Troglitazone对人腹膜间皮细胞TGF-β_1和纤维连接蛋白表达的影响

目的:研究过氧化物增生体激活受体γ(PPAR-γ)激动剂troglitazone(曲格列酮,TGZ)对高浓度葡萄糖刺激下体外培养的人腹膜间皮细胞(HPMCs)中TGF-β1和细胞外基质纤维连接蛋白(Fn)表达的影响。方法:采用胰蛋白酶消化法从人大网膜组织中分离间皮细胞,建立稳定的体外培养模型。根据细胞增殖与毒性实验选择troglitazone的最佳浓度15μmol/L,干预高浓度葡萄糖(30mmol/LD-葡萄糖)刺激下的人腹膜间皮细胞。采用逆转录多聚酶链式反应(RT-PCR)半定量检测HPMCs中PPAR-γ,TGF-β1以及Fn的 mRNA表达;采用双抗夹心法酶联免疫吸附实验检测HPMCs培养液中TGF-β1蛋白质水平;Western印迹检测Fn蛋白质水平。结果:高糖(30mmol/LD-葡萄糖)刺激可在 mRNA和蛋白质水平明显上调HPMCs表达TGF-β1和Fn(P<0.01),troglitazone干预高糖孵育的HPMCs,可使TGF-β1和Fn mRNA和蛋白质的表达明显下调(P<0.05)。结论:Troglitazone能够明显抑制在高糖刺激下的HPMCsTGF-β1和Fn的表达,这可能为临床防治长期腹膜透析患者的腹膜纤维化提供一种较为有效的方法。

Troglitazone对高糖和LPS诱导的人肾小球mPGEs蛋白表达的抑制作用

目的 :研究PPAR γ激动剂troglitazone对LPS或高浓度葡萄糖作用的体外培养的人肾小球mPGEs蛋白表达水平的影响。方法 :用LPS或高浓度葡萄糖分别与不同浓度troglitazone作用体外培养的人肾小球 ,ELISA法检测上清液中mPGEs浓度。结果 :LPS(10 μg/ml)或 5 %葡萄糖均可使体外培养的人肾小球mPGEs明显增高。 10～ 2 0nmol/L的troglitazone可明显抑制LPS(10 μg/ml)和 5 %葡萄糖诱导的大鼠系膜细胞mPGEs蛋白表达水平的增高 (P <0 .0 5 )。结论 :LPS和 5 %葡萄糖可诱导体外培养的人肾小球mPGEs蛋白表达水平明显增高 ,PPAR γ激动剂troglita zone可明显抑制两者所诱导的体外培养的人肾小球mPGEs蛋白表达水平的增高。

Troglitazone对小鼠Ⅱ型葡萄糖载体和Ⅰ型葡萄糖载体mRNA表达的影响

目的探讨Troglitazone对小鼠Ⅱ型葡萄糖载体(mGLUT2)和I型葡萄糖载体(mGLUT1)mRNA表达的影响。方法采用分子克隆技术,将过氧化物酶体增殖物受体γ(PPARγ)和维甲酸类受体X(RXRα)及mGLUT2和mGLUT1cDNA分别克隆到表达载体pCMX和pGL3b上。PPARγ与RXRα及mGLUT2与mGLUT1克隆载体转染NIH3T3细胞,处理或不处理Troglitazone,应用荧光素酶活性测定法及RNA印迹等方法测定Troglitazone对mGLUT2和mGLUT1重组体荧光素酶活性调节及对mRNA表达的影响。结果Troglitazone可激活mGLUT2和mGLUT1重组体荧光素酶的活性,并且可增加mR-NA的表达水平。结论Troglitazone可增强mGLUT2和mGLUT1的表达,可能参与PPARγ对mGLUT2和mGLUT1的调节过程。

Troglitazone对小鼠Ⅰ型葡萄糖载体表达的影响

目的:探讨Troglitazone对小鼠I型葡萄糖载体(mGLUT1)表达的影响。方法:采用分子克隆技术,将过氧化物酶体增殖物受体γ(PPARγ)和维甲酸类受体X(RXRα)及mGLUT1 cDNA分别克隆到表达载体pCMX和pGL3b上。PPARγ与RXRα及mGLUT1克隆载体转染NIH 3T3细胞,处理或不处理Troglitazone,应用荧光素酶活性测定法及RNA印迹等方法测定Troglitazone对mGLUT1重组体荧光素酶活性调节及对mRNA表达的影响。结果:Troglitazone可激活mGLUT1重组体荧光素酶的活性,并且可增加mGLUT1的mRNA表达水平。结论:Troglitazone可增强mGLUT1的表达,可能参与PPARγ对mGLUT1的调节过程。

