白藜芦醇抑制巨噬细胞细胞外基质金属蛋白酶诱导物的表达

目的探讨白藜芦醇对细胞外基质金属蛋白酶诱导物(EMMPRIN)表达的影响。方法将人类单核细胞系THP-1和MCF-7细胞共培养,测定上清液中MMP-9活性。用PMA诱导THP-1为巨噬细胞，加入白藜芦醇，观察EMMPRIN表达和MMP-9活性变化。细胞共转染实验测定白藜芦醇对PPARγ的激动作用。用PPARγ的拮抗剂GW9662预处理细胞后，测定白藜芦醇对EMMPRIN表达的影响。结果PMA诱导使单核细胞EMMPRIN表达明显增强，与EMMPRIN高表达的MCF-7细胞共培养显著增加单核细胞表达MMP-9。白藜芦醇显著抑制EMMPRIN和MMP-9生成。白藜芦醇明显激动PPARγ，GW9662大幅减弱白藜芦醇对EMMPRIN和MMP-9的作用。结论单核细胞向巨噬细胞分化过程中表达明显增强的EMMPRIN可能是促进MMPs表达的主要因子。白藜芦醇通过激动PPARγ抑制EMMPRIN的表达，可能是其抑制巨噬细胞MMPs产生的机制。     

GW9662, a potent antagonist of PPARgamma, inhibits growth of breast tumour cells and promotes the anticancer effects of the PPARgamma agonist rosiglitazone, independently of PPARgamma activation

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is activated by several compounds, including the thiazolidinediones. In addition to being a therapeutic target for obesity, hypolipidaemia and diabetes, perturbation of PPARgamma signalling is now believed to be a strategy for treatment of several cancers, including breast. Although differential expression of PPARgamma is observed in tumours compared to normal tissues and PPARgamma agonists have been shown to inhibit tumour cell growth and survival, the interdependence of these observations is unclear. This study demonstrated that the potent, irreversible and selective PPARgamma antagonist GW9662 prevented activation of PPARgamma and inhibited growth of human mammary tumour cell lines. Controversially, GW9662 prevented rosiglitazone-mediated PPARgamma activation, but enhanced rather than reversed rosiglitazone-induced growth inhibition. As such, these data support the existence of PPARgamma-independent pathways and question the central belief that PPARgamma ligands mediate their anticancer effects via activation of PPARgamma.     

Decursin inhibits induction of inflammatory mediators by blocking nuclear factor-kappaB activation in macrophages

In the course of screening inhibitors of matrix metalloproteinase (MMP)-9 induction in macrophages, we isolated decursin, a coumarin compound, from the roots of Angelicae gigas. As a marker for the screening and isolation, we tested expression of MMP-9 in RAW264.7 cells and THP-1 cells after treatment with bacterial lipopolysaccharide (LPS), the TLR-4 ligand. Decursin suppressed MMP-9 expression in cells stimulated by LPS in a dose-dependent manner at concentrations below 60 microM with no sign of cytotoxicity. The suppressive effect of decursin was observed not only in cells stimulated with ligands for TLR4, TLR2, TLR3, and TLR9 but also in cells stimulated with interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha, indicating that the molecular target of decursin is common signaling molecules induced by these stimulants. In addition to the suppression of MMP-9 expression, decursin blocked nitric oxide production and cytokine (IL-8, MCP-1, IL-1beta, and TNF-alpha) secretion induced by LPS. To find out the molecular mechanism responsible for the suppressive effect of decursin, we analyzed signaling molecules involved in the TLR-mediated activation of MMP-9 and cytokines. Decursin blocked phosphorylation of IkappaB and nuclear translocation of NF-kappaB in THP-1 cells activated with LPS. Furthermore, expression of a luciferase reporter gene under the promoter containing NF-kappaB binding sites was blocked by decursin. These data indicate that decursin is a novel inhibitor of NF-kappaB activation in signaling induced by TLR ligands and cytokines.     

