The flavonoid glycoside vaccarin inhibits adipogenesis and stimulates lipolysis via Hedgehog signaling in 3T3-L1 adipocytes

Vaccarin,a flavonoid glycoside isolated from Vaccaria segetalis,is non-toxic to 3T3-L1 cells up to concentrations of 200μM.Accordingly,we investigated the effects of this natural product on adipogenesis and lipolysis in 3T3-L1 adipocytes.Our results revealed that vaccarin significantly inhibited lipid accumulation by suppressing the adipogenesis-related transcription factors peroxisome proliferator-activated receptorγ(PPARγ)and the CCAAT/enhancer-binding proteinα(C/EBPα).Specifically,lipid accumulation decreased by up to 27.7±2.7%when 3T3-L1 adipocytes were treated with a 10μM concentration of vaccarin.Mechanistic studies showed that the compound inhibited adipogenesis through activation of the Hedgehog(Hh)signaling pathway and so restoring Smo and Gli1 expression at an early stage of differentiation.In mature 3T3-L1 cells,vaccarin significantly increased the secretion of glycerol into the surrounding medium and thus indicating that it accelerated the degradation of triglycerides.In addition,vaccarin,was shown to enhance lipolysis through stimulation of the transcription levels of lipoprotein lipase,monoglycerides lipase,adipose triacylglyceride lipase,hormone-sensitive lipase and adipose differentiated-related protein.All told,vaccarin suppressed lipid accumulation and enhanced lipolysis during adipocyte differentiation by restoring Hh signaling.As such,it is a phytochemical capable of halting adipocyte hyperplasia and,thereby,ameliorating the effects of obesity.

Chemical composition and glucose uptake effect on 3T3-L1 adipocytes of Ligustrum lucidum Ait. flowers

Ligustri lucidi Fructus is a traditional Chinese medicine and possesses various bioactivities,including hypoglycemic effect.Ligustrum lucidum Ait flowers are poorly investigated.Thus,we hypothesized that L.lucidum flowers also could have hypoglycemic effect.Chemical composition and glucose uptake effect of L.lucidum flowers on 3T3-L1 adipocytes were investigated.In this study,the components of L.lucidum flowers were investigated by various chromatographic and spectroscopic methods and the effects of L.lucidum flowers on the induction of glucose uptake were investigated by 3T3-L1 adipocytes.Seven compounds were isolated and identified from L.lucidum flowers,including ursolic acid(1),kaempferol-7-O-α-Lrhamnoside(2,KR),β-sitosterol(3),β-daucosterol(4),kaempferitrin(5,KF),10-hydroxy oleuropein(6,HO),and kaempferol-3-O-β-D-glucopyranoside-7-α-L-rhamnopyranoside(7,KGR).The results of glucose uptake showed that total extract(TE),KR,KF and HO from L.lucidum flowers significantly enhanced glucose uptake.This study demonstrated that compounds 2,5-7 were isolated for the first time from this plant and compounds 2 and 7 were isolated for the first time in genus Ligustrum,L.lucidum flowers,KR,KF and HO may possess potential hypoglycemic effect.

Effect of Tumor Necrosis Factor-αon Resistin Expression in 3T3-L1 Adipocytes and Its Mechanism

Summary: In order to investigate the effect of tumor necrosis factor-α (TNFα) on resistin expression in 3T3-L1 adipocytes, and further explore its mechanisms, the differentiated 3T3-L1 adipocytes were incubated with 0, 1, 10, 100 ng/mL TNFα respectively for 24 h, and then the expression of resistin was determined. The differentiated 3T3-L1 adipocytes were incubated with 100 ng/mL TNFα for 3, 6, 24 h respectively, and then the expression of resistin mRNA was analyzed. 3T3-L1 adipocytes were induced to differentiate into mature adipocytes. The cells were randomly divided into 4 groups for culture. In the control group, no drugs were added. Cells of TNFα group were treated with 100 ng/mL TNFα. In Ro-31-8220 group, 5 μmol/L protein kinase C inhibitor Ro-31-8220 was added. With TNFα+Ro-31-8220 group, 100 ng/mL TNFα were added 1 h after the addition of 5 μmol/L Ro-31-8220. All adipocytes were cultured for 24 h. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were employed to detect the expression of resistin gene. Our results showed that resistin protein and mRNA in 3T3-L1 adipocytes were inhibited by TNFα at different concentrations (P<0.01), and the inhibitory effect increased with the concentration (P<0.01). At the same concentrations, the inhibitory effect increased with time (P<0.01). Ro-31-8220 could inhibit its expression and the inhibitive effect remained unchanged with addition of TNFα(P>0.05). It was concluded that TNFα could inhibit the expression of resistin in 3T3-L1 adipocytes. The mechanism may be that the expression of resistin is partly controlled by protein kinase C signal conduction pathway.

