【作者】 王捷思；

【导师】 瞿伟菁；

【作者基本信息】 华东师范大学 ， 植物学， 2011， 博士

【摘要】 沙棘(Hippophae rhanmoides L.)系胡颓子科(Elaeagnaceae)沙棘属植物,不仅具有良好的改良土壤和水土保持功能,而且有很高的药用价值。沙棘籽渣是沙棘籽榨油后的残渣,也是沙棘初级利用后的弃物,沙棘籽渣黄酮(flavonoids from seed residues of Hippophae rhamnoides L., FSH)是其所含的生物活性成分之一。课题组前期研究显示FSH具有干预糖、脂代谢异常的生物学活性,本文在此基础之上,通过观察FSH对脂肪细胞糖代谢及分化的影响,结合高脂饮食小鼠模型,进一步探讨了FSH调节糖、脂代谢的作用机理及靶点。研究首先分离大鼠脂肪细胞,通过检测细胞葡萄糖消耗量,对实验室几种植物有效成分进行了筛选,结果显示FSH促进大鼠脂肪细胞消耗葡萄糖的作用最为明显,200-1000μg/mL的FSH均可显著促进脂肪细胞消耗葡萄糖,并且FSH与胰岛素联合作用时,细胞葡萄糖消耗量显著高于胰岛素单独作用。根据上述结果,实验进一步观察了FSH对3T3-L1脂肪细胞糖代谢的影响。结果显示,10-1000μg/mL的FSH对前脂肪细胞消耗葡萄糖的能力无明显影响,但200-800μg/mL的FSH能显著促进经诱导分化成熟的脂肪细胞消耗葡萄糖。同时FSH在200μg/mL浓度下可有效提高GLUT4 mRNA的表达,提示FSH可能通过上调GLUT4 mRNA的表达来促进脂肪细胞消耗葡萄糖。另外,通过油红O染色和甘油三酯含量测定观察FSH干预脂肪细胞分化的程度。结果显示,50和100μg/mL的FSH能够显著降低油红O洗脱液吸光度值,25、50和100μg/mL的FSH可使脂肪细胞内甘油三酯含量显著降低,说明FSH能够抑制3T3-L1脂肪细胞的分化。同时,MTT实验结果显示,5-100μg/mL的FSH可对3T3-L1前脂肪细胞的增殖产生抑制作用。以上细胞试验结果表明,FSH具有促进脂肪细胞消耗葡萄糖,抑制脂肪细胞分化及前脂肪细胞增殖的作用,提示脂肪细胞可能是FSH发挥作用的潜在靶细胞,FSH在体内的降糖调脂活性可能是通过对脂肪细胞功能的调节而实现的。为进一步验证和探寻FSH发挥药效的作用途径,本研究利用高脂饮食小鼠模型观察FSH对模型鼠糖、脂代谢的影响并探索其作用机理及靶点。结果显示：连续灌胃FSH(50、100和150mg/kg剂量)12 w,与高脂对照组相比,模型小鼠体重、肝脏系数、附睾处脂肪垫系数下降,但平均饮食量和饮水量、心脏系数、脾脏系数、肾脏系数无明显变化：血清TC、LDL-C、LDL-C/TC和血糖水平降低,HDL-C/TC升高,口服糖耐量得到改善,而胰岛素水平和胰岛素敏感指数无显著性变化。FSH处理组小鼠肝脏TC、TG含量降低,透射电镜观察显示FSH组小鼠肝细胞中脂滴数量减少,而骨骼肌中TC、TG含量无明显变化。此外,FSH组小鼠肝脏LPL和HL活性及骨骼肌LPL活性均无显著性差异,而脂肪组织LPL活性显著降低。同时,FSH组小鼠肝脏GSH-PX和SOD活性提高,GSH、MDA和T-AOC无明显变化。根据上述实验结果并结合细胞试验,推测肝脏和脂肪组织可能是FSH发挥药效作用的主要靶组织,因此试验进一步检测了肝脏和脂肪组织中与糖、脂代谢相关基因表达的变化。结果显示,与高脂对照组相比,FSH组小鼠肝脏组织中LDLR和PPARαmRNA表达量增加,G-6-Pase mRNA表达下降,并且低剂量的FSH(50 mg/kg)能够增加GK mRNA的表达。在脂肪组织中,高剂量的FSH(150 mg/kg)提高了GLUT4和PPARγmRNA的表达,另外,FSH降低IRS-2 mRNA表达,但能不同程度的增加IR mRNA和PI3K、Akt蛋白的表达。综上所述,FSH能够降低高脂饮食小鼠的血脂血糖水平,肝脏和脂肪组织是FSH调节糖、脂代谢的主要靶组织。FSH提高肝脏LDLR和PPARαmRNA的表达可能是FSH调节血脂水平的部分机理,而FSH对前脂肪细胞增殖和分化的抑制作用可能是其减轻体重的关键机制。FSH的降血糖作用一方面可能与降低肝脏G-6-Pase mRNA表达而升高GK mRNA表达有关,另一方面,可能是通过增加脂肪组织GLUT4、PPARγ、IR mRNA和PI3K、Akt蛋白的表达,促进脂肪细胞消耗葡萄糖来实现的。此外,FSH能降低脂肪组织LPL活性,减少脂质在组织中的积累,也可能是FSH维持糖、脂代谢稳定的一个重要原因。更多还原

