【作者】 屠迪；

【导师】 薛立群； 文利新；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2012， 博士

【摘要】 花青素(Anthocyanidin),属黄酮类(Flavonoids)化合物是植物花瓣中的主要呈色物质。自然界有300多种不同的花青素,在这些花青素的非配糖体中,矢车菊素(Cyanidin, CY)的化学结构最为简单,且其他各种花青素分子结构多含有矢车菊素的基本结构,部分花青素还可由矢车菊素在动物体内代谢形成。目前研究发现矢车菊素具有多种生物活性,如预防癌症、保护心血管系统、保护上皮细胞、调节脂肪代谢、保肝护肝、对抗霉菌毒素等,这些生物学活性与矢车菊素的吸收、转运和代谢密切相关；动物试验证实,矢车菊素具有改善动物体内氧化应激状态和调节脂代谢的生物学活性。然而有关矢车菊素的药理作用、药代动力学和动物试验相关数据却相对匮乏,对于其在畜牧生产中的应用潜力也缺乏探索。为了填补矢车菊素对肝细胞脂代谢调节的部分理论基础空白,本论文以肝细胞为研究对象,通过体外建立肝细胞脂肪变性模型,并以不同浓度的CY进行干预,对肝细胞脂代谢状况、氧化应激水平、肝细胞凋亡及部分调节肝内脂代谢部分相关信号进行了体外研究。相关实验结果显示如下：1.细胞的选择：在体外,低浓度矢车菊素(40μg/mL)的处理对人肝癌细胞系HepG2的生长产生抑制作用,而相同条件下L02细胞系的生长并未受到影响；通过RT-PCR方法检测发现,在L02细胞中可检出全部(PPARα、PPARγ、ABCA1、 LXRα、CYP7A1、RXRα)脂代谢相关信号,而HepG2细胞中无法检出CYP7A1和RXRα的表达。以上结果表明：L02细胞比HepG2细胞更适合作为肝细胞脂肪变性的模型细胞进行进一步的研究。2.肝细胞脂肪变性模型的建立：50mg/mL软脂酸抑制L02细胞增殖。用不同剂量的软脂酸(Pamitic acid, PA)处理L02细胞24h,可引起L02细胞内甘油三酯(Triglycerides, TG)和胆固醇(Cholesterol, TC)的蓄积,且TG和TC的蓄积程度随PA浓度的升高而增加；细胞内脂代谢相关信号的检测结果显示,当L02细胞发生上述脂代谢障碍时,细胞内RXRα和ABCA1的基因和蛋白表达水平都显著上调,PPARα、PPARγ和CYP7A1的水平呈现下调。PA还能引起L02细胞的氧化损伤,主要表现在导致丙二醛(Malondialdehyde, MDA)水平上升以及过氧化氢酶(Hydrogen peroxide enzyme, CAT)、超氧化物歧化酶(superoxide dismutase, SOD)和谷胱甘肽(glutathione, GSH)水平下降,其氧化损伤程度与PA的剂量正相关；而且,L02细胞受到PA影响发生氧化损伤后会异常激活Caspase9和Caspase3,经线粒体通路使L02细胞发生凋亡,其细胞凋亡率也与PA剂量呈正相关；发生凋亡的细胞主要处于早期凋亡阶段。以上结果表明软脂酸(本实验中相对的最大无毒浓度30mg/mL)作为诱导剂,培养24h,可造成明显的L02细胞脂代谢障碍和细胞的氧化损伤,但对细胞活率没有统计学意义上的影响,以上条件可以作为本实验中肝细胞脂肪变性模型的造模条件。3.矢车菊素对肝细胞脂肪变性模型的影响：CY干预后能够显著降低细胞模型内TG、TC浓度,改善脂代谢紊乱的状况。当CY剂量>5μg/mL时,改善模型组细胞脂代谢障碍的作用较为明显。CY可以通过下调模型细胞内LXRα,并抑制ABCA1基因和蛋白的表达水平；CY通过上调PPARα、PPARγ和RXRα并及下游信号CYP7A1促进胆固醇和TG, TC代谢,改善模型细胞内脂质蓄积状况,上述作用与CY的剂量呈正相关。此外,CY还可以降低模型细胞MDA水平,并增加CAT/SOD和GSH的浓度缓解细胞模型的氧化损伤,并通过抑制Caspase9和Caspase3激活抑制细胞模型凋亡。综上所述,本实验以PA诱导的L02细胞为肝细胞脂肪变性模型,并证实CY可以通过多种途径改善PA所引起的L02细胞模型脂代谢紊乱,缓解脂质蓄积所造成的细胞氧化损伤和细胞凋亡。更多还原