Valsartan and telmisartan abrogate angiotensin II-induced downregulation of ABCA1 expression via AT1 receptor, rather than AT2 receptor or PPARγ activation

The possible pharmacological mechanism by which partial PPARγ-activating angiotensin II (Ang II) type 1 receptor blocker (ARB) telmisartan and non-PPARγ-activating ARB valsartan reverse Ang II-suppressed ABCA1 expression is still unclear. In this study, human monocyte-derived THP-1 cells were differentiated into macrophages. Cells were treated with various concentrations of Ang II alone or with Ang II and various drugs including highly selective ARB valsartan, partial PPARγ-activating ARB telmisartan, angiotensin II type 2 (AT2) receptor blocker PD123319, full PPARγ agonist pioglitazone, and PPARγ antagonist GW9662, respectively. After treatment, messenger RNA and protein expressions of ABCA1 and ABCG1 were analyzed by real-time polymerase chain reaction and Western blotting, respectively. ABCA1 expression was remarkably suppressed by Ang II at both messenger RNA and protein levels in a dose-dependent manner in THP-1-derived macrophages, whereas ABCG1 expression was not affected. Valsartan and telmisartan could both reverse the downregulation of Ang II on ABCA1 expression. Such effects were not affected by either AT2 receptor blocker PD123319 or PPARγ antagonist GW9662. Our findings suggest that the effect of Ang II on ABCA1 expression should be mediated by the AT1 receptor. Both valsartan and telmisartan abrogate Ang II-induced downregulation of ABCA1 expression mainly through AT1 receptor rather than through AT2 receptor or PPARγ-dependent pathway.     

Pioglitazone improves visceral sensation and colonic permeability in a rat model of irritable bowel syndrome

Visceral hypersensitivity and impaired gut barrier with minor inflammation are considered to play an important role in the pathophysiology of irritable bowel syndrome (IBS). Since pioglitazone is known to have anti-inflammatory property, we hypothesized that pioglitazone is beneficial for treating IBS. In this study, the effect was tested in rat IBS models such as lipopolysaccharide or repeated water avoidance stress-induced visceral allodynia and increased colonic permeability. Pioglitazone blocked these visceral changes, and GW9662, a peroxisome proliferator-activated receptor gamma (PPAR-γ) antagonist fully reversed the effect by pioglitazone. These results suggest that PPAR-γ activation by pioglitazone may be useful for IBS treatment.     

吡格列酮对HepG2细胞增殖和凋亡的影响及机制研究

目的观察吡格列酮对体外培养的HepG2细胞增殖和凋亡的影响,并探讨其是否通过PPARγ依赖途径发挥上述药理作用。方法将不同浓度的吡格列酮作用于体外培养HepG2细胞,以MTT比色法检测HepG2细胞增殖情况,以3H-TdR参入实验检测细胞DNA合成速率,采用RT-PCR和Western blot检测PPARγmRNA和蛋白的表达,以流式细胞术检测细胞凋亡和细胞周期;同时观察PPARγ特异性拮抗剂GW9662和(或)瞬时转染pSG5-PPARγ真核表达质粒对吡格列酮细胞增殖作用的影响;并将PPARγ小干扰RNA(pGCsi-PPARγ)表达质粒稳定转染HepG2细胞,观察PPARγ沉默后吡格列酮对HepG2细胞增殖作用的影响。结果吡格列酮作用于HepG2细胞后,导致HepG2细胞的增殖受到抑制、DNA合成速率减慢,并诱导细胞凋亡,呈一定的剂量依赖关系;在此过程中,G0/G1期细胞比例明显增加,S期细胞比例明显减少,但PPARγmRNA和蛋白的表达没有变化;GW9662部分拮抗吡格列酮的增殖抑制作用,但转染pSG5-PPARγ真核表达质粒可以逆转GW9662的作用;吡格列酮在高浓度(20μmol.L-1)时对pGCsi-PPARγ表达质粒稳定转染的HepG2细胞仍表现出增殖抑制作用。结论吡格列酮能够抑制HepG2细胞的增殖并诱导凋亡,具有潜在的抗瘤作用,这种作用与其诱导细胞G0/G1期的停滞有关,PPARγ依赖和非依赖途径参与上述过程。     

芍药苷对THP-1细胞CD147表达及MMP-9分泌的影响

目的 观察芍药苷干预THP-1细胞CD147表达及MMp-9分泌的影响.方法 用THP-1细胞作为模型,采用West Blot检测THP-1细胞CD147蛋白的表达,酶联免疫吸附试验（ELISA法）检测MMp-9分泌的变化,以探讨芍药苷治疗狼疮肾炎的可能机制.结果 芍药苷可抑制THP-1细胞CD147蛋白的表达（P＜0.05）,同时减少MMP-9分泌（P＜ 0.05）,差异具有统计学意义.结论 芍药苷可有效抑制THP-1细胞CD147表达及减少MMP-9分泌.     