Photoactivation of GLUT4 translocation promotes glucose uptake via PI3-K/Akt2 signaling in 3T3-L1 adipocytes

Insulin resistance is a hallmark of the metabolic syndrome and type 2 diabetes.Dysfunction of PI-3K/Akt signaling was involved in insulin resistance.Glucose transporter 4(GLUT4)is a keyfactor for glucose uptake in muscle and adipose tissues,which is closely regulated by Pi-3K/Aktsignaling in response to insulin treatment.Low-power laser irradiation(LPLI)has been shown toregulate various physiological processes and induce the synthesis or release of multiple moleculessuch as growth factors,which(especially red and near infrared light)is mainly through theactivation of mitochondrial respiratory chain and the initiation of intracellular signaling path-ways.Nevertheless,it is unclear whether LPLI could promote glucose uptake through activationof PI-3K/Akt/GLUT4 signaling in 3T3L-1 adipocytes.In this study,we investigated how LPLIpromoted glucose uptake through activation of PI-3K/Akt/GLUT4 signaling path way.Here,we showed that GLUT4 was localized to the Golgi apparatus and translocated from cytoplasm tocytomembrane upon LPLI treatment in 3T3L-1 adipocytes,which enhanced glucose uptake.Moreover,we found that glucose uptake was mediated by the PI3-K/Akt2 signaling,but notAkt1 upon LPLI treatment with Akt isoforms gene silence and PI3-K/Akt inhibitors.Collec-tively,our results indicate that PI3-K/Akt2/GLUT4 signaling act as the key regulators forimprovement of glucose uptake under LPLI treatment in 3T3L-i adipocytes.More importantly,our findings suggest that activation of PI3-K/Akt2/GLUT4 signaling by LPLI may provideguidance in practical applications for promotion of glucose uptake in insulin-resistant adiposetissue.

A Triterpenoid Inhibited Hormone-Induced Adipocyte Differentiation and Alleviated Dexamethasone-Induced Insulin Resistance in 3T3-L1 adipocytes

6a-Hydroxylup-20(29)-en-3-on-28-oic acid(1),a natural triterpenoid,was found to possess the ability in a dose-dependent manner inhibiting hormone-induced adipocyte differentiation in 3T3-L1 preadipocytes,and restoring glucose consuming ability in dexamethasone(DXM)-induced insulin resistant 3T3-L1 adipocytes.Compound 1 was also found to ameliorate DXM-induced adipocyte dysfunction in lipolysis and adipokine secretion.Mechanistic studies revealed that 1 inhibited adipocyte differentiation in 3T3-L1 preadipocytes via down-regulating hormone-stimulated gene transcription of peroxisome proliferator-activated receptor c and CCAAT-enhancer-binding protein alpha which are key factors in lipogenesis,and restored DXM-impaired glucose consuming ability in differentiated 3T3-L1 adipocytes via repairing insulin signaling pathway and activating down-stream signaling transduction by phosphorylation of signaling molecules PI3K/p85,Akt2 and AS160,thus leading to increased translocation of glucose transporter type 4 and transportation of glucose.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

Effect of mango seed kernel extract on the adipogenesis in 3T3-L1 adipocytes and in rats fed a high fat diet