Troglitazone Induced Apoptosis via PPARγ Activated POX-induced ROS Formation in HT29 Cells

Objective In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells,the effects of PPARγ and POX-induced ROS were explored.Methods [3-（4,5）-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） assay,Annexin V and PI staining using FACS,plasmid transfection,ROS formation detected by DCFH staining,RNA interference,RT-PCR RT-QPCR,and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells.Results Troglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis.During this process,mitochondria related pathways including ROS formation,POX expression and cytochrome c release increased,which were inhibited by pretreatment with GW9662,a specific antagonist of PPARγ.These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern.Furthermore,the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone.Conclusion The findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation,at least partly,via PPARγ activation.

Reversal of P-glycoprotein-mediated Multidrug Resistance in SGC7901/VCR Cells by PPARγ Activation by Troglitazone

Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma(PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Western blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR) alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

Troglitazone induced apoptosis of human pterygium fibroblasts through a mitochondrial-dependent pathway

AIM: To study the effect of troglitazone on primary culture human pterygium fibroblast (HPF). METHODS: Cell viability loss and apoptosis were quantified by cell counting kit-8, AnnexinV-FITC/PI double staining, caspases activity test and western blotting. Flow cytonnetry was used to detect mitochondrial membrane potential. RESULTS: Peroxisome proliferator-activated receptor gamma (PPAR-gamma) was positively expressed in pterygium specimens (n = 5). Troglitazone showed dose-dependent inhibition of cell survival, induced phospholipids redistribution, activated caspase-3, -9, and altered mitochondrial potential. Western blot assay demonstrated the increase of Bax/Bcl-2 protein ratio. CONCLUSION: Troglitazone induced apoptosis of HPF through a mitochondrial-dependent pathway.

Effect of Troglitazone on Expression of Adhesion Molecules and eNOS in Human Saphenous Vein Gaft

To investigate whether peroxisome proliferators-activated receptor-γ （PPARγ） ligand Troglitazone can reduce endothelial injury and activation during storage of harvested saphenous vein grafts. Segments of human saphenous vein graft were collected from 9 patients undergoing coronary bypass surgery and then divided into two equal parts of control and test specimens, were stored in ei- ther heparinized blood （control group） or heparinized blood containing 20 μmol/L troglitazone （test group） for 1 h at room temperature. Tissue distribution and protein expression of VCAM-I, ICAM-I, and endothelial nitric oxide synthase （eNOS） were compared using immunohistochemistry and Western blot analysis. Myeloperoxidase （MPO） activity, a marker of neutrophil sequestration in human saphenous vein grafts, was also measured in each group. The expression of ICAM-1 （753±132 versus 7201±934; P〈0.01） , VCAM-1 （3731±294 versus 8292±793; P〈0.01）, and MPO activity （1.52±0.42 U/g, 5.04±1.26 U/g P〈0.01） were significantly lower in test group. In contract, eNOS expression （7983±834 versus 3989±1008; P〈0.01） was significantly higher in test group. PPARγ ligand troglitazone might reduce endothelial injury during the storage period of human saphenous vein grafts.

PPAR-γ激动剂Troglitazone对高糖诱导的大鼠肾小球系膜细胞mPGEs蛋白表达的影响

目的 研究PPAR -γ激动剂Troglitazone对高浓度葡萄糖作用的大鼠肾小球系膜细胞mPGEs蛋白表达水平的影响。方法 用高浓度葡萄糖和不同浓度Troglitazone作用大鼠肾小球系膜细胞 ,ELISA法检测上清液中mPGEs浓度。结果 5 %葡萄糖可使体外培养的大鼠系膜细胞mPGEs蛋白表达水平明显增高 ( P <0 0 5 ) ;10～ 2 0nmol/L的Troglitazone可明显抑制 5 %葡萄糖诱导的大鼠系膜细胞mPGEs蛋白表达水平的增高 (P <0 0 5 )。结论 高浓度葡萄糖可诱导体外培养的大鼠系膜细胞mPGEs蛋白表达水平明显增高 ,PPAR

Boiogito Increases the Synthesis and Secretion of Adiponectin by Promoting Differentiation in Cultured Human Adipocytes