大鼠脑缺血再灌注早期过氧化物酶体增殖物激活受体γ 活化与细胞焦亡的关系

目的 探讨过氧化物酶体增殖物激活受体γ（PPARγ）活化在大鼠脑缺血再灌注损伤中与细胞焦亡的关 系。方法 40只SPF级雄性SD 大鼠随机分为假手术组（sham组）、模型组（MCAO组）、给药组（PGZ组、PGZ+GW9662 组）,每组10只;采用Zea-Longa评分法进行神经功能评分,TTC染色测量脑梗死面积,运用Western blot 技术分析大 鼠脑组织中PPARγ,焦亡关键蛋白半胱氨酸天冬氨酸蛋白酶-1（caspase-1）、Gasdermin D（GSDMD）及炎症因子白细 胞介素-1β（IL-1β）、白细胞介素-18（IL-18）的表达变化。结果 与 sham 组比较,缺血再灌注 24 h 后 MCAO 组中 PPARγ 表达明显降低（P＜0.05）,焦亡关键蛋白 caspase-1、GSDMD 及炎症因子 IL-1β、IL-18 水平显著增高（P＜ 0.05）,神经功能评分明显增加,脑梗死面积增大（P＜0.01）;与MCAO组比较,PGZ组中PPARγ显著升高（P＜0.05）, 焦亡关键蛋白caspase-1、GSDMD及炎症因子IL-1β、IL-18水平降低（P＜0.05）,神经功能评分显著下降,脑梗死面积 减少（P＜0.01）;而PGZ+GW9662组中,PPARγ的作用被逆转。结论 在脑缺血再灌注损伤早期PPARγ激活可通过 抑制细胞焦亡产生从而减轻神经细胞损伤。     

Pioglitazone inhibits androgen production in NCI-H295R cells by regulating gene expression of CYP17 and HSD3B2

Thiazolidinediones (TZDs) such as pioglitazone and rosiglitazone are widely used as insulin sensitizers in the treatment of type 2 diabetes. In diabetic women with polycystic ovary syndrome, treatment with pioglitazone or rosiglitazone improves insulin resistance and hyperandrogenism, but the mechanism by which TZDs down-regulate androgen production is unknown. Androgens are synthesized in the human gonads as well as the adrenals. We studied the regulation of androgen production by analyzing the effect of pioglitazone and rosiglitazone on steroidogenesis in human adrenal NCI-H295R cells, an established in vitro model of steroidogenesis of the human adrenal cortex. Both TZDs changed the steroid profile of the NCI-H295R cells and inhibited the activities of P450c17 and 3betaHSDII, key enzymes of androgen biosynthesis. Pioglitazone but not rosiglitazone inhibited the expression of the CYP17 and HSD3B2 genes. Likewise, pioglitazone repressed basal and 8-bromo-cAMP-stimulated activities of CYP17 and HSD3B2 promoter reporters in NCI-H295R cells. However, pioglitazone did not change the activity of a cAMP-responsive luciferase reporter, indicating that it does not influence cAMP/protein kinase A/cAMP response element-binding protein pathway signaling. Although peroxisome proliferator-activated receptor gamma (PPARgamma) is the nuclear receptor for TZDs, suppression of PPARgamma by small interfering RNA technique did not alter the inhibitory effect of pioglitazone on CYP17 and HSD3B2 expression, suggesting that the action of pioglitazone is independent of PPARgamma. On the other hand, treatment of NCI-H295R cells with mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) enhanced promoter activity and expression of CYP17. This effect was reversed by pioglitazone treatment, indicating that the MEK/ERK signaling pathway plays a role in regulating androgen biosynthesis by pioglitazone.     