Mangoes (Mangifera indica L.) are one of the most important tropical foods. The seed is one of the main by-products of mango processing. Therefore, it is important to find an economically viable use for this waste (e.g., as a food additive or supplement with high nutraceutical value). We investigated the anti-obesity effects of mango seed kernel extract with hot water (MSKE-W) in 3T3-L1 adipocytes and in a high fat diet (HFD)-induced obesity rat model. MSKE-W caused a significant decrease in the activity of glycerol 2-phosphate dehydrogenase in 3T3-L1 adipocytes without eliciting cell cytotoxicity and inhibited cellular lipid accumulation through down-regulation of transcription factors such as PPARγ and C/EBPα. In the animal model, rats fed an HFD containing 1% MSKE-W gained less weight than rats fed an HFD alone. The visceral fat mass in rats fed an HFD containing 1% MSKE-W tended to be lower than that in rats fed an HFD alone. Furthermore, histological examination of rat livers from an HFD showed steatohepatitis. However, rats on an HFD containning 1% MSKE-W showed no histopathological changes in liver tissue. Our results indicate that MSKE-W influences anti-obesity effects, both in vitro and in vivo, and suggest that MSKE-W provides a novel preventive potential against obesity.

<i>Tephrosia purpurea</i>Fraction Attenuates Lipid Accumulation and Adipogenesis in 3T3-L1 Adipocytes and Reduces Body Weight in High Fat Diet Induced Obese Rats

The anti-adipogenic and anti-obesity activity of chloroform fraction of Tephrosia purpurea (CFTp) on 3T3-L1 adipocytes and high fat diet (HFD)-fed obese rats was evaluated in this study. A substantial and dose dependent inhibition of α-glucosidase (81%) and lipase (75%) activities by CFTp was noticed. Treatment with CFTp (250 μg/mL) significantly inhibited 3T3-L1 adipocytes differentiation and lipid accumulation. A semi-quantitative RT-PCR analysis of 3T3-L1 cells revealed down regulation of mRNA expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS) and acetyl CoA carboxylase-2 (ACC-2), while glucose transporter type-4 (GLUT-4) expression was up-regulated in a dose dependent manner with CFTp. Further, oral administration of CFTp (200 mg/kg.b.wt.) significantly reduced body weight gain, fat mass, blood glucose and leptin levels in high fat diet (HFD)-induced obese rats. Taken together, these findings demonstrate that CFTp possesses potent anti-obesity activities.

Bis(acetylacetonato)-oxidovanadium(Ⅳ) inhibits isoproterenol-induced lipolysis in insulin-resistant 3T3-L1 adipocytes by reducing phosphorylation of perilipin and hormone-sensitive lipase

Insulin resistance is characterized as one of crucial pathological changes in type 2 diabetes mellitus（T2DM）, and dyslipidaemia is frequently detected in T2DM. A variety of vanadium compounds have been studied as drug candidates for diabetes based on their insulin-like action. However, few studies focus on their antilipolytic effect. In the present study, we established an insulin-resistant model in 3T3-L1 adipocytes to mimic pathological conditions of T2DM according to a well-established method by the treatment of high concentrations of glucose and insulin, which was validated by oil red O staining and the decreased levels of phosphorylated Akt, AS160 and GSK3 after insulin treatment. The results demonstrated that bis（acetylacetonato）-oxidovanadium（Ⅳ）（VO（acac）_2） could inhibit isoproterenol-stimulated lipolysis through the reduction of the phosphorylated HSL and perilipin levels in both insulin-sensitive and insulin-resistant 3T3-L1 adipocytes. Moreover, although the levels of phosphorylated Akt induced by VO（acac）_2 were decreased, the rates of lipolytic inhibition were not significantly altered compared with those under insulin-sensitive condition, indicating that the anti-lipolytic effect of VO（acac）_2 might also function in an Akt-independent way in insulin-resistant adipocytes. Our work here help elucidate the anti-diabetic effects of vanadium compounds. It may not only shed light on the utility of vanadium-based compounds as potential anti-diabetic drugs but also serve as a useful screening model for new anti-diabetic drugs.

