小檗碱与梓醇及其配伍对胰岛素抵抗3T3-L1脂肪细胞葡萄糖转运子4蛋白及C-Cb1相关蛋白表达的影响

目的观察梓醇与小檗碱及其配伍对胰岛素抵抗3T3-L1脂肪细胞葡萄糖消耗及这一过程中葡萄糖转运子4(Glut4)和C-Cb1相关蛋白(CAP)表达的影响。方法采用高糖联合高胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗(IR),分别给予小檗碱、梓醇、小檗碱 梓醇、盐酸罗格列酮进行干预,以葡萄糖氧化酶法检测培养液中葡萄糖消耗量,以Western Blotting法检测Glut4和CAP蛋白的表达。结果与模型组相比,小檗碱能增加培养液中葡萄糖的消耗,但对Glut4蛋白的表达无影响;梓醇、小檗碱 梓醇均能显著增加培养液中葡萄糖的消耗,并使细胞中Glut4蛋白的表达增强,且小檗碱 梓醇组的效应优于梓醇组及小檗碱组;与模型组相比,小檗碱与梓醇及其配伍对CAP的表达没有显著性影响。结论小檗碱、梓醇及其配伍能改善IR 3T3-L1脂肪细胞的胰岛素活性,其作用机制与罗格列酮不同。     

梓醇对3T3-L1脂肪细胞糖脂代谢的影响及其机制研究

目的 观察梓醇对3T3-L1脂肪细胞糖脂代谢及过氧化物酶体增长因子活化受体(PPAR-γ)蛋白表达的影响。方法 采用3T3-L1前脂肪细胞诱导分化为成熟脂肪细胞,以葡萄糖氧化酶法检测细胞的葡萄糖消耗,以油红O染色法检测梓醇、罗格列酮对前脂肪细胞分化的影响,以Western Blot法检测各组PPAR-γ蛋白的表达。结果 与空白对照组比较,梓醇组、罗格列酮组3T3-L1脂肪细胞的葡萄糖消耗量均明显增高(P < 0.01),梓醇组与罗格列酮组比较差异无统计学意义(P ＞ 0.05);罗格列酮组OD值及PPAR-γ蛋白的表达较空白对照组明显升高(P < 0.05);梓醇组OD值及PPAR-γ蛋白的表达较空白对照组明显降低(P < 0.05)。结论 梓醇在抑制PPAR-γ蛋白表达的同时能够促进葡萄糖的消耗,初步表明其具有体外调节脂肪细胞糖脂代谢的作用。     

小檗碱对3T3-L1脂肪细胞增殖及糖代谢的影响

目的:研究小檗碱对脂肪细胞增殖、糖代谢及地塞米松诱导3T3-L1脂肪细胞中葡萄糖转运蛋白4(GLUT4)mRNA表达的影响。方法:用四甲基偶氮唑盐(MTT)方法检测3T3-L1前脂肪细胞及脂肪细胞的增殖情况,检测小檗碱对3T3-L1前脂肪细胞及脂肪细胞培养基中葡萄糖浓度的影响;采用地塞米松诱导胰岛素抵抗细胞模型,分别给予罗格列酮、小檗碱进行干预,用RT-PCR检测GLUT4 mRNA的表达。结果:在DMEM高糖培养基中,小檗碱可促进3T3-L1前脂肪细胞增殖,抑制3T3-L1脂肪细胞增殖,作用呈明显量效关系;使3T3-L1前脂肪细胞及3T3-L1脂肪细胞葡萄糖消耗量增加,呈明显量效关系,同时小檗碱组GLUT4 mRNA的表达升高。结论:小檗碱可以显著促进前脂肪细胞的增殖,抑制脂肪细胞的增殖;同时具有显著的降糖作用。且小檗碱能上调脂肪细胞GLUT4 mRNA的表达,这提示小檗碱改善胰岛素抵抗的作用机制可能与罗格列酮不同。     

