TLR4/Akt相关分子在DMF暴露小鼠肝损伤中的作用
Role of TLR4/Akt Related Molecules in Liver Injury of Mice Exposed to DMF
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摘要: 二甲基甲酰胺（N，N-dimethylformamide，DMF）可通过呼吸，皮肤及消化道等途径进入机体，经肝脏细胞色素P450家族成员2E1的代谢，生成活性代谢产物发挥多系统毒性。建立DMF暴露ICR小鼠模型，观察DMF对肝脏影响及潜在机制。80只雌雄各半ICR小鼠适应性喂养后随机分为对照组、低剂量组、中剂量组和高剂量组，分别以0、350、700和1 400 mg·kg-1·d-1的DMF灌胃90 d。记录体质量，苏木精-伊红（HE）、油红O染色法观察小鼠肝脏病理学改变，化学比色法测量肝脏谷丙转氨酶（alanine aminotransferase，ALT）、谷草转氨酶（aspartate aminotransferase，AST）、碱性磷酸酶（alkaline phosphatase，ALP）、总胆固醇（total cholesterol，TC）和甘油三酯（triglyceride，TG）含量变化。蛋白质免疫印迹法（Western blotting，WB）检测Toll样受体4（toll-like receptors 4，TLR4）、蛋白激酶B （protein kinase B，Akt）、核因子κB （nuclear factor kappa-B，NF-κB）活性。ELISA法检测小鼠肝脏中白细胞介素1（interleukin-1，IL-1）、白介素6（interleukin-6，IL-6）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）等炎性因子的水平。结果表明，高剂量组小鼠体质量从第5周开始显著降低，不同剂量组小鼠肝脏系数均显著增加（P<0.05），暴露组HE染色结果可见不同程度炎性损伤，油红O染色结果可见暴露组小鼠肝脏充斥着大量脂肪滴。和对照组相比，DMF引起肝脏ALT、AST和ALP含量显著上升（P<0.05），TG和TC的水平均在高剂量组出现显著增高。WB结果显示，TLR4水平及其下游分子Akt、NF-κB磷酸化活性显著增高（P<0.05）。ELISA结果显示，与对照组相比，暴露组小鼠IL-1、IL-6、TNF-α水平随着DMF暴露剂量的增加而显著增加（P<0.05）。以上结果表明，DMF亚慢性暴露通过诱导TLR4/Akt信号通路激活，影响肝脂代谢，促进NF-κB活化并介导免疫和炎症因子应答，诱导肝细胞凋亡，最终引起肝脏损伤。Abstract: N,N-dimethylformamide (DMF) can enter the body through ingestion, respiration and skin contact, and is oxidized by CYP2E1 into toxic intermediates in liver. The aim of this study was to establish an effective ICR mouse model of DMF exposure and to identify potential mechanism of its hepatotoxicity. 80 ICR mice were randomly divided into a control group and three exposure groups (350, 700 and 1 400 mg·kg-1·d-1). DMF was administered by oral gavage for 90 d. Body weight was recorded weekly. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol (TC) and triglyceride (TG) of each group were measured. The hematoxylin-eosin (H&E) and oil red O staining were performed. Total protein was extracted from the liver tissues. The expression of toll-like receptors 4 (TLR4), protein kinase B (Akt), and nuclear factor kappa-B (NF-κB) was tested by Western blotting (WB). The contents of inflammatory factors such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The results showed that the body weight of the 1 400 mg·kg-1·d-1 group was significantly decreased from the 5th week and the liver coefficient increased significantly in all DMF exposure groups (P<0.05). The H&E staining showed different degrees of inflammatory damage in the exposed groups. The accumulation of lipid droplets was observed in the livers of all DMF exposure groups. In addition, a significant increase was recorded in AST, ALT and ALP levels in DMF groups (P<0.05). Compared with the control, the levels of TG and TC increased significantly in the 1 400 mg·kg-1·d-1 group. The Western blot results showed that the expression of TLR4, p-Akt and p-NF-κB was significantly increased after exposure (P<0.05). Moreover, the levels of IL-1, IL-6, and TNF-α increased significantly in all exposed groups compared with the control group (P<0.05). Above results indicate that subchronic exposure to DMF leads to liver damage by inducing the activation of TLR4/Akt signaling pathway, interfering with lipid metabolism and promoting inflammatory responses.
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Key words:
- N,N-dimethylformamide /
- ICR mouse /
- liver injury /
- Toll-like receptor
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