红景天苷促进MC3T3-E1细胞成骨分化能力的体外实验

背景:骨缺损可直接影响牙齿种植的成功率及种植体的长期稳定性。研究显示红景天苷可促进成骨细胞增殖、分化,但对于其成骨分化相关通路具体研究不多。目的:体外细胞实验研究红景天苷对MC3T3-E1细胞增殖和分化的影响,并探究其对相关基因和蛋白表达的影响。方法:采用CCK-8实验和碱性磷酸酶实验筛选红景天苷(0.5,1,5,10,50μmol/L)促进MC3T3-E1细胞增殖和分化的最佳浓度。实验分为4组:对照组、红景天苷组、红景天苷+LY294002组、LY294002组,分别用成骨诱导液、含10μmol/L红景天苷、10μmol/L红景天苷+10μmol/L LY294002、10μmol/L LY294002的成骨诱导液进行培养,观察红景天苷及PI3K/Akt信号通路抑制剂LY294002对成骨相关基因和蛋白表达的影响。结果与结论:①CCK-8实验和碱性磷酸酶实验显示:红景天苷促进MC3T3-E1细胞增殖的作用在10μmol/L时最显著;②与对照组相比,红景天苷可以促进矿化、促进细胞黏附、减少细胞死亡,提高Runx-2、骨钙素、骨桥蛋白的mRNA表达(P<0.01),提高Runx-2和p-Akt蛋白表达(P<0.01);而添加PI3K/Akt信号通路抑制剂LY294002则可逆转以上结果;③结果表明,红景天苷能促进MC3T3-E1细胞矿化,并能促进成骨相关基因、蛋白的表达,这可能与PI3K/Akt信号通路的激活有关。

黄芪甲苷可缓解MC3T3-E1细胞氧化应激损伤并促进成骨

背景:氧化应激是导致骨质疏松症的主要原因之一,减少氧化应激水平与增加抗氧化防御是治疗骨质疏松症的重要研究方向。研究证实黄芪甲苷具有抗骨质疏松作用,但其作用机制尚不明确。目的:探讨黄芪甲苷对氧化应激条件下MC3T3-E1细胞成骨的作用。方法:将MC3T3-E1细胞分4组培养:对照组加入完全培养基,模型组加入含过氧化氢的完全培养基干预24 h后更换为完全培养基,黄芪甲苷组加入含过氧化氢、黄芪甲苷的完全培养基干预24 h后更换为含黄芪甲苷的完全培养基,抑制剂组加入含过氧化氢、黄芪甲苷、细胞外信号调节激酶(extracellular signal-regulated kinases,ERK)抑制剂的完全培养基干预24 h后更换为含黄芪甲苷、ERK抑制剂的完全培养基。过氧化氢干预48 h后,通过丙二醛含量检测黄芪甲苷对MC3T3-E1细胞氧化应激的缓解作用;过氧化氢干预48 h后进行成骨诱导培养,通过碱性磷酸酶染色、茜素红染色验证MC3T3-E1细胞成骨及矿化能力,通过RT-qPCR检测成骨相关基因的表达,Western blot检测成骨相关蛋白及ERK/腺苷酸激活蛋白激酶(AMP-activated protein kinase,AMPK)信号通路蛋白的表达。结果与结论:(1)模型组细胞内碱性磷酸酶含量与矿化结节形成均少于对照组(P<0.05),黄芪甲苷组细胞内碱性磷酸酶含量与矿化结节形成均多于模型组(P<0.05)。(2)与对照组比较,模型组细胞内丙二醛含量增加(P<0.05),骨钙素、RUNX2、Ⅰ型胶原蛋白的mRNA与蛋白表达均降低(P<0.05),AMPK mRNA与p-AMPK蛋白表达升高(P<0.05);与模型组比较,黄芪甲苷组细胞内丙二醛含量减少(P<0.05),骨钙素、RUNX2、Ⅰ型胶原蛋白的mRNA与蛋白表达均升高(P<0.05),ERK1/2、AMPK mRNA表达升高(P<0.05),p-AMPK、p-ERK1/2蛋白表达均升高(P<0.05);ERK抑制剂可部分抑制黄芪甲苷的上述作用。(3)结果表明,黄芪甲苷可通过激活ERK/AMPK信号通路促进氧化应激条件下MC3T3-E1细胞的成骨分化。

Effect of dexamethasone on peroxisome proliferator activated receptor-gamma mRNA expression in 3T3-L1 adipocytes with the human recombinant adiponectin