梓醇和小檗碱及其配伍对脂肪细胞糖代谢和分化的影响

目的:观察中药提取物梓醇和小檗碱及其配伍对3T3-L1脂肪细胞增殖,葡萄糖代谢及细胞分化的影响,初步探讨药物改善胰岛素抵抗(IR)的可能机制。方法:采用四甲基偶氮唑盐(MTT)方法检测3T3-L1前脂肪细胞的增殖;以葡萄糖氧化酶法检测药物干预后培养液中葡萄糖消耗量,并以油红O染色检测细胞分化过程中胞浆脂质的堆积。结果:与对照组比较,小檗碱组使前脂肪细胞MTT值增加29%,而小檗碱加梓醇组、梓醇组均无差异;梓醇、小檗碱及其配伍组分别使成熟脂肪细胞的葡萄糖消耗增加64%、76.5%和98%;小檗碱处理脂肪细胞胞浆脂质堆积明显低于对照组,梓醇组与对照组无差异。结论:小檗碱能促进前脂肪细胞增殖,增加葡萄糖消耗,抑制脂肪细胞分化;梓醇能促进葡萄糖吸收;其作用均不依赖胰岛素的存在。直接增加葡萄糖的吸收可能是这两种药物降低血糖,改善胰岛素抵抗的机制之一。     

小檗碱对白介素-6诱导胰岛素抵抗 3T3-L1脂肪细胞脂联素表达的影响

目的:观察小檗碱对白介素-6(IL-6)诱导胰岛素抵抗的3T3-L1脂肪细胞脂联素表达的影响。方法:选用IL-6诱导3T3-L1脂肪细胞胰岛素抵抗(IR)模型。以20μg·L-1IL-6培养48 h,将3T3-L1脂肪细胞随机分为正常对照组、模型组、吡格列酮组(50μmol.L-1)和小檗碱高、中、低剂量组(10,20,50μmol.L-1),以葡萄糖氧化酶法测定葡萄糖消耗量,观察小檗碱对脂肪细胞葡萄糖摄取的影响,鉴定IR模型;采用实时荧光定量PCR技术测定脂肪细胞脂联素基因mRNA水平。结果:模型组葡萄糖消耗量及脂联素基因表达水平与正常对照组比较,均显著降低(P<0.05);小檗碱高、中、低剂量组及吡格列酮组均能显著增加葡萄糖消耗量及脂联素基因表达水平(P<0.05)。结论:小檗碱可增加白介素-6诱导胰岛素抵抗的3T3-L1脂肪细胞脂联素基因mRNA的表达,改善胰岛素抵抗状态。     

降脂平肝汤含药血清对大鼠3T3-L1脂肪细胞胰岛素抵抗的影响

目的观察降脂平肝汤含药血清对大鼠3T3-L1脂肪细胞胰岛素受体底物1(IRS-1)mRNA和过氧化物酶增殖体激活受体(PPAR)-γmRNA表达的影响,探讨其对脂肪细胞胰岛素抵抗影响的可能机制。方法选择Wistar大鼠16只,诱导分化3T3-L1脂肪前体细胞为成熟的脂肪细胞并以肿瘤坏死因子α(TNFα)诱导建立脂肪细胞的胰岛素抵抗模型,取诱导成功的胰岛素抵抗模型细胞分为对照组(基础培养液加入20%大鼠空白血清)、模型组(基础培养液加入20%大鼠空白血清和20ng/mlTNF-α)、罗格列酮组(基础培养液加入20%大鼠含药血清和20ng/mlTNF-α)、降脂平肝汤组(基础培养液加入20%大鼠含药血清和20ng/mlTNF-α)每组4只,培养48h,观察细胞IRS-1mRNA和PPAR-γmRNA表达水平的变化。结果模型组IRS-1mRNA和PPAR-γmRNA表达明显降低,与对照组比较差异有统计学意义(P<0.05);罗格列酮组与降脂平肝汤组IRS-1mRNA和PPAR-γmRNA表达均明显增高,与模型组比较差异有统计学意义(P<0.05)。结论降脂平肝汤可能通过增强大鼠IRS-1、PPAR-γ基因的表达起到抑制脂肪细胞胰岛素抵抗的作用。     

