基于临床用药特点的补骨脂斑马鱼肝毒性确证与机制探讨

Clinical characterization and mechanism exploration of Fructus Psoraleae-induced hepatotoxicity in zebrafish

目的遵循补骨脂临床用药特点,基于蛋白质组学探究其对于斑马鱼肝脏影响及其潜在肝毒性机制。方法以低、中、高浓度补骨脂水煎液(0.025、0.050、0.100 mg·mL-1)连续对成年斑马鱼给药21 d,比较斑马鱼体重变化、肝脏病理切片及酶联免疫吸附试验(ELISA)测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)的含量变化;基于鉴定的肝脏蛋白质组学,通过聚类分析和通路富集分析研究潜在肝毒性机制,并结合分子对接探究补骨脂主要肝毒性成分的潜在毒性靶点。结果补骨脂水煎液给药组斑马鱼与空白组相比,给药组斑马鱼体重明显下降,且随着给药浓度的增加体重下降更加明显。补骨脂给药组的ALT、AST含量升高,差异具有统计学意义(P<0.05),且具有明显的剂量依赖性。苏木精一伊红(HE)染色结果显示,补骨脂给药组的肝脏组织与对照组相比肝细胞排列混乱、间隙增加,伴有脂肪变性、免疫细胞侵润等现象,且病变程度随着给药剂量的升高肝脏病变越发严重。基于蛋白质组学的聚类和功能富集分析表明,补骨脂影响斑马鱼肝脏中脂质和能量代谢相关的蛋白质乙酰辅酶A乙酰转移酶1(acat1)、3-羟基酰基辅酶A脱氢酶(ehhadh)、琥珀酸脱氢酶(sdha与sdhb)的表达,且分子对接发现补骨脂中主要肝毒性成分具有与此类蛋白结合的潜能。结论长时间补骨脂给药造成肝脏脂质代谢紊乱,最终产生以脂肪变性为代表的肝毒性,其潜在毒性机制为补骨脂主要肝毒性成分介导的脂质和能量代谢相关蛋白的表达异常。

Objective To investigate the effects of Fructus Psoraleae(FP)in accordance with clinical medicinal characteristics on the liver of zebrafish based on the proteome and its potential hepatotoxic mechanism.Methods Adult zebrafish were continuously treated with low,medium,and high concentrations of FP decoction(0.025,0.050,0.100 mg·mL-1)for 21 days.Changes in zebrafish body weight,liver histopathology,and levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum detected by enzyme-linked immunosorbent assay(ELISA)were compared.Potential hepatotoxic mechanisms were studied based on identified liver proteome through cluster and pathway enrichment analysis,and molecular docking was used to explore potential toxic targets of the main hepatotoxic components of FP.Results Zebrafish treated with FP decoction showed a significant decrease in body weight compared to the control group,with a more pronounced weight loss as the concentration increased.The levels of ALT and AST were significantly elevated in the Angelica sinensis treated group,with a dose-dependent effect(P<0.05).Hematoxylin-eosin(HE)staining results showed disordered arrangement of liver cells,increased gaps,and phenomena such as steatosis and immune cell infiltration in the liver tissues of the FP-treated group compared to the control group,with the severity of the lesions worsening with increasing concentration.Cluster and functional enrichment analysis of the proteome indicated that FP affected the expression of lipid and energy metabolism-related proteins,including acetyl-CoA acetyltransferase 1(acat1),3-hydroxyacyl-CoA dehydrogenase(ehhadh),succinate dehydrogenase(sdha)and succinate dehydrogenase(sdhb)in the zebrafish liver,and molecular docking revealed the potential of the main hepatotoxic components in FP to bind to these proteins.Conclusion Long-term Fructus Psoraleae administration causes hepatic lipid metabolism disorders,ultimately resulting in hepatotoxicity characterized by steatosis,with the potential toxicity mechanism involving abnormal expression of lipid and energy metabolism-related proteins induced by main hepatotoxic compounds in FP.