目的 基于脂多糖(LPS)诱导大鼠肝脏损伤建立何首乌肝毒性模型,探究何首乌乙醇提取液(AEP)对大鼠细胞色素P450(CYP450)酶主要亚型活性及蛋白表达的影响。方法 雄性SD大鼠100只,随机分为6组:对照组、LPS组、对乙酰氨基酚(APAP)组、(LPS+APAP)组、AEP组和(LPS+AEP)组。尾iv 4 mg/kg LPS,2 h后,按组别分别ig 625 mg/kg APAP和6 g/kg AEP,每天1次,连续给药7 d,每天观察大鼠体质量变化。在建模过程中,取第2、14小时和5、8天4个不同时间点,分别对各组别动物麻醉后取血,检测肝功能生化指标丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)和碱性磷酸酶(ALP)活性;解剖,记录肝脏质量;苏木精-伊红(HE)染色进行组织病理学检查;试剂盒法测定肝细胞中细胞色素CYP1A2、CYP2E1、CYP3A1活性的变化;提取肝脏蛋白,应用Western blotting法检测CYP1A2、CYP2E1、CYP3A1蛋白表达情况。结果 与对照组比较,第2和14小时,LPS组、(LPS+APAP)组和(LPS+AEP)组ALT、AST和ALP活性显著升高;组织病理学检查发现,肝细胞灶状坏死,伴炎细胞浸润;给药后第8天,LPS组组织病理学检查正常,但(LPS+AEP)组可见显著肝细胞变性,局部慢性炎性灶。第8天,AEP组大鼠肝脏CYP1A2、CYP2E1、CYP3A1的活性明显降低;Western blotting法检测发现,AEP能显著降低大鼠肝脏CYP1A2蛋白的表达,而对CYP2E1和CYP3A1蛋白表达没有显著影响。结论 经LPS诱导,AEP对SD大鼠产生明显的肝脏毒性,毒性的发生及LPS诱发的免疫作用与抑制CYP1A2、CYP2E1、CYP3A1的活性和抑制CYP1A2蛋白表达有关。

Objective Based on LPS-induced animal model of the hepatotoicity of Polygonum multiflorum in rat liver, we aimed to study the influence of alcohol extract of P. multiflorum (AEP) on the activity of main subtypes of CYP450s and the expression of those protein during activated in the rats. Methods Male SD rats (120 rats) were randomly divided into 6 groups: control, LPS, acetaminophen (APAP), LPS + APAP, AEP, and LPS + AEP group, iv injected LPS (4 mg/kg). After 2 h, the rats in different groups were orally administered with APAP (625 mg/kg) and AEP (6 g/kg) respectively, once daily, continuous administration for 7 d. We observed the change in their weight every day and collected their abdominal aortic blood respectively at 2 h, 14 h, 5 d, and 8 d for each group. Then the biochemical indicators were detected and the body weight ratio of liver was recorded using histopathological HE staining. Furthermore, we detected the activities of CYP1A2, CYP2E1, and CYP3A1 cytochrome in liver cells; Western blotting method was used to analyze the expression of CYP1A2, CYP2E1, and CYP3A protein. Results Compared with the control group, the level of ALT, AST, and ALP in LPS group, LPS + APAP group, and LPS + AEP group increased significantly at 2 h and 14 h after injection of LPS; Histopathological examination revealed that LPS group, LPS + APAP group, and LPS + AEP group showed focal necrosis of liver cells, with inflammatory cell infiltration 2 h and 14 h after injection of LPS, 8 d after the administration, In LPS group, histopathological examination was normal, but LPS + AEP group showed significant liver cell degeneration and local chronic inflammatory lesions. Fluorescence assay found in rats induced by LPS in 8 d, AEP could significantly reduce the activities of CYP1A2, CYP2E1, and CYP3A1 in rat liver; Western blotting method was used to detect the protein expression of CYP1A2, CYP2E1, and CYP3A1, AEP can significantly reduce the protein expression of CYP1A2 in rat liver, whereas without significant effect on the expression of CYP2E1 and CYP3A1. Conclusion After induction by LPS, AEP produces significant liver toxicity to SD rats, the occurrence of toxicity and LPS-induced immune function were related to inhibition of CYP1A2, CYP2E1, and CYP3A1 activity and inhibition of CYP1A2 protein expression.

重大新药创制科技重大专项(2013ZX09302303);重大新药创制科技重大专项(2012ZX09301-001-008);北京市科委基金项目(Z131100006513010)