黄芪多糖和小檗碱对3T3-L1脂肪细胞糖代谢及细胞分化的影响

目的比较黄芪多糖和小檗碱对脂肪细胞糖代谢和细胞分化的影响,并分析其改善糖代谢的可能机制。方法检测黄芪多糖和小檗碱干预的脂肪细胞对^3H-葡萄糖的摄取,对黄芪多糖和小檗碱干预分化的细胞进行油红O染色并通过比色定量分析脂肪细胞分化程度。逆转录多聚酶联反应(RT—PCR)检测脂肪细胞分化相关基因过氧化物体增殖剂活化受体γ(PPARγ)、CAAT／增强子结合蛋白α(C／EBPα)mRNA的表达。结果黄芪多糖和小檗碱组葡萄糖摄取率分别为正常组的109．3％和182．7％;黄芪多糖明显促进分化及其PPARγmRNA表达,与对照组比较,差异有显著性(P<0．01);小檗碱则明显抑制细胞分化及PPARγ,C／EBPα mRNA表达,与对照组比较,差异有显著性(P<0．01)。结论黄芪多糖促进脂肪细胞葡萄糖摄取及细胞分化和PPARγ mRNA表达,而小檗碱促进葡萄糖摄取但抑制脂肪细胞分化和PPARγ及C／EBPα mRNA的表达。     

三黄消渴汤对胰岛素抵抗3T3-L1脂肪细胞的影响

目的探讨三黄消渴汤配伍对胰岛素抵抗3T3-L1脂肪细胞糖脂代谢的调控作用,阐明其降糖机理。方法培养3T3-L1前脂肪细胞并采用以地塞米松、3-异丁基-1-甲基黄嘌呤和胰岛素诱导分化为成熟的脂肪细胞,采用地塞米松诱导成熟的脂肪细胞建立胰岛素抵抗3T3-L1细胞模型。以该模型为手段,以葡萄糖消耗量、细胞游离脂肪酸的溢出水平为主要指标,通过拆方、组方对三黄消渴汤的降糖作用进行研究。结果与模型组相比,黄连组和配伍组均可降低培养液中葡萄糖的含量(P0.01),提高葡萄糖的利用率,配伍组效果优于单药组。且含或不含10 nmol·L-1胰岛素的条件下,各组培养上清液中游离脂肪酸浓度均明显低于模型组(P0.01),与胰岛素(10 nmol·L-1)无依存关系。结论三黄消渴汤配伍能显著增加3T3-L1脂肪细胞的葡萄糖消耗量,同时可减少游离脂肪酸的溢出,通过调节糖代谢和脂代谢改善胰岛素抵抗。     

胰岛素抵抗3T3-L1脂肪细胞模型及中药有效成分的干预作用

高糖高胰岛素、游离脂肪酸、炎症因子、地塞米松、金属铬、雌激素、葡糖胺等均可诱导3T3-L1脂肪细胞建立胰岛素抵抗模型,且具有稳定、可靠、易于重复等优点。目前已证实小檗碱+梓醇、小檗碱、熊果酸、西洋参茎叶总皂苷及其他活性成分、蒲黄总黄酮、灵芝多糖、附子多糖、地黄寡糖、积雪草酸、白藜芦醇、葛根素等中药有效成分具有改善胰岛素抵抗及调节糖代谢作用。     

宫颈CIN免疫相关转录因子T-bet、GATA3、Foxp3的表达

目的 研究各级宫颈上皮内瘤变(CIN)组织中T-bet、GATA3、Foxp3的表达.方法 将宫颈组织按病变程度分为宫颈炎症组、CIN Ⅰ级组、CIN Ⅱ级组和CIN Ⅲ级组,组织包埋、切片后进行镜下观察及免疫组化检测.结果 各组病变组织中T淋巴细胞数量无显著性差异(P>0.05);T-bet阳性细胞数量随病变进展增多...     