Boiogito (BOT) ameliorates insulin resistance and diabetes in several animal models;however, the underlying mechanisms for these in vivo effects remain unclear. Thiazolidine derivatives, which are peroxisome proliferator-activated receptor γ (PPARγ) agonists for the treatment of type II diabetes, promote adiponectin production by inducing adipocyte differentiation, thereby reducing insulin resistance. This study aimed to evaluate the effect of BOT on adipocyte differentiation using cultured human visceral preadipocytes (HVPAds) compared with the thiazolidine derivative troglitazone (TRG). We investigated the effects of BOT (0.125 - 1 mg/mL) and TRG (10 μM) on the differentiation of adipocytes treated with or without tumor necrosis factor-α (TNF-α: 5 ng/mL). On day 14 of culture, the following adipocyte differentiation marker levels were measured: intracellular lipids, extracellular (i.e., medium) adiponectin, and intracellular differentiation-related genes (PPARγ, CCAAT/enhancer binding protein, adiponectin, differentiation cluster 36, glucose transporter type 4). BOT and TRG increased factors associated with differentiation including lipid, adiponectin, and differentiation-related gene expression levels compared with the controls. The increases in these differentiation markers were inhibited by the PPARγ antagonist GW9662 (20 μM). Furthermore, TNF-α decreased all differentiation marker levels. The decreases in differentiation markers were inhibited by BOT and TRG;however, these inhibitory effects were blocked by GW9662. The results suggest that BOT increases the synthesis and secretion of adiponectin by promoting differentiation similar to TRG. This study is the first to demonstrate that adipocyte differentiation-promoting activity is a mechanism for the beneficial effects of BOT on diabetes and insulin resistance.

Src与胶原同源插头蛋白(Shc)调控曲格列酮引起的PAE细胞自噬

【目的】明确Src与胶原同源插头蛋白(Src homology and collagen homology,Shc)调控曲格列酮(troglitazone,TZ)引起的猪血管内皮(porcine aortic endothelial,PAE)细胞自噬的机制。【方法】我们先利用激光共聚焦显微镜、蛋白免疫杂交检测了TZ引起的PAE细胞自噬;然后通过siRNA干扰敲降Shc,转染wtShc、3mShc等质粒的方法确定了p52Shc参与自噬的调控;最后通过siRNA干扰敲降Ulk1得到最终结论。【结果】通过研究发现,敲降Shc,会增加细胞的自噬;而过量表达p52Shc抑制了TZ引起的细胞自噬;p52Shc抑制自噬与其自身的酪氨酸磷酸化位点Tyr239、Tyr240和Tyr317相关;同时发现,p52Shc能抑制自噬调节分子磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase,AMPK)及其下游底物Unc51样激酶1(UNC-51-like kinase-1,Ulk1)的活性。【结论】Shc通过调控AMPK与Ulk1的磷酸化调节TZ引起的细胞自噬。

Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis

Mitochondrial fatty acid oxidation(mtFAO)is a key metabolic pathway required for energy production in the liver,in particular during periods of fasting.One major consequence of drug-induced impairment of mtFAO is hepatic steatosis,which is characterized by an accumulation of triglycerides and other lipid species,such as acyl-carnitines.Actually,the severity of this liver lesion is dependent on the residual mitochondrial b-oxidation flux.Indeed,a severe inhibition of mtFAO leads to microvesicular steatosis,hypoglycemia and liver failure.In contrast,moderate impairment of mtFAO can cause macrovacuolar steatosis,which is a benign lesion in the short term.Because some drugs can induce both microvesicular and macrovacuolar steatosis,it is surmised that severe mitochondrial dysfunction could be favored in some patients by non-genetic factors(e.g.,high doses and polymedication),or genetic predispositions involving genes that encode proteins playing directly or indirectly a role in the mtFAO pathway.Example of drugs inducing steatosis include acetaminophen(APAP),amiodarone,ibuprofen,linezolid,nucleoside reverse transcriptase inhibitors,such as stavudine and didanosine,perhexiline,tamoxifen,tetracyclines,troglitazone and valproic acid.Because several previous articles reviewed in depth the mechanism(s)whereby most of these drugs are able to inhibit mtFAO and induce steatosis,the present review is rather focused on APAP,linezolid and troglitazone.These steatogenic drugs are indeed rarely discussed in the literature as regards their ability to impair mtFAO.