【Abstract】 Hippophae rhamnoides L., of the Elaeagnaceae family, not only has good soil improvement and soil conservation function, but also has a high medicinal value. After oil extraction, a large amount of seed residue remains with bioactive substances. In our previous study, the flavonoids from seed residues of Hippophae rhamnoides L (FSH) exhibited hypoglycemic and hypolipidemic properties, however, its efficacy in modulating blood lipid and glucose levels has not been investigated in depth. Therefore, the effects of FSH on glucose metabolism and differentiation of adipocytes were investigated in the present study. Furthermore, high-fat diet fed mice were used to evaluate the hypoglycemic and hypolipidemic effects of FSH and its mechanisms.First, isolated rat adipocytes were used to screening several plants’active ingredients by glucose consumption test, and FSH showed the strongest ability to increase glucose consumption.200～1000μg/mL FSH significantly increased the glucose consumptions in rat adipocytes, moreover, FSH could enhance the effect of insulin.Further observations were carried out in 3T3-L1 adipocytes by glucose consumption test. The results showed that 10-1000μg/mL FSH had no obvious effects on glucose consumptions of 3T3-L1 preadipocytes. however,200～800μg/mL FSH could increase glucose consumptions in differentiated 3T3-L1 adipocytes. Meanwhile, treatment of differentiated adipocytes with 200μg/mL FSH could upregulate GLUT4 mRNA expression. These results suggested that FSH could enhance glucose consumption in 3T3-L1 adipocytes. which may related to upregulation of GLUT4 mRNA expression.3T3-L1 preadipocytes were induced under standard differentiation process in the presence of various concentrations of FSH. Oil red O staining and measurement of triglyceride contents were performed to investigate the effect of FSH on adipocytes differentiation. The results showed that FSH could inhibit adipogenesis of 3T3-L1 adipocytes as well as inhibit the proliferation of 3T3-L1 preadipocytes detected by MTT assay.In vitro experiments showed that FSH could increase glucose consumption and inhibit adipogenesis of adipocytes, suggesting that adipocytes may be a target of FSH, and its ability to regulating lipid and glucose metabolism may due to its effect on adipocytes. Thus, a high-fat diet fed mice model was used to investigate the effects of FSH on glucose and lipid metabolism and its mechanisms. Oral administration with FSH (50,100 and 150 mg/kg) for 12 w reduced the body, liver and epididymal fat pad weights of mice when compared with high-fat diet control group, without obvious effects on average food intake, water intake, heart, spleen and kidney weights. As compared with high-fat diet control group, treatment with FSH decreased serum TC, LDL-C, LDL-C/TC and glucose levels and increased levels of HDL-C/TC, without obvious changes in serum insulin level and insulin sensitivity index while improving oral glucose tolerance. FSH also lowered TC and TG concentrations in liver, results corroborated by transmission electron microscope findings, however, there was no significant changes in muscle TC and TG levels. Although FSH had no obvious effects on liver LPL, HL activities and muscle LPL activity, it could decrease the LPL activity in adipose tissue. Additionally, FSH could increase GSH-PX and SOD activities in liver but had no obvious effects on GSH, MDA and T-AOC. These results suggested that liver and adipose tissue may be the major target tissues of FSH, therefore, we detected the expressions of several genes that related to lipid and glucose metabolism in liver and adipose tissue. Compared with high-fat diet control group, the LDLR and PPARa mRNA expressions in liver were upregulated by FSH treatment, but the G-6-Pase mRNA expression was downregulated by FSH, and GK mRNA expression was increased by 50 mg/kg FSH. In adipose tissue,150 mg/kg FSH increased GLUT4 and PPARγmRNA expressions. Moreover, FSH decreased IRS-2 mRNA expression while increased IR mRNA and PI3K, Akt protein expressions.These results suggested that FSH possess hypolipidemic and hypoglycemic properties in mice fed high-fat diet. Its lipid lowering action may be partly related to upregulated LDLR and PPARαmRNA expressions in liver. The inhibition effects of FSH on adipocytes differentiation and preadipocytes proliferation may be a mechanism to control body weight gain. Downregulated G-6-Pase mRNA expression and upregulated GK mRNA expression in liver, increased GLUT4, PPARγ, IR mRNA and PI3K, Akt protein expressions in adipose tissue may collectively contribute to the hypoglycemic effect of FSH. Moreover, decreasing LPL activity in adipose tissue and preventing excess lipids accumulation in tissues may contribute to the ability of FSH to maintain lipid and glucose homeostasis.更多还原