【作者】 郑斌；

【导师】 许建华；

【作者基本信息】 福建医科大学 ， 药理学， 2010， 博士

【摘要】 第一部分目的研究姜黄素及其类似物L3对体外培养的人脐静脉内皮细胞氧化损伤的作用,并探讨其可能的调节作用机制。方法以过氧化氢(H2O2)诱导HUVEC损伤,体外模拟自由基损伤模型。采用MTT法以细胞存活率为指标评价姜黄素及其类似物L3对氧化损伤的HUVEC的保护作用。测定H2O2损伤HUVEC后细胞超氧化物歧化酶(SOD),谷胱甘肽-S-转移酶(GST)、谷胱甘肽过氧化物酶(GSH-PX)和过氧化氢酶(CAT)活性和还原型谷胱甘肽(GSH)及丙二醛(MDA)含量的变化。DCF-DA荧光试剂法检测胞内活性氧(ROS)的含量。DAF-FM DA荧光试剂法检测胞内一氧化氮(NO)的含量,硝酸还原酶法检测胞外NO的含量。Western blotting检测内皮细胞一氧化氮合酶eNOS的表达。分光光度法检测Caspase 3和Caspase 9的活性,Western blotting检测其蛋白表达。罗丹明123荧光试剂法检测线粒体膜电位。Western blotting检测ERK1/2磷酸化、NF-кB核转位及p53蛋白表达。结果200μM H2O2作用36 h,细胞存活率显著降低。H2O2诱导产生显著的细胞凋亡特征,包括线粒体去极化及Caspase 3和Caspase 9的活性和蛋白表达显著增加。另外,H2O2还诱导丙二醛(MDA)含量增加、谷胱甘肽-S-转移酶(GST)活性和胞内活性氧(ROS)产生增强,诱导还原型谷胱甘肽(GSH)和胞内/胞外一氧化氮(NO)含量降低,细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)和过氧化氢酶(CAT)活性降低及内皮细胞一氧化氮合酶eNOS表达减少。12.5-50μM L3与H2O2损伤的HUVEC共孵育,细胞存活率显著增加,具有浓度依赖性;而12.5-25μM姜黄素对H2O2损伤的HUVEC无保护作用。L3可以抑制H2O2诱导的细胞凋亡,减轻H2O2诱导的其它损伤,且具有浓度依赖性。L3还可以抑制H2O2诱导的p53和NF-кB活化,促进ERK1/2磷酸化。结论姜黄素类似物L3可以浓度依赖性地减轻H2O2诱导的HUVEC损伤,保护血管内皮细胞。其保护作用机制可能与减少ROS生成,增强抗氧化酶活性,改善细胞氧化/抗氧化状态,提高内皮细胞eNOS的表达,激活内皮细胞NO的合成,维持线粒体膜电位,抑制细胞凋亡有关。第二部分目的研究姜黄素及其类似物L3对链脲菌素(STZ)+高脂饲料诱导的高脂血症糖尿病小鼠的作用,并探讨其对糖尿病小鼠相关器官损伤的保护作用。方法雄性ICR小鼠以隔天一次腹腔注射50 mg/kg体重STZ溶液共五次诱导糖尿病模型,随后饲以高脂饲料,并分别给予不同剂量的姜黄素或L3进行干预。各组小鼠分别在给药4周后和给药16周后处死,测定小鼠血糖、糖耐量、糖化血红蛋白、胰岛素、血脂、肝脂、肝脏GSH和MDA含量,SOD、GST、GSH-PX和CAT活性。DCF-DA荧光试剂法检测胰腺ROS的含量。DAF-FM DA荧光试剂法检测主动脉弓NO的含量,硝酸还原酶法检测血浆NO的含量。免疫组化检测主动脉弓LOX-1的表达。肝脏和主动脉弓组织切片行病理检查。结果腹腔注射STZ溶液五次后成功诱导小鼠糖尿病模型。饲以高脂饲料4周后和16周后,小鼠血脂、肝脂显著升高。各种氧化应激参数和抗氧化酶活性发生明显变化。胰腺ROS生成显著增加。血浆和主动脉弓NO含量显著降低。主动脉弓LOX-1表达显著升高。肝脏和主动脉弓发生明显的脂肪变性和动脉粥样硬化变性。姜黄素高剂量组和L3中、高剂量组小鼠糖、脂代谢明显改善,氧化应激参数和抗氧化酶活性也明显改善。胰腺ROS生成显著减弱。血浆和主动脉弓NO含量显著增加。主动脉弓LOX-1表达显著降低。肝脏和主动脉弓的脂肪变性和动脉粥样硬化变性明显改善。结论姜黄素及其类似物L3对链脲菌素(STZ)+高脂饲料诱导的高脂血症糖尿病小鼠及相关器官损伤具有保护作用。更多还原