Vitexin, orientin and other flavonoids from Spirodela polyrhiza inhibit adipogenesis in 3T3-L1 cells

To investigate the adipogenesis inhibitory effect on lipid accumulation, 3T3-L1 cells were treated with fractions and isolated flavonoids of Spirodela polyrhiza. An ethanol extract of S. polyrhiza was fractionated into three fractions. The butanol soluble fraction (SPB) exhibited potent antiadipogenesis activity and decreased C/EBPα and PPARγ protein expression level in 3T3-L1 cells without significant cytotoxicity. The flavonoids were isolated from SPB and their chemical structures were identified as chrysoeriol (1), apigenin (2), luteolin (3), vitexin (4), cosmosin (5), orientin (6) and luteolin-7-O-β-d-glucoside (7) by spectroscopic analysis. Studies on the adipogenesis and intracellular triglyceride accumulation inhibitory effect showed that compounds 4 and 6 had the highest activity and decreased C/EBPα and PPARγ protein expression level in 3T3-L1 cells. These results suggest that the flavonoids isolated from SPB, especially compounds 4 and 6, contribute to the inhibitory activity of S. polyrhiza in 3T3-L1 cells.     

竹节参对MC3T3-E1成骨样细胞增殖与分化的影响

目的：研究竹节参不同提取物对体外培养成骨样细胞（MC3T3-E1）增殖、分化作用的影响。方法：采用MC3T3-E1细胞为体外药物筛选的细胞模型,用MTT法测定药物对成骨细胞的增殖作用,碱性磷酸酶（ALP）试剂盒测定ALP的活性,考察竹节参不同提取物对MC3T3-E1细胞增殖、分化作用的影响。结果：10^-1mg／mL的竹节参水提物和10^-4mg／mL的95％乙醇提取物能显著促进MC3T3-E1细胞的增殖和分化（P〈0．05）。结论：一定浓度的竹节参水提物和95％乙醇提取物能显著促进MC3T3-E1细胞的增殖和分化,该药物具有开发抗骨质疏松药物的潜力。     

Anti‐diabetic Activity of Swertiamarin is due to an Active Metabolite,Gentianine, that Upregulates PPAR‐γ Gene Expression in 3T3‐L1 cells

We have previously shown the anti‐diabetic effects of swertiamarin; however, pharmacokinetic analysis showed that swertiamarin had a plasma half‐life of 1.3 h. Gentianine is an active metabolite of swertiamarin that possesses a pharmacophoric moiety. The aim of this study was to explore the possibility whether the anti‐diabetic effect of swertiamarin is due to gentianine. Swertiamarin treatment had no significant effect on adipogenesis, or the mRNA expression of PPAR‐γ and GLUT‐4; however, there was a significant increase in the mRNA expression of adiponectin. On the other hand, treatment with gentianine significantly increased adipogenesis, which was associated with a significant increase in the mRNA expression of PPAR‐γ, GLUT‐4 and adiponectin. These findings suggest, for the first time, that the anti‐diabetic effect of swertiamarin is due to gentianine, an active metabolite of swertiamarin. Copyright © 2012 John Wiley & Sons, Ltd.     

Gastroprotective effect of barbatusin and 3-beta-hydroxy-3-deoxibarbatusin,quinonoid diterpenes isolated from Plectranthus grandis, in ethanol-induced gastric lesions in mice

Ethnopharmacological relevance

Validate the popular use of Plectranthus grandis in gastric disorders through the active components.

Aims

Isolation of barbatusin (BB) and 3β-hydroxy-3-deoxibarbatusin (BBOH), diterpenes from Plectranthus grandis, and evaluation of their gastroprotective effect and possible mechanisms of action.

Materials and methods

Isolation and chemical characterization of diterpenes from Plectranthus grandis by chromatographic and spectroscopic methods and evaluation of gastroprotective action of the diterpenes through ethanol-induced gastric injury in mice model. It was evaluated the effect of capsazepine, indomethacin and the role of nitric oxide and K_ATP− channels on the gastroprotective effect of BBOH and BB. Additionally it was measured the concentrations of gastric mucus, non-proteic-sulfhydryl groups and total thiobarbituric acid-reactive substances.

Results

Orally administered BBOH and BB at doses of 5 and 10 mg/kg, markedly reduced the gastric lesions by 59 and 96%, and 32 and 76%, respectively, with superior results as compared to N-acetylcysteine (150 mg/kg, i.p.), reference compound that caused 85% lesion suppression. Although BBOH presented a higher gastroprotection than BB they act by similar mechanisms in relation to N-acetylcysteine, and prevent the depletion of gastric mucus, gastric mucosal non-proteic-sulfhydryl groups as well as the increase in thiobarbituric acid-reactive species. Moreover, the gastroprotective effect of BB was effectively blocked in mice pretreated with TRPV1 antagonist capsazepine, by the non-selective cyclooxygenase inhibitor indomethacin, or by the nitric oxide synthase inhibitor l-NAME but not by K⁺_ATP channel inhibitor glibenclamide. In contrast, the gastroprotective effect of BBOH was blocked only by indomethacin and glibenclamide pretreatments.