【Abstract】 Anthocyanin, also known as Anthocyanidin, is a flavonoid compound. It is a water-soluble natural pigment widely present in plants, it is also the main coloring material in petals. There are more than three hundred anthocyanins and cyanidin (CY) is the simplest chemical form of anthocyanin glycosides. All other anthocyanins contain the molecular structures of cyaniding and one part of anthocyanins can be formed in vivo during the metabolism of cyanidin. It has been found that cyanidin has different biological activities such as cancer prevention, cardiovascular and epithelial cell protection, fat metabolism regulation, and liver protection against mycotoxins. These biological activities of cyanidin are closely related to its absorption, transportation and metabolism. Prior experiments have shown that CY can improve the oxidative stress status of animals and regulate the biological activity of lipid metabolism. These data indicated that the application of CY on animal production may significantly improve animal performance and promote their resistance to diseases. However, reports regarding its pharmacological effects, pharmacokinetics and in vivo animal studies are relatively limited. In this study, we explored the effects of cyanidin on liver cell lipid metabolism in vitro which may provide theoretical basis to support the potential application of CY in livestock production. In vitro hepatic steatosis model was developed, and effects of CY on liver cell lipid metabolism, oxidative stress, lipo-apoptosis, intrahepatic lipid metabolism and in vitro signal pathway were studied.1. Selection of the optimal cell line:L02and HepG2were used in this study, results showed that low concentration of cyanidin (40μg/mL) could inhibit the HepG2cell growth but no effects was observed in L02cells which indicated that L02was more tolerable to CY than HepG2. Further detections using RT-PCR showed that all target pathway signaling designed in the present study including PPARa, PPARy, ABCA1, CYP7A1and RXRa could be detected in L02cells, but only PPARa, PPARy, ABCA1except CYP7A1and RXRa could be detected in HepG2cells. All these data indicated that L02cell is more optimal than HepG2used to develop a hepatic steatosis model.2. Establishment of the hepatic steatosis model:Palmitic acid (PA) was used to establish the hepatic steatosis model. L02cells were treated using different doses of PA,50mg/mL PA could inhibit the proliferation of the L02. Thirty mg/mL PA could induce the accumulation of triglycerides (TG) and cholesterol (TC) with dose-dependent pattern in cells. mRNA and preotein Levels of RXRa and ABCA1showed a significant increase in L02hepatic steatosis cell model, but PPARa, PPARy and CYP7A1presented a decrease. Furthermore, PA caused oxidative damage with accumulation of malondialdehyde (MDA) and reduction of hydrogen peroxide enzyme (CAT), superoxide dismutase (SOD) and glutathione (GSH) in L02cells. The intensity of the damage was also dependent on the PA dose. Moreover. Caspase-3and Caspase-9were activated and increased in L02cells after oxidative damage which resulted in cell apoptosis. Most of the apoptotic cells were limited to the early stage and the rate of cell apoptosis was positively correlated with PA dose. All above data indicated that using PA as an inducing agent (the maximum non-toxic concentration is30mg/mL) could cause abnormal liver metabolism and oxidative demage in L02cells with no effect on its vitality which means these conditions are suitable to establish the hepatic steatosis cell model..3. Effects of cyaniding on the L02hepatic steatosis model:L02hepatic steatosis model was treated with different dose of CY, data showed that CY could significantly decreased the intercellular TG and TC to improve the status of lipid accumulation in model cell. When the CY dose was≥5μg/mL, the effect was obvious. CY could down-regulated the intercellular LXRa and inhibited the expression of ABCAl in both mRNA and protein levels; CY also increased the levels of PPARa, PPARy and RXRa as well the down-stream signaling CYP7A1which promoted the metabolism of TG and TC with dose-dependent manner. Moreover, CY could increase the level of MDA and increase CAT, SOD and GSH which helped to relief the oxidative damage and inhibit the cell apoptosis through inhibition the activation of caspase-3and caspase-9in this cell model.In conclusion, the presnet study confirmed that the CY, through a variety of ways, regulated lipid metabolism, alleviated cellular oxidative damage caused by lipid accumulation and reduced fat apoptosis rate through inhibiting caspase-3and caspase-9in the L02hepatic steatosis cell model induced by PA.更多还原