死亡数，计算出幼鱼死亡率初步评估补骨脂水提物和醇提物的毒性大小。药浴后的斑马鱼幼鱼进行组织匀浆，通过生化仪测定谷丙转氨酶（ALT），谷草转氨酶（AST）和乳酸脱氢酶（LDH）的酶活。斑马鱼成鱼给药24h后冰浴处死，进行HE染色观察肝脏病理变化。采用qPCR和Western Blot法检测成鱼肝脏中固醇调节元件结合蛋白-1c（SREBP-1c），脂肪酸合成酶（FAS），乙酰辅酶A羧化酶（ACC1）以及微粒体甘油三脂转移蛋白（MTP）的mRNA表达和蛋白表达。结果 与正常组相比，补骨脂药浴组的斑马鱼幼鱼在2dpf时出现死亡。并且补骨脂对幼鱼的毒性具有一定的浓度依赖性。相同浓度下，醇提`物的毒性大于水提物。HE染色结果可观察到补骨脂醇提和水提给药组的成鱼中肝脏发生脂变，同时给药组的斑马鱼幼鱼匀浆组织液中ALT，AST和LDH含量明显高于空白组。通过qPCR实验发现，补骨脂水提物和醇提物给药组的斑马鱼肝脏中SREBP-1c，FAS，ACC1的表达升高，MTP的表达降低。Western Blot检测结果与qPCR结果一致。结论 浓度为300μg/mL和600μg/mL的补骨脂水提物和浓度为40μg/mL和80μg/mL的补骨脂醇提物药浴给药48h会导致斑马鱼的肝脏产生病变，甚至死亡，且机制可能与肝脂变有关。 

The Liver Toxicity and Mechanism of Psoralen Based on Zebrafish

OBJECTIVE Using zebrafish model to explore the toxic of Psoralen on liver METHODS Recording the number of the survival and the death of zebrafish larval in different concertrations of ethanol extract and water extract， to calculate the mortality and evaluate their toxicity. Adult zebrafish were sacrificed after 24h of treatment and remove the liver， Ultrasonic homogenization treatment of zebrafish larval and collect the tissue fluid to detect the ALT， AST and LDH. HE staining was used to observe the change of liver. Meanswhile qPCR was used to detect the mRNA expressions of SREBP-1c，FAS，ACC1 and MTP， At last， Western Blot assay was used to detect the protein expression of SREBP-1c，FAS，ACC1 and MTP. RESULTS The result shows that compared with the control group， the treatmeat groups lead zebrafish larval to death obviously. And the LD₅₀ decreased with the increase of the concentration of the extractive， besides， the toxicity of ethanol extract was greater than water extract at the same concentration. The liver had a degree of hepatic steatosis in treatment groups， and the level of ALT， AST and LDH of tissue fluid in treatments were higher than control group. qPCR results showed that the mRNA of SREBP-1c，FAS and ACC1 level were significantly increased while the level of MTP level was decreased after treatmeat， Western Blot results were the same with qPCR. CONCLUSION The experiment shows that at the concentration of 40μg/mL and 80μg/mL ethanol extract of Psoralen and at the concentration of 300μg/mL and 600μg/mL water extract for 48hours will make zebrafish liver bad even to death. And the possible mechanism is related to hepatic steatosis.