白藜芦醇对U937细胞基质金属蛋白酶-9转录的抑制作用

目的:观察白藜芦醇对佛波酯诱导的U937细胞中基质金属蛋白酶-9活性的影响,并从蛋白、mRNA及核转录因子激活蛋白-1(AP-1)水平对其影响进行分析。方法:酶谱法测定U937细胞培养上清中MMP-9的活性;Western blot法考察MMP-9蛋白的生成;RT-PCR法检测MMP-9 mRNA的表达;电泳迁移率变动分析法(EMSA)研究核转录因子SP-1的活性。结果:PMA 10nmol/L 可显著诱导无血清培养的U937细胞中MMP-9活性(P<0.01);白藜芦醇在1和10 μmol/L浓度下可抑制 PMA 10 nmol/L诱导的MMP-9活性(P<0.05 和P<0.01);PMA 10 nmol/L可显著诱导U937细胞中MMP-9蛋白生成(P<0.01)和MMP-9 mRNA的表达(P<0.01),白藜芦醇在1、10μmol/L浓度下可抑制PMA 10 nmol/L诱导的MMP-9蛋白生成和MMP-9 mRNA的表达(P<0.05);白藜芦醇在10、1和0.1μmol/L浓度下可抑制PMA诱导的U937细胞中AP-1的活化。结论:白藜芦醇可有效地抑制PMA诱导的U937细胞中MMP-9的活性,其作用可能是通过抑制PMA诱导的U937细胞核转录因子AP-1活化,进而降低MMP-9 mRNA表达,减少MMP-9蛋白生成而实现的。     

葛根素对巨噬细胞基质金属蛋白酶9及其组织抑制物1表达的影响

目的 观察葛根素对巨噬细胞分泌和表达基质金属蛋白酶9(MMP-9)及其组织抑制因子1(TIMP-1)的影响.方法 将人单核细胞系THP-1来源的巨噬细胞与不同浓度的葛根素进行培养,采用RT-PCR和ELISA方法 分别检测巨噬细胞的MMP-9、TIMP-1的基凶和蛋白质表达.结果 葛根素对氧化低密度脂蛋白(ox-LDL)诱导THP-1细胞MMP-9 mRNA及其蛋白质表达作用呈浓度依赖性地减少MMP-9 mRNA及其蛋白的表达,但对TIMP-1的表达无影响.结论 葛根素可以通过调节巨噬细胞分泌MMP-9途径发挥稳定动脉粥样硬化斑块的作用.     

PPARγ dependence of cyclosporine-isoprenaline renovascular interaction: roles of nitric oxide synthase and heme oxygenase

We previously showed that cyclosporine (CSA) impairs renal vasodilations caused by β-adrenoceptor activation. This study investigated whether the peroxisome proliferator-activated receptor gamma (PPARγ) and related nitric oxide synthase (NOS)/heme oxygenase (HO) signaling mediates the CSA-β-adrenoceptor interaction. The vasodilatory response elicited by a bolus injection of isoprenaline (1 μmole) in phenylephrine-preconstricted perfused kidneys of rats was reduced after prior infusion of zinc protoporphyrin IX (ZnPP, HO inhibitor) or GW9662 (PPARγ antagonist), suggesting the involvement of PPARγ and HO-derived CO in the isoprenaline response. In contrast, the inhibition of NOS activity by N-nitro-l-arginine methyl ester had no effect on isoprenaline responses. CSA (5 μM) significantly attenuated isoprenaline vasodilations, an effect that was abolished in the presence of GW9662 and accentuated by ZnPP. Also, supplementation with the PPARγ agonist pioglitazone or with l-arginine or hemin, substrates for NOS and HO, respectively, eliminated the unfavorable effect of CSA on isoprenaline vasodilations. The protection conferred by pioglitazone against CSA-evoked attenuation of isoprenaline vasodilations was maintained in N-nitro-l-arginine methyl ester-treated kidneys and disappeared after treatment with ZnPP or GW9662. In conclusion, the activation of the HO/CO/PPARγ cascade is probably the cellular mechanism that underlies the beneficial effect of pioglitazone on the CSA-isoprenaline interaction. Further, the facilitation of the HO/CO or NOS/NO pathway seems to offset this harmful effect of CSA.     

Interferon gamma induced by resveratrol analog,HS-1793, reverses the properties of tumor associated macrophages

Macrophages are capable of both inhibiting and promoting the growth and spread of cancers, depending on their activation state. Tumor-associated macrophages (TAM) are a kind of alternatively activated M2 macrophage, which may contribute to tumor progression. Following our previous study to evaluate the anti-tumor effect of a synthetic resveratrol analog HS-1793, the current study demonstrated that HS-1793 treatment significantly increased IFN-γ secreting cells in splenocytes and decreased CD206 + macrophage infiltration compared to CD68 + cells in the tumor site with a higher expression of IFN-γ. As these results suggested that IFN-γ increased locally at the tumor sites could modulate the status of TAM, we designed an in vitro model to study macrophage morphology and functions in relation to the tumor microenvironment. Human monocytic cell line THP-1 cells stimulated with phorbol-12-myristate-13-acetate (PMA) differentiated to macrophages with M2-like phenotypes. TAM-like properties of CD206