【作者】 陈霞；

【导师】 孙慧君；

【作者基本信息】 大连医科大学 ， 药理学， 2008， 硕士

【摘要】 目的:研究短期内应用茶多酚（tea polyphenols, TPs）对小鼠细胞色素P450 （CYP450）和b5含量及CYP2E1和CYP1A2表达的影响。方法:1.测定CYP450和b₅含量:雄性小鼠分别按小剂量（25 mg/kg﹒d^-）、中剂量（50 mg/kg﹒d^-）和高剂量（100 mg/kg﹒d^-）TPs腹腔注射给药两天。氯霉素为阳性对照组,在取肝前1小时以50 mg/kg﹒d^-腹腔注射给药;阴性对照组给等量生理盐水,在第三天所有小鼠断头处死取肝,制备肝微粒体,用紫外分光光度计测定小鼠肝脏CYP450和b5含量。2. TPs对扑热息痛所致小鼠死亡率的影响:雄性小鼠分别按100 mg/kg﹒d^-、200 mg/kg﹒d^-和400 mg/kg﹒d^- TPs灌胃给药6天。阳性对照组给予900 mg/kg的N-乙酰半胱氨酸,阴性对照组给予等量的生理盐水。末次给药后1小时,所有小鼠灌胃扑热息痛1000 mg/kg,记录72小时内各组小鼠的死亡数量并计算出药物的保护率。3. TPs对CYP2E1和CYP1A2表达的影响:雄性小鼠分别按100 mg/kg﹒d^-、200 mg/kg﹒d^-和400 mg/kg﹒d^- TPs灌胃给药5天。阳性对照组和阴性对照组分别给予等量的生理盐水。末次给药后1小时,除生理盐水组外,所有小鼠灌胃扑热息痛500 mg/kg,24小时后断头处死立即取肝,用冷的生理盐水冲洗,把部分肝脏标本放于10%福尔马林固定和-80℃中冻存。用病理切片观察肝脏病变,用免疫组化、Western blot和RT-PCR方法检测CYP2E1和CYP1A2表达情况。结果:与阴性对照组相比,TPs三个剂量组小鼠CYP450和b₅含量明显降低,差异显著（P<0.01）,且有剂量依赖性;TPs能显著降低扑热息痛所致小鼠的死亡率和改善扑热息痛所致肝损伤病理改变;与阴性对照组相比,TPs中剂量组和低剂量组能显著降低CYP2E1蛋白和mRNA表达（P<0.01）,而TPs各剂量组能显著降低CYP1A2蛋白和mRNA表达（P<0.05）。结论:1.短期应用TPs对CYP450和b5有抑制作用并呈剂量依赖性。2.在扑热息痛诱发的小鼠肝损伤模型中,TPs显示了显著的肝脏保护作用,表现为小鼠的死亡率明显降低和肝脏病变明显改善。3.在扑热息痛诱发的小鼠肝损伤模型中,TPs对CYP450同功酶CYP2E1和CYP1A2蛋白和mRNA表达有显著抑制作用且有剂量依赖性,这一结果提示TPs通过抑制CYP2E1和CYP1A2基因转录,使扑热息痛代谢降低,毒性产物N-乙酰-P-苯醌亚胺生成减少,这可能是TPs对扑热息痛肝损伤保护作用的机制之一。更多还原

【Abstract】 Objective: To investigate the effect of tea ployphenols （TPs） on the level of hepatic cytochrome P450 （CYP450） and b₅ along with the expression of CYP2E1 and CYP1A2 in mice after short time taking TPs.Methods: 1. Kunming male mice were intraperitoneally injected with TPs at the doses of 25, 50 and 100 mg/ kg﹒d^- for two days. The positive control group was given chloramphenicol at the dose of 50 mg/kg﹒d^- 1h before killing the mice. The negative control group was given the same volume of saline solution. After the mice were killed by decapitation, The livers were removed and microsomes were isolated and then, the contents of CYP450 and b5 were measured by UV-spectrophotometry and compared with control group mice. 2. To evaluate the detoxification effect of TPs, the male mice were intragastricly pre-treated with TPs at the doses of 100, 200 and 400 mg/ kg﹒d^- for six days, The positive control group was given N-Broncholysin at the dose of 900 mg/kg﹒d^-. The negative control group was given the same volume of saline solution and then, all the mice were given paracetamol at the dose of 1000 mg/kg. The acute mortality was compared with control mice. 3. To examine the expression of CYP2E1 and CYP1A2, male mice were pre-treated with TPs （100, 200, and 400 mg/kg﹒d^-） for five days. The model group and the negative control group were given the same volume of saline solution. Then the mice were given paracetamol at the dose of 500 mg/kg 1 hour later after the last time TPs administration except the mice in negative control group. The livers were removed immediately and washed with cold saline solution .Then all specimens were fixed in formalin for histological examinations or snap-frozen in liquid nitrogen and stored at -80℃for protein and RNA extraction. CYP2E1 and CYP1A2 protein and mRNA expression levels in the liver were evaluated by western blotting, immunohistochemical staining and RT-PCR.Results: The contents of CYP450 and b5 in the livers of mice in three TPs-treated groups were dose-dependently decreased compared with the negative control mice （P<0.01） after short time taking TPs. TPs treatment significantly improved hepatic lesions and led to a dramatic reduction of the mortality of paracetamol-treated mice. TPs treatment resulted in a significant reduction of the CYP2E1 and CYP1A2 expression at both protein and mRNA levels in a dose-dependent manner （P<0.01）.Conclusions:1. TPs could suppress both CYP450 and b5 in a dose dependent manner after short time administration.2. In the paracetamol induced mouse liver injury model, TPs demonstrated obvious hepato-protective effects dose-dependently which were shown by substantially lowering the mortality of mice and obviously improving hepatic lesions.3. In the paracetamol induced mouse liver injury model, TPs could supressed CYP450 isoform CYP2E1 and CYP1A2 expression to defend paracetamol-induced liver damage. This result suggested TPs could reduced paracetamol’s metabolism and decrease the production of the hepatotoxic N-acetyl-p-benzoquinonimine by suppressing genetic transcription of CYP2E1 and CYP1A2. This might be one of the mechanisms to hepato-protective effects of TPs.更多还原