Background The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin （ADPN） function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma （PPAR-y） mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed. Methods The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1^＋ were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1^＋ were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1^＋-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent （Qiagen）. Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone （0.5 mmol/L） for 24 hours, cells were collected and total RNA was extracted. The PPAR-γ mRNA expression was quantified by semiquantitative reverse transcription-polymerase chain reaction （RT-PCR）. Results After eukaryotic recombinant was digested by Hind Ⅲ and EcoR Ⅰ, fragments of 800 bp and 5.4 kb were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone （0.5 mmol/L） decreased PPAR-y mRNA expression compared to untreated controls （P〈0.01）. Effect of dexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin. Conclusion The 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-γ mRNA expression. Dexamethasone suppressed PPAR-γ mRNA expression in the 3T3-L1 cells. Effect of dexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.

Anti-adipogenic effect of flavonoids from Chromolaena odorata leaves in 3T3-L1 adipocytes

Objective： The leaves of Chromolaena odorata, a highly invasive shrub found growing wild worldwide, are traditionally used for wound healing. Due to its high flavonoid contents, we aimed to find a new appli- cation for this plant. Preliminary tests using its ethanolic leaf extract showed that it could suppress the accumulation of lipids in adipocytes. We therefore studied the anti-adipogenic effect of several C. odorata leaf extracts and the relationship between molecular structure and bio-activity of its isolated flavonoid constituents using 3T3-LI preadipocytes/adipocytes as a model. Methods： Three leaf extracts and thirteen flavonoids isolated from C odorata were tested for their effect on lipid accumulation in 3T3-L1 adipocytes using AdipoRed reagent, with quercetin as the positive con- trol. The effects of active flavonoids on the adipocytes were confirmed by oil red O staining and visualized under a light microscope.Results： n-Hexane and ethyl acetate extracts of C odorata leaves displayed anti-adipogenic activity. The latter extract was the more potent one, especially at 40 μg/mL. Four flavonoids, pectolinarigenin, kaemp- feride, 4,2＇-dihydroxy-4＇,5＇,6＇-trimethoxychalcone and dillenetin, exhibited significant, concentration- dependent inhibitory effects on lipid accumulation in 3T3-L1 adipocytes. The most potent flavonoid obtained in this study was 4,2＇-dihydroxy-4＇,5＇,6＇-trimethoxychalcone, which caused 75% and 90% inhi- bition of cellular lipid accumulation at 30 and 50μmol/L, respectively. Both kaempferide and 4,2＇-dihy droxy-4＇,5＇,6＇-trimethoxychalcone were major constituents in the ethyl acetate extract of this plant.Conclusion： C odorata leaves contained several flavonoids with anti-adipogenic effects against lipid accu- mulation in 3T3-L1 adipocytes. The plant, normally considered a useless weed, may actually provide an abundant source of biologically active flavonoids.

bta-miR-27a-5p的生物信息学分析及其对3T3-L1前脂肪细胞分化的影响

为了探究bta-miR-27a-5p的生物学特性及其对3T3-L1前脂肪细胞分化的影响,试验利用miRBase数据库对不同物种间miR-27a-5p的保守性进行分析,并构建了miR-27a-5p的系统进化树;利用TargetScan和miRWalk在线网站预测bta-miR-27a-5p的靶基因,并对靶基因进行GO功能注释和KEGG信号通路分析;利用实时荧光定量PCR方法检测在不同月龄夏南牛不同器官中bta-miR-27a-5p基因的相对表达量;最后通过实时荧光定量PCR方法、Western-blot检测、三酰甘油浓度测定和油红O染色探究过表达bta-miR-27a-5p后对3T3-L1前脂肪细胞分化的影响。结果表明:不同物种miR-27a-5p的成熟序列保守性较高;牛miR-27a-5p基因与山羊的亲缘关系最近,与非洲爪蟾的亲缘关系最远;bta-miR-27a-5p靶基因主要在细胞过程、生物过程、细胞、细胞部分、结合等GO条目上富集,并富集在胰岛素抵抗、细胞衰老、脂肪细胞因子等信号通路中;bta-miR-27a-5p基因可在夏南牛的多个器官中表达,且12月龄夏南牛脂肪组织中的相对表达量均显著低于0月龄和24月龄(P<0.05);过表达bta-miR-27a-5p基因可显著或极显著抑制C/EBPα、PPARγ和FABP4基因和蛋白质表达(P<0.05或P<0.01),并显著或极显著降低3T3-L1前脂肪细胞内脂滴和三酰甘油浓度(P<0.05或P<0.01)。说明bta-miR-27a-5p在不同物种间高度保守,在不同月龄夏南牛脂肪组织中显著差异表达,并且能抑制3T3-L1前脂肪细胞分化,bta-miR-27a-5p基因可能是调控牛脂肪生成的候选miRNA。

