【作者】 刘云云；

【导师】 杨瑞仪；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2012， 硕士

【摘要】 目的：研究牛蒡子苷(Arctiin)及牛蒡子苷元(Arctigenin)对3T3-L1小鼠前脂肪细胞增殖分化及糖脂代谢的影响,并初步探讨其作用机制。方法：采用不同剂量Arctiin和Arctigenin作用于3T3-L1前脂肪细胞,四甲基偶氮唑盐(MTT)法检测其对细胞增殖的影响；油红0染色法及分光光度法分析脂肪细胞的分化程度及脂肪含量；通过甘油磷酸氧化酶法和比色法分别检测细胞培养液中甘油和游离脂肪酸含量反映脂肪分解状况；通过葡萄糖氧化酶法检测细胞培养液中葡萄糖含量反映细胞对葡萄糖的消耗量；实时荧光定量PCR和Western-blot法在mRNA和蛋白质水平检测细胞过氧化物增殖物激活受体Y (peroxisome proliferators activated receptor γ, PPAR γ)和CCAAT/增强子结合蛋白a (CCAAT enhancer binding protein a, C/EBP α)的表达情况；紫外分光光度法检测脂肪酸合酶(fatty acid synthase, FAS)的活性。结果：Arctiin和Arctigenin在12.5-100μmol/L浓度范围内对3T3-L1前脂肪细胞增殖无显著影响(P>0.05),但显著抑制其向脂肪细胞的分化以及脂肪的合成(P<0.01),且随剂量增加而增强。相同浓度下,Arctigenin抑制细胞分化和成脂的能力优于Arctiin(P<0.01或P<0.05)。Arctigenin在25-100μ mol/L浓度范围内能显著抑制成熟脂肪细胞甘油三酯分解产生甘油和游离脂肪酸(P<0.01或P>0.05),且随剂量增加而抑制作用增强,最高剂量(100μ mol/L)组细胞培养液中甘油含量仅为对照组的28.7%,游离脂肪酸含量为对照组的40.6%。而Arctiin则对脂肪细胞分解无显著影响(P>0.05)。Arctigenin和Arctiin在12.5-100μ mol/L浓度范围内能显著增加成熟脂肪细胞对葡萄糖的消耗(P<0.01)。实时荧光定量PCR结果显示Arctigenin和Arctiin均能降低3T3-L1前脂肪细胞分化过程中的PPARγ和C/EBP a mRNA表达水平(P<0.01或P<0.05),且高浓度抑制作用优于低浓度。Arctigenin最高剂量(100μmol/L)组PPARγ和C/EBPα mRNA水平仅为对照组的13.5%和6.3%；Arctiin最高剂量(100μmol/L)组PPARγ和C/EBP α mRNA水平仅为对照组的33.9%和8.4%。Western-blot结果同样显示,与对照相比,Arctigenin和Arctiin可降低PPARγ和C/EBP α蛋白表达水平且呈剂量依赖关系。表明Arctigenin和Arctiin均能在转录和翻译水平上抑制PPARγ和C/EBP α表达。Arctigenin和Arctiin在12.5-100μ mol/L浓度范围内能有效降低脂肪细胞FAS的活性(P<0.01或P<O.05)。相同浓度下,Arctigenin抑制FAS活性的作用强于Arctiin(P<0.01或P<0.05)。结论：Arctiin与Arctigenin对3T3-L1前脂肪细胞无明显毒性,可抑制脂肪细胞分化,促进脂肪细胞对葡萄糖的消耗；Arctigenin可抑制脂肪细胞的脂肪分解。其作用机制可能与下调PPARγ和C/EBP a基因表达、抑制FAS活性有关。更多还原

【Abstract】 ObjectiveTo investigate the effects of arctiin and arctigenin on proliferation/differentiation and glucose/lipid metabolism of murine3T3-L1preadipocytes and study their possible mechanism.Methods3T3-L1preadipocytes were cultured with arctiin and arctigenin in different concentrations. Cell proliferation was analyzed by MTT method. The degree of differentiation was evaluated by oil red0staining and spectrophotography. To unravel the lipolysis and glucose consumption of3T3-L1preadipocytes were induced into adipocytes, the glycerol, free fatty acid and glucose in the supernatants were quantitatively assayed by glycerol phosphate oxidase-peroxidase method, colorimetry and glucose oxidase method respectively. The transcriptional and translational expression of peroxisome proliferators activated receptor γ (PPAR γ) gene and CCAAT enhancer binding protein α (C/EBPα) gene were detected by real time fluorescence quantitative-polymerase chain reaction (RTFQ-PCR) and western blot. The activity of fatty acid synthase (FAS) was determined by ultraviolet spectrophotography.ResultsIn a concentration range of12.5～100μ mol/L, arctiin and arctigenin did not exhibit remarkable influence on proliferation of3T3-L1preadipocytes (P>0.05), but significantly inhibited the cell differentiation and adipogenesis in a dose-dependent manner (P<0.01). At the same concentration, the inhibitory effect of arctigenin was stronger than that of arctiin (P<0.01or P<0.05) The release of glycerol and free fatty acid was obviously decreased with increasing dose of arctigenin within the range of25-100μ mol/L (P<0.01或P<0.05). In the highest concentration (100μmol/L) group, the content of glycerol and free fatty acid were28.7%and40.6%of that in control. Arctiin had no significant effect on the lipolysis in adipocytes (P>0.05)The data also showed that arctigenin and arctiin promoted the glucose consumption of adipocytes with the concentration from12.5to100μmol/L (P<0.01)The PPARγ and C/EBPa mRNA levels during differentiation of3T3-L1preadipocyte were decreased after the treatment with arctiin and arctigenin (F<0.01or P<0.05), and the inhibitory action accompanied with the dose effect relation. At the highest dosage (100μmol/L) of arctigenin and arctiin groups, the PPARγ and C/EBP a transcript level were just13.5%/6.3%and33.9%/8.4%of those in control respectively. Western blot gave the results similar to the RTFQ-PCR. It was indicated that arctiin and arctigenin could down-regulate the expression of PPARγ and C/EBPα gene both in the transcriptional and translational level.At the doses of12.5～100μ mol/L, arctiin and arctigenin effectively reduced the FAS activity of adipocytes (P<0.01or P<0.05). The FAS activities were lower in the arctigenin groups than in the arctiin groups at the same dosage (P<0.01or P<0.05)ConclusionArctiin and arctigenin inhibit the differentiation of3T3-L1preadipocytes, and increase the consumption of glucose in3T3-L1preadipocytes without cytotoxicity. Arctigenin suppresses the lipolysis in adipocytes. The decreased expression of PPARγ and C/EBP a, and depressed activity of FAS may be involved in the possible mechanism of arctiin and arctigenin.更多还原