过氧化物酶增殖物激活受体γ抑制大鼠肝星状细胞中转化生长因子-β1诱导的结缔组织生长因子表达

目的 探讨过氧化物酶增殖物激活受体γ(PPARγ)对大鼠肝星状细胞(HSC)中转化生长因子-β1(TGF-β1)诱导的结缔组织生长因子(CTGF)表达的抑制作用.方法 常规培养大鼠HSC,预先给予或不给予PPARγ特异性拮抗剂GW9662处理,再经系列浓度的PPARγ天然配体(15-d-PGJ2)或合成配体(GW7845)及TGF-β1序贯作用后,通过半定量RT-PCR及Western-blotting分析CTGF表达状况,透射电镜观察HSC形态学变化.结果 15-d-PGJ2和GW7845可显著抑制HSC中TGF-β1诱导的CTGF表达(同时在转录和转录后水平),且PPARγ配体对CTGF表达的抑制作用可被GW9662部分或完全逆转,说明此种抑制效应确由PPARγ所介导;电镜观察亦显示HSC由活化态向静息态的形态学转变.结论 PPARγ活化可显著抑制HSC中TGF-β1诱导的CTGF表达,提示PPARγ可作为逆转肝纤维化治疗的新的有效靶点.

Abstract：

Objective To investigate the effect of peroxisome proliferator-activated receptor gamma(PPARγ)activation on transforming growth factor betal(TGF-β1)-induced connective tissue growth factor(CTGF)expression in rat hepatic stellate cells(HSCs).Methods Cultured HSCs with or without PPARγ-specific antagonist GW9662 treatment prior to the addition of an increasing amount of PPARγ natural ligand(15-d-PGJ2)or synthetic ligand(GW7845)were stimulated with TGF-β1.The mRNA and protein levels of CTGF expression were detected by semi-quantitative RT-PCR and Western blotting,respectively.The morphological changes of the HSC were obgerved by electron microscope.Results 15-d-PGJ2 and GW7845 significantly inhibited TGF-β1-induced CTGF expression at both mRNA and protein levels in HSCs, and the inhibitory effect was dramatically,if not completely,abolished by pretreatment with GW9662,suggesting that the inhibition was mediated by PPARγ. Morphological observation revealed that PPARγactivation caused obvious changes of HSCs from activated to quiescent phenotypes.Conclusion PPARγ ligand shows potent inhibitory effect on TGF-β1-induced CTGF expression in rat HSCs,suggesting its potential as a candidate agent for treatment and prevention of hepatic fibrosis.     

Effects of curcumin on peroxisome proliferator-activated receptor γ expression and nuclear translocation/redistribution in culture-activated rat hepatic stellate cells

Background The function of peroxisome proliferator-activated receptor γ （PPARγ） in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells （HSCs） through PPARγ signaling were investigated. Methods HSCs were isolated from the normal Sprague Dawley rats through in situ peffusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy Isulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARγ subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay. Results Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARγ nuclear expression level decreased. Curcumin up-regulated PPARγ expression and significantly inhibited the production of a-SMA and collagen Ⅰ. PPARγ is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFKB p65 protein and TGFβR-Ⅰ protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin. Conclusions Curcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARγ sianal transduction pathway and associated with PPARγ nuclear translocation/redistribution.     

Shp-2／NF-κB Pathway Mediates the Inhibition of Lipoxin A4 onIL-1β-induced Synthesis of IL-6 in Glomerular Mesangial Cells

Objective: To examine whether lipoxin A4 (LXA4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β with or without preincubation with LXA4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzymelinked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2( SH2 ) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results:IL-1β- snulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA4 in a dose-dependent manner. LXA4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β Conclusion: LXA4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cellsthrough the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.     

Regulation of the expression and function of glucose transporter-1 by TGF-β1 and high glucose in mesangial cells

Objective To study the effects of high glucose and transforming growth factor-β1 (TGF-β1) on the expression and function of glucose transporter-1 (GLUT1) in mouse mesangial cells.Methods Cultured mouse mesangial cells were used.The expression of GLUT1 mRNA was detected by Northern Blot; glucose uptake and its kinetics were determined with a 2-Deoxy-［(3)H］-D-glucose uptake assay.Results Mesangial cells exposed to enriched glucose medium (20 mmol/L) for 72 hours demonstrated a decrease in both GLUT1 mRNA and V(max) for uptake of the glucose analog, 2-deoxy-D-glucose　(2DOG), as compared to mesangial cells cultured in physiologic glucose concentrations(5.5 mmol/L).In contrast, hypertonic mannitol had no effect on GLUT1 mRNA levels.TGF-β1 treatment for 10 hours stimulated 2DOG uptake, both in 5.5 mmol/L and 20 mmol/L glucose medium, by approximately 4.28-fold in a dose-dependent manner (2 ng/ml maximum).Kinetic analysis of 2DOG uptake revealed an increase in V(max) and a decrease in K(m) in the presence of TGF-β1. TGF-β1 also up-regulated the expression of GLUT1 mRNA in mesangial cells.The addition of anti-TGF-β neutralizing antibody (30 μg/ml) in mesangial cells cultured in enriched glucose medium (20 mmol/L) led to a 40% decrease in 2DOG uptake.Conclusions The expression of GLUT1 can be suppressed by exposure of mesangial cells to high glucose medium, which may serve as a protective mechanism against possible adverse effects of excessive glucose flux into cells.TGF-β1 stimulates glucose uptake by enhancing the expression and function of GLUT1 in mesangial cells.This effect is independent of the glucose milieu in the cultured medium.     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective： Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin （IL） -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells （HMCs）. Methods： The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay （RPA）. Activity of nuclear factor-kappa B （NF-κB） and activa- tor protein-1 （AP-1） was examined by electrophoretic mobility shift assay （EMSA）. NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase （JNK） activity were assayed by immunoblot. Results： Recombinant IL-13 inhibited tumor necrosis factor-α （TNF-u）, IL-1α, IL-1β, monocyte chemoattractant protein-1 （MCP-1）, IL-8, and transforming growth factor-β1 （TGF-β1） mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides （LPS） dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion： IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.     

Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL-60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL-60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragmem analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL-60 and U937cells effectively in a dose-dependent manner. After incubation with FTY720 for 24 h, apoptosis wasobserved in HL-60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down-regulated and the distribution of ERK1/2 protein in cell‘ nuclear was reduced during FTY720-induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720-induced apoptosis and proliferation inhibition of leukemia cells.     

Progress of Targeting Transforming Growth Factor-β1 Small Interfering RNA in Liver Fibrosis

Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells （HSCs） remains a central event in fibrosis. HSCs are the main source of extracellular matrix （ECM）. Transforming growth factor-beta （TGF-β）, which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference （RNAi） is a novel gene disruption technology. Studies have shown that small interfering RNA （siRNA） targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 siRNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 siRNA in liver fibrosis.     

Different effects of telmisartan and valsartan on human aortic vascular smooth muscle cell proliferation

Background  Vascular smooth muscle cell proliferation is an important process in the development of atherosclerosis and is associated with other cellular processes in atherogenesis. Telmisartan is reported to have partial peroxisome proliferator-activated receptor (PPAR)-γ activating properties and has been referred to as selective PPAR modulators, but valsartan just blocks angiotensin II (AngII) type 1 (AT1) receptors. This study aimed to compare the different effects of telmisartan and valsartan on human aortic smooth muscle cells (HASMCs) proliferation.

Methods  Ability of telmisartan and valsartan to inhibit proliferation of HASMCs was evaluated by the Cell Counting Kit-8 (CCK-8) in continuous cell culture. Whether the antiproliferative effects of telmisartan and valsartan depend on their effects on AngII receptors or activating the peroxisome PPAR-γ was also investigated in this study.

Results  Telmisartan inhibited proliferation of HASMCs by 52.4% (P <0.01) at the concentration of 25 µmol/L and the effect depended on the dose of telmisartan, but valsartan had little effect on HASMCs proliferation (P >0.05) and no dose response. When tested in cells stimulated with AngII, telmisartan had the same inhibition of HASMCs by 59.2% (P <0.05) and valsartan also inhibited it by 41.6% (P <0.05). Telmisartan and valsartan had the same effect on down-regulating AT1 receptor expression and telmisartan was superior to valsartan up-regulating AngII type 2 (AT2) receptor expression. Antiproliferative effects of telmisartan were observed when HASMCs were treated with the PPAR-γ antagonist GW9662 but antiproliferative effects of the PPAR-γ activator pioglitazone were not observed.

Conclusions  Telmisartan, but not valsartan, inhibits HASMCs proliferation and has dose-dependent response without stimulation of AngII. AT2 receptor up-regulation of telmisartan contributes to its greater antiproliferative effects than valsartan. Its PPAR-γ activation does not play a critical role in inhibiting HASMCs proliferation.

     

Visfatin Protects Rat Pancreatic β-cells against IFN-γ-Induced Apoptosis through AMPK and ERK1/2 Signaling Pathways

ObjectiveInterferon-γ (IFN-γ) plays an important role in apoptosis and was shown to increase the risk of diabetes.Visfatin, an adipokine, has anti-diabetic, anti-tumor, and regulating inflammatory properties. In this study we investigated the effect of visfatin on IFN-γ-induced apoptosis in rat pancreatic β-cells. MethodsThe RINm5F (rat insulinoma cell line) cells exposed to IFN-γ were treated with or without visfatin. The viability and apoptosis of the cells were assessed by using MTT and flow cytometry. The expressionsof mRNA and protein were detected by using real-time PCR and western blot analysis. ResultsThe exposure of RINm5F cells to IFN-γ for 48 h led to increased apoptosis percentage of the cells. Visfatin pretreatment significantly increased the cellviability and reduced the cell apoptosis induced by IFN-γ. IFN-γ-induced increase in expression of p53 mRNA and cytochrome c protein, decrease in mRNA and protein levels of anti-apoptotic protein Bcl-2 were attenuated by visfatin pretreatment. Visfatin alsoincreasedAMPK and ERK1/2phosphorylation and the anti-apoptotic action of visfatin was attenuated by the AMPK and ERK1/2 inhibitor. ConclusionThese results suggested that visfatin protected pancreatic islet cells against IFN-γ-induced apoptosis via mitochondria-dependent apoptotic pathway. The anti-apoptotic action of visfatin is mediated by activation of AMPK and ERK1/2 signaling molecules.