Conclusion

The protective role for BBOH and BB affording gastroprotection against gastric damage induced by ethanol indicates that these compounds contribute for the activity of Plectranthus species. The different modes of action are probably related to differences in their chemical structure.     

Cecropia obtusifolia Bertol and its active compound, chlorogenic acid, stimulate 2-NBDglucose uptake in both insulin-sensitive and insulin-resistant 3T3 adipocytes

Ethnopharmacological importance

Cecropia obtusifolia Bertol (Cecropiaceae) is a plant extensively used for the empirical treatment of type 2 diabetes in México. Although some of its hypoglycemic principles have been described, their mechanisms of action remain unclear.

Aim of the study

To investigate the anti-diabetic mechanisms of Cecropia obtusifolia aqueous extract (CAE) and its active compound chlorogenic acid (CGA).

Materials and methods

Non-toxic concentrations of CAE and CGA were assayed on the adipogenesis and 2-NBDglucose uptake in 3T3-F442A murine adipocytes.

Results

Added to adipogenic medium, CAE 70 μg/ml induced a modest increment (20%) in 3T3 adipogenesis whereas CGA did not affect adipogenesis at any of the tested concentrations (0.1–100 μM). Both preparations stimulated 2-NBDG uptake in adipocytes by 51% (CAE) and 176% (CGA) in the absence of insulin, and by 174% (CAE) and 404% (CGA) in the presence of the hormone. CAE and CGA also stimulated the 2-NBDG uptake in insulin-resistant 3T3 adipocytes by 35% and 141%, respectively, compared with the incorporation shown by insulin-sensitive adipocytes stimulated by the hormone. The potency of CGA to stimulate 2-NBDG uptake was comparable to the anti-diabetic drug rosiglitazone.

Conclusion

Cecropia obtusifolia and CGA exert their anti-diabetic effects stimulating glucose uptake in both insulin-sensitive and insulin-resistant adipocytes without appreciable pro-adipogenic effects.     

黄芩水提液对3T3-L1脂肪细胞增殖、诱导分化及脂联素启动子荧光素酶活性的影响

目的：本研究旨在观察黄芩水提液（Scutellaria Baicalensis Water Extract,SBWE）对3T3-L1前体细胞增殖、分化,对脂肪细胞因子脂联素表达以及脂联素（Adiponectin,ADP）启动子荧光素酶活性的影响,从分子生物学角度阐述SBWE降脂作用的可能机理。方法：通过体外培养3T3-L1细胞,采用MTT法检测SBWE对3T3-L1细胞增殖能力的影响;通过诱导脂肪细胞分化成为成熟脂肪细胞,观察SBWE对脂肪形成的影响;化学发光法检测脂联素启动子双荧光素酶报告基因活性;荧光定量PCR法检测脂联素mRNA（Adipoq）表达。结果: 与正常组相比,给予3T3-L1细胞0.01、0.1、1 mg?mL^-1浓度的SBWE 24 h,可显著抑制细胞的增殖活性（P<0.05）;0.1、1 mg?mL^-1浓度的SBWE能够降低3T3-L1细胞分化为脂肪细胞的数量,并减少细胞内脂滴聚集,但无明显剂量依赖性;0.01、0.1 mg?mL^-1浓度SBWE能显著提高脂联素基因启动子荧光素酶活性,与空载体比较差异有统计学意义（P<0.05）;与正常组相比,给予3T3-L1细胞0.1 mg?mL^-1 SBWE 24 h,诱导前后的脂肪细胞Adipoq表达均明显增加 （P<0.05）。结论: SBWE可有效抑制3T3-L1脂肪细胞的增殖、分化,同时增加脂联素基因表达,这可能是通过增强脂联素基因启动子荧光素酶活性实现,这些为黄芩水提液减肥的作用机制提供一定的基础。