初级纤毛介导成骨前体细胞系MC3T3-E1传代衰老减低成骨分化能力

背景:在大段骨缺损修复中,由于种子细胞传代等多种因素可引起成骨类细胞衰老,造成组织工程骨植入体内后成骨分化活性降低。近年来,初级纤毛介导细胞衰老的机制已被广泛研究,但“传代衰老-成骨活性减低”的初级纤毛相关机制尚未完全明了。目的:探讨初级纤毛调控成骨性MC3T3-E1细胞传代衰老的可能机制。方法:成骨前体细胞系MC3T3-E1细胞传代至第10代(早期传代)及第40代(晚期传代),同时使用siRNA沉默IFT88阻碍初级纤毛生成,即将细胞分为第10代组、第40代组、第10代+siRNA IFT88-组、第40代+siRNA IFT88-组。各组细胞成骨诱导3 d,采用RT-PCR和Western blot检测衰老标志物CDKN2A(P16)的表达、成骨活性标志物骨形态发生蛋白2和碱性磷酸酶的表达、Hedgehog通路IHH的表达;茜素红染色和初级纤毛免疫荧光染色分析初级纤毛形态;对初级纤毛阳性率与IHH、骨形态发生蛋白2的表达进行Spearman相关性分析。结果与结论:①成骨诱导3 d,第40代组MC3T3-E1细胞的CDKN2A(P16)表达显著高于第10代组,而在siRNA IFT88-干预后差异消失;②第10代组的初级纤毛细胞阳性率高于第40代组,siRNA IFT88-则显著性抑制第10代与第40代细胞的初级纤毛表达;③第10代组MC3T3-E1细胞的碱性磷酸酶及骨形态发生蛋白2的转录活性和蛋白表达均高于第40代组,通过siRNA抑制初级纤毛表达后,上述差异减低或者消失;④初级纤毛细胞阳性率与IHH蛋白表达及骨形态发生蛋白2蛋白表达呈正相关。结果表明:初级纤毛介导了成骨性MC3T3-E1细胞的传代衰老,可调控成骨分化能力。

Effects of Rosiglitazone on Adiponectin Expression in 3T3-L1 Adipocytes at High Levels of Both Testosterone and Insulin In Vitro Culture

Objective To evaluate effects of rosiglitazone （RSG） on the expression of adiponectin in mature adipocytes at high levels of both testosterone （T） and insulin in vitro culture. Methods Mouse 3T3-L1 preadipocytes were induced to be mature adipocytes and used in this study. According to RSG concentrations, the cells added with T （10^-5 mol/L） and insulin （10^-4 mol/L） were divided into 4 groups： free-RSG group （0 mol/L RSG, FR-TI）, low-dose group （10^-9 mol/L RSG, LR-TI）, middle-dose group （lO-7mol/L RSG, MR-T1） and high-dose group （10^-6 mol/L RSG, HR-TI）. Besides, the cells added with RSG without T and insulin were also divided into 4 groups： FR, LR, MR, and HR. These 8 groups were incubated for 42 h. Cell viability was determined by MTT assay. Expression of adiponectin was detected by Western blotting. Results The maximum viability in FR-TI group was observed at point of 42 h. The growth of the adipocytes was significantly inhibited in MR-TI group compared with FR-TI （P〈0.01）. The level of adiponectin in MR-TI group was higher than that in LR- TI group （P〈0.01）. However, with RSG increasing; HR-TI group showed the lowest level of adiponectin among three treatment groups （P〈0.01）. In addition, adiponectin expression in MR-TI group was significantly higher than that in MR group （P〈0.01）. Conclusion RSG could increase the expression of adiponectin in 3T3-L1 adipocytes under high levels of both T and insulin, but it acts in a narrow concentration range.

