【作者】 张瑜；

【导师】 许建华；

【作者基本信息】 福建医科大学 ， 药理学， 2008， 硕士

【摘要】 目的:通过复合方式建立脂肪肝体外模型和动物模型,观察并探讨姜黄素和四氢姜黄素对体内外脂肪肝模型的干预作用。方法:(1)采用0.8%乙醇和5μg/ml软脂酸共同作用于人正常肝细胞株L02的复合方式在体外建立脂肪肝细胞模型,并应用MTT法筛选姜黄素及四氢姜黄素作用于肝细胞的适合浓度,经油红O染色,观察不同给药组细胞内脂滴形成情况,并检测细胞内甘油三酯(TG)水平变化。从而观察姜黄素及四氢姜黄素在体外抗脂肪肝的作用效果。(2)通过给予高脂饲料、30%乙醇灌胃、40%CCl4葵花籽油溶液皮下注射的复合方式建立小鼠脂肪肝的模型,同时给予不同剂量的姜黄素和四氢姜黄素固体分散剂,于实验第16天,取血及肝组织,计算肝指数、检测血脂:血清TG、CHO、LDL-C,肝脏内脂质:肝匀浆TG、CHO、LDL-C,以及观察肝脏病理组织形态学改变。从而观察姜黄素及四氢姜黄素固体分散剂在小鼠体内抗脂肪肝的疗效。结果:1.(1)通过MTT法筛选出姜黄素的作用浓度为2.5μM、5.0μM、10μM,四氢姜黄素的作用浓度为5.0μM、10μM、20μM、25μM;(2)油红O染色后,在光镜下观察发现:模型组细胞内有大量的桔红色或红色脂滴积聚,而空白对照组L02细胞几乎未发现脂滴的存在。姜黄素及四氢姜黄素随着作用浓度的增加细胞内的脂滴逐渐减少;(3)根据油红O染色的结果,选择姜黄素10μM组及四氢姜黄素25μM给药浓度进一步通过检测细胞内TG的变化以便更客观地说明姜黄素及四氢姜黄素抗脂肪肝的作用效果,实验结果发现:模型组与空白对照组相比,细胞内TG含量显著增加(P<0.01)。与模型组相比,姜黄素10μM组及四氢姜黄素25μM组均能显著地降低细胞内TG的含量,分别下降了25.6%、41.3%( P<0.05)。2.(1)通过给予高脂饲料、30%乙醇灌胃、40%CCl4葵花籽油溶液皮下注射的复合方式成功建立了小鼠脂肪肝的模型,与正常组相比,模型组小鼠的肝湿重、肝指数、血清TG、CHO、LDL-C以及肝匀浆TG、CHO、LDL-C均显著升高(P<0.01)。肝脏病理组织也证实其发生了弥漫性的脂肪肝病变;(2)不同剂量姜黄素固体分散剂给药组与模型组相比,随着给药剂量的增加,对于血清以及肝匀浆中TG、CHO、LDL-C的含量均有不同程度的降低,其中血清TG、CHO以及肝匀浆TG、LDL-C的差异具有统计学意义。病理组织学检查也有改善,其中姜黄素300mg/kg组的作用最为显著;(3)与模型组相比,随着四氢姜黄素固体分散剂的浓度的增加,其降低肝湿重、肝指数、血清及肝匀浆中的TG、CHO、LDL-C作用也出现剂量依赖性增强。除了肝湿重和病理组织改变外,四氢姜黄素300mg/kg组对各指标的改善均具有统计学意义。结论:1.姜黄素及四氢姜黄素能防治由0.8%乙醇和5μg/ml软脂酸诱导的体外脂肪肝,有效地减少细胞内脂滴的形成和甘油三酯的蓄积。2.姜黄素及四氢姜黄素固体分散剂对由高脂饲料喂养、30%乙醇灌胃、40% CCl4葵花籽油溶液皮下注射的复合方式诱导的小鼠脂肪肝均具有一定的预防作用,且这种作用呈现出一定的剂量依赖性。更多还原

【Abstract】 Objectives: The study aims is to establish a compound fatty liver model in vitro and in vivo, and then investigate the effects of curcumin (Cur) and tetrahydrocurcumin (THCUR) on the fatty liver.Methods: (1) The fatty liver model was established by treating human liver cell L02 with 0.8% ethanol and 5μg/ml palmitic acid in vitro. The MTT assay was used to choose the appropriate concentration of Cur and THCUR which would be used to treat fatty liver. Oil red O staining was used to observe the intracellular lipid droplet under light microscope. The content of intracellular TG was detected by TG assay kit. (2) The compound fatty liver models were induced by high-fat feed, intragastric administration with the alcohol and subcutaneous injection with CCl4. Sixty mice were randomly divided into six groups, including normal group, model group and three different doses groups of Cur or THCUR. On the 16th day, all the mice were killed, and then the contents of TG, CHO and LDL-C in serum and liver tissue were determined. The wet weight of liver and the liver index (liver/body) were measured. The change of liver histology was observed.Result: 1. (1) MTT assay showed out the appropriate concentration of Cur and THCUR on L02 cells: 2.5μM, 5.0μM, 10μM and 5.0μM, 10μM, 20μM, 25μM separately. (2) Oil red O staining presented a large quantity of orange-red or red lipid droplet in cytoplasm of L02 cells of model group under light microscope. As the management concentration of Cur and THCUR increasing, the amount of lipid droplet in the cells was lessening, especially in the cells that treated with 10μM of Cur and 25μM of THCUR. (3) According to the result of the oil red O staining, the 10μM of Cur and 25μM of THCUR were chose to treat the L02 cells in order to observe the effect of Cur and THCUR on cellular fatty liver model by detecting the intracellular TG. Compared with normal group, the intracellular TG level of the model group significant increased(P < 0.01). Compared with the model group, the intracellular TG level of the groups that dealt with Cur and THCUR were notably decreased by 25.6%, 41.3% separately, P<0.05. 2.(1) The compound fatty liver model in mice was established successfully by high-fat feed, intragastric administration with the alcohol and subcutaneous injection with CCl4. The contents of TG, CHO and LDL-C in the serum and the liver in the model group were significant increased compared with the normal group, P<0.01. The wet weight of liver and the liver index were also increased. The livers of the model group were confirmed as fatty liver in histology. (2) The contents of TG, CHO and LDL-C in the serum and the liver were decreased in the groups of Cur compared with the model group,especially the group of 300mg/kg of Cur. The liver histology and liver index of the Cur groups were better than that of the model group. (3) Compared with the model group, THCUR caused a significant reduction in the level of TG, CHO and LDL-C in the serum and the liver tissue. And the effect may depend on its dose. Especially,the amendment of them in the groups of 300mg/kg of THCUR was significant statistically.Conclusion: 1. Both Cur and THCUR were able to prevent fatty liver induced by 0.8% ethanol and 5μg/ml palmitic acid in vitro. They both decreased lipid droplet and intracellular TG efficiently. 2. Both solid dispersion of Cur and THCUR showed preventive effect on experimental fatty liver induced by high-fat feed, intragastric administration with the alcohol and subcutaneous injection with CCl4 in a dose-dependent manner.更多还原