小柴胡汤对D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的保护作用

Protective effect of Xiaochaihu Decoction on D-GalN/LPS-induced hepatocyte injury in hybrid grouper(Epinephelus lanceolatusy♂×E.fuscoguttatus♀)

为探讨小柴胡汤对虎龙杂交石斑鱼(Epinephelus lanceolatus♂×E.fuscoguttatus♀)化学性肝细胞损伤的免疫功能影响,用D-氨基半乳糖(D-GalN,20 mmol·L^(–1))和脂多糖(LPS,1μg·mL^(–1))作用肝细胞24 h构建虎龙杂交石斑鱼肝细胞损伤模型,用不同质量浓度的小柴胡汤(100、200和400μg·mL^(–1))对肝细胞进行前处理[D-氨基半乳糖联合脂多糖(DGalN/LPS)损伤前加药],通过CCK-8法检测肝细胞活力,HE染色观察肝细胞形态,收集细胞上清液检测炎症因子INFγ、COX-2和PGE2的含量,以及细胞沉淀中凋亡和免疫相关基因的表达。结果表明,小柴胡汤可以提高肝细胞的成活率,不同程度地抑制D-GalN/LPS损伤引起的肝细胞培养上清液中INF-γ活性以及COX-2和PGE2含量的升高,降低细胞内乳酸脱氢酶(LDH)、谷草转氨酶(AST)和谷丙转氨酶(ALT)活性以及细胞凋亡率,对肝细胞结构具有明显的保护作用;同时显著抑制凋亡相关基因caspase-3、caspase-9和P53的上调,促进免疫相关基因TLR3的上调。数据统计分析显示,添加200μg·mL^(–1)小柴胡汤组的抑制效果最明显。综合以上结果,小柴胡汤对虎龙杂交石斑鱼化学性肝损伤有一定的保护作用,可预防肝脏疾病的发生和发展,为鱼类保肝药物研究提供了新的见解。

The study aims to evaluate the protective effect of Xiaochaihu Decoction(XCHD)on chemical hepatocyte injury.We treated the hepatocytes with D-GalN(20 mmol·L^(–1))and LPS(1μg·mL^(–1))for 24 h to build a hybrid grouper(Epinephelus lanceolatus♂×E.fuscoguttatus♀)hepatocyte injury model,then pretreated the liver cells with different concentrations of XCHD(100,200 and 400μg·mL^(–1))(D-GalN/LPS damage),and measured the hepatocyte viability by the CCK-8 assay.Hepatocyte morphology was visualized by HE staining,and cell supernatants were collected to detect the content of the inflammatory factors INF-,COX-2,and PGE2,as well as the expression of apoptotic and immune-related genes in the cell cryoprecipitate.The results show that XCHD can improve the survival rate of hepatocytes,inhibit INF-γactivity and coX-2 and PGE2 contents in the supernatant of hepatocytes induced by D-GalN/LPS injury to a different extent,reduce LDH,AST and ALT contents and apoptosis rate of hepatocytes.Besides,it had a significant protective effect on the structure of hepatocytes,significantly inhibiting the upregulation of apoptosis-related genes caspase-3,caspase-9 and P53,and enhancing the up-regulation of immune-related genes TLR3.According to the data from the statistical analysis,the inhibition effect was most obvious in the 200μg·mL^(–1) XCHD group.In conclusion,XCHD has a protective effect on the chemical hepatocyte injury of Epinephelus lanceolatus♂×E.fuscoguttatus♀,and can prevent the occurrence and development of liver disease,which provides a new insight for the study of liver protection drugs in fish.