【Abstract】 PartⅠObjective: To explore the effects of curcumin and its analogue L3 on human umbilical vein endothelial cell injury induced by oxidative stress in vitro and to investigate their possible regulatory mechanisms.Methods: Peroxide hydrogen (H2O2) was used to induce human umbilical vein endothelial cells (HUVECs) injury. The cell survival rate was evaluated with MTT assay to explore the protective effect of curcumin and its analogue L3 on vascular endothelial cells injury. The activities of cytosolic superoxide dismutase (SOD),glutathione S transferase (GST), glutathione peroxidase (GSH-PX) and catalase (CAT) and the levels of cytosolic glutathione (GSH) and malondialdehyde (MDA) were determined in H2O2-treated HUVECs. The fluorescence agent DCF-DA was used to detect intracellular generation of reactive oxygen species (ROS), and DAF-FM DA to detect intracellular generation of nitric oxide (NO). Level of extracellular nitric oxide (NO) was measured by nitrate reductase method. Protein expression of endothelial nitric oxide synthase (eNOS) in H2O2-treated HUVECs was detected by Western blotting analysis. Activities and expressions of caspase 3 and caspase 9 were determined by spectrophotometric and Western blotting assay. The fluorescence agent rhodamine 123 (RH 123) was used to detect mitochondrial membrane potential (MMP). Phosphorylation of ERK1/2, translocation of nuclear factor-kappa B (NF-кB) and protein expression of p53 were investigated by Western blotting analysis.Results: Thirty six-hour treatment with 200μM H2O2 significantly decreased the viability of HUVEC cells, which was accompanied with apparent apoptotic features including the mitochondrial depolarization and increased activities and expressions of caspase 3 and caspase 9. In addition, it is observed that H2O2 increased the amount of malondialdenhyde (MDA),the activity of glutathione S transferase (GST) and the generation of reactive oxygen species (ROS), and decreased the levels of glutathione (GSH) and intra/extracellular nitric oxide (NO), the activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-PX) and catalase (CAT) and the expression of endothelial nitric oxide synthase (eNOS) in HUVEC cells. Co-incubation with 12.5-50μM L3 resulted in a significant increase of cell viability in a concentration-dependent manner, while 12.5-25μM curcumin didn’t exhibit any protective effect. Also, L3 resulted in a significant recovery from H2O2-induced cell apoptosis and decreased other H2O2-induced damage in a concentration-dependent manner, which might be achieved via decreasing of p53 expression and NF-кB translocation and increasing of ERK1/2 phosphorylation.Conclusion: L3 could attenuate H2O2-induced injury in HUVECs in a concentration-dependent manner and showed protective effect on vascular endothelial cells. The probable mechanisms closely associated with its protective effect might include repressing ROS generation, increasing the activities of antioxidant enzymes, improving redox status, upregulating eNOS expression, promoting NO synthesis, maintaining mitochondrial membrane potential and inhibiting cell apoptosis in H2O2-exposed HUVECs.PartⅡObjective: To evaluate the effects of curcumin and its analogue L3 on streptozotocin and high cholesterol diet-induced diabetes and hyperlipidemia in mice and their possible protection of multiple organ involvement under diabetic condition.Methods: Male ICR mice were injected intraperitoneally streptozotocin solution (50mg/kg body weight)once every other day for a total of five times to induce diabetes and then were fed either only high cholesterol diet or additional intragastrically administrated curcumin or L3 of different doses. Each half of the mice were sacrificed at the end of four and sixteen weeks, the levels of plasma glucose, glucose tolerance, glycated hemoglobin, insulin,plasma and liver lipids (TG, TC, HDL-C and LDL-C), glutathione (GSH) and malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) , glutathione S transferase (GST), glutathione peroxidase (GSH-PX) and catalase (CAT) were detected. The fluorescence agent DCF-DA was used to detect reactive oxygen species (ROS) generation in pancreas, and DAF-FM DA to detect nitric oxide (NO) generation in aortic arch. Level of plasma nitric oxide (NO) was measured by nitrate reductase method. Protein expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in aortic arch was detected by immunohistochemistry analysis. Histopathological sections of the liver and aortic arch were examined.Results: After a total of five STZ injections diabetic status was induced successfully in mice. Plasma and liver lipids increased significantly four or sixteen weeks after the mice were fed high cholesterol diet. Oxidative stress parameters and activities of antioxidant enzymes altered significantly. The generation of ROS was elevated in pancreas. The level of NO was depressed significantly both in aortic arch and in plasma. Expression of LOX-1 increased significantly in aortic arch. Apparent steatosis and artherosclerosis were observed in liver and aortic arch histopathological sections. The glucose and fatty metabolisms were improved after treatment with high-dose curcumin and medium/high-dose L3. Oxidative stress parameters and activities of antioxidant enzymes were also improved. The generation of ROS was attenuated significantly in pancreas. The level of NO increased significantly both in aortic arch and in plasma. Expression of LOX-1 was downregulated significantly in aortic arch. Steatosis and artherosclerosis were attenuated in liver and aortic arch histopathological sections.Conclusion: Curcumin and its analogue L3 appear to be beneficial in improving diabetic status and in protecting multiple organ involvement in STZ and HCD-induced diabete and hyperlipidemia in mice.更多还原