葛根素对3T3-L1脂肪细胞胰岛素抵抗的影响及机制研究

目的:探讨葛根素对3T3-L1脂肪细胞胰岛素抵抗(IR)的影响及可能的作用机制。方法:将3T3-L1脂肪细胞分为7组,即对照组(control组)、葛根素3μmol/L组、葛根素10μmol/L组、葛根素30μmol/L组、葛根素100μmol/L组、葛根素300μmol/L组和阳性对照组(罗格列酮10μmol/L组,RGZ组),每组6孔。采用MTT法检测细胞增殖,油红O染色法检测细胞分化。利用地塞米松诱导建立3T3-L1脂肪细胞IR模型,并给予不同浓度的葛根素进行干预,测定葡萄糖利用情况,利用细胞转染过表达TLR2(命名为Pue+oe-TLR2组);蛋白质免疫印迹法(western blotting)测定TLR2蛋白水平;酶联免疫吸附试验测定干扰素-γ(IFN-γ)水平。结果:与control组相比,葛根素各剂量组3T3-L1脂肪细胞的增殖率均无显著变化(P>0.05)。与control组相比,葛根素各剂量组3T3-L1脂肪细胞的分化明显增加(P<0.05)。与control组相比,葛根素各剂量组IR 3T3-L1脂肪细胞的葡萄糖消耗量显著提高(P<0.05);葛根素各剂量组IR 3T3-L1脂肪细胞中TLR2表达量、IFN-γ分泌量降低,GLUT4和PPARγ的水平升高(P<0.05)。Pue+oe-TLR2组TLR2的相对表达量及IFN-γ分泌量显著高于Pue+oe-NC组,PPARγ、GLUT4的相对表达量显著低于Pue+oe-NC组(P<0.01)。结论:葛根素可提高IR 3T3-L1脂肪细胞葡萄糖消耗,缓解IR,其机制可能与抑制TLR2表达进而降低IFN-γ分泌有关。

木犀草苷对3T3-L1前脂肪细胞分化和脂代谢的影响

目的:研究木犀草苷对3T3-L1前脂肪细胞分化和脂代谢的影响及其作用机制。方法:采用3T3-L1前脂肪细胞,利用四甲基偶氮唑蓝法检测不同质量浓度木犀草苷对3T3-L1前脂肪细胞毒性的影响;使用鸡尾酒诱导法诱导细胞分化,用油红O染色观察脂滴形成情况;测定分化后细胞中甘油三酯、总胆固醇的含量以及相关炎症因子如肿瘤坏死因子(tumor necrosis factor,TNF)-α、白细胞介素(interleukin,IL)-6、IL-10、瘦素(leptin,LEP)和脂联素(adiponectin,ADPN)的分泌量;利用Western Blot法检测细胞过氧化物酶体增殖物激活受体(peroxisome proliferators activated receptor,PPAR)γ、CCAAT增强子结合蛋白(CCAAT-enhancer-binding proteins,C/EBP)-α、固醇调节元件结合蛋白1(sterol regulatory element-binding protein 1,SREBP1)、PPARα、解偶联蛋白1(uncoupling protein 1,UCP-1)、肉碱棕榈酰转移酶1(carnitine palmitoyltransferase 1,CPT-1)蛋白的相对表达水平。结果:3T3-L1前脂肪细胞存活率随木犀草苷质量浓度的升高整体呈降低趋势,木犀草苷质量浓度在5~60μg/mL时细胞存活率均在80%以上。木犀草苷处理组可显著抑制3T3-L1前脂肪细胞的分化,减少脂质积累;木犀草苷可下调成脂分化后细胞中总胆固醇、甘油三酯的含量以及炎症因子TNF-α、IL-6和LEP的分泌量,上调IL-10和ADPN的分泌量;木犀草苷能下调PPARγ、C/EBP-α和SREBP1蛋白的相对表达水平,上调PPARα、UCP-1和CPT-1蛋白的相对表达水平。结论:木犀草苷可以抑制3T3-L1前脂肪细胞分化和影响脂代谢,其机制一方面是下调PPARγ、C/EBP-α和SREBP1蛋白表达,抑制脂肪合成;另一方面是激活PPARα上调下游蛋白,促进脂肪消耗;木犀草苷还可能通过调节细胞因子的分泌量,促进细胞脂解,从而改善脂代谢失衡。

山药多糖对地塞米松诱导IR-3T3-L1脂肪细胞的降血糖作用及机制研究

目的探究山药多糖(Chinese yam polysaccharide,CYPS)在地塞米松(dexamethasone,DEX)诱导的3T3-L1胰岛素抵抗(IR-3T3-L1)细胞模型中的降血糖作用。方法将IR-3T3-L1脂肪细胞随机分为对照组(Con)、DEX组(DEX)、Met组(Met,阳性药组)、CYPS组(0.05、0.15、0.45 mg/mL),通过DEX(1μmol/L)诱导建立IR-3T3-L1细胞模型,进行给药后细胞对葡萄糖的消耗量和细胞中血脂(总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C))与丙二醛(malondialdehyde,MDA)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、己糖激酶(hexokinase,HK)、丙酮酸激酶(pyruvate kinase,PK)含量的检测,并采用qRT-PCR对AMPK-PI3K/Akt信号通路中相关基因表达进行检测。结果DEX组与Con组相比,1μmol/L DEX处理48 h显著降低了IR-3T3-L1细胞的葡萄糖消耗量(P<0.01),且在60 h内维持稳定。0.05、0.15、0.45 mg/mL CYPS处理IR-3T3-L1细胞显著增加了细胞中葡萄糖的消耗(P<0.01)、HK、PK、GSH-Px酶活性(P<0.01)、HDL-C含量(P<0.01);降低了MDA酶活性(P<0.01)、T-CHO、TG、LDL-C含量(P<0.01)。同时,CYPS可以显著回调PI3K、Akt、GLUT-4、AMPK、IRS-2、PPARa、Adiponectin的mRNA水平(P<0.05),降低IRS-1、GSK-3β、ACC、FAS的mRNA表达水平(P<0.01)。结论CYPS能够改善DEX诱导的IR-3T3-L1细胞对葡萄糖的消耗,调节脂代谢紊乱,缓解氧化应激,其作用机制可能通过调控IR-3T3-L1脂肪细胞的AMPK-PI3K/Akt信号通路来实现糖脂代谢紊乱和胰岛素抵抗调节来实现。

紫苏葶对3T3-L1细胞和黄羽肉鸡脂质沉积的影响

试验旨在探究紫苏葶对3T3-L1前脂肪细胞以及肉鸡脂肪沉积的影响。通过体外诱导3T3L-1前脂肪细胞分化为成熟脂肪细胞,检测紫苏葶(0、10、25、50、75、100μmol/L)对细胞增殖、脂质积累和能量代谢、脂质代谢相关基因表达的影响。选择30只1日龄雄性黄羽肉鸡按体重随机分为对照组(CON组)、高脂组(HFD组,基础日粮中添加猪油14%、胆固醇6%、蔗糖5%)、紫苏葶处理组(HFD+PER组,HFD组日粮中添加500 mg/kg紫苏葶),试验期15 d,研究紫苏葶对肉鸡腹部脂肪沉积的影响。结果表明:10~100μmol/L紫苏葶对3T3-L1细胞没有毒性作用;与对照组相比,75μmol/L紫苏葶组3T3-L1细胞中甘油三酯含量以及脂滴积累显著降低,细胞线粒体数量和三磷酸腺苷含量显著增加,线粒体棕色脂肪解偶联蛋1、过氧化物酶体增殖物激活受体-γ共激活因子-1α、脂肪酸结合蛋白4的mRNA表达水平显著升高,脂肪酸转位酶的mRNA表达水平显著降低;与对照组相比,HFD组黄羽肉鸡腹脂率显著升高,HFD+PER组腹脂率显著降低;通过RNA-seq发现,紫苏葶改变了肉鸡脂肪组织中与脂质沉积相关的256个基因的表达水平,这些基因主要富集在碳水化合物代谢、二磷酸腺苷代谢等途径。可见,紫苏葶是一种潜在的、可抑制脂肪沉积的天然小分子化合物或营养补充剂,该研究结果可为畜牧生产行业改善畜禽脂质沉积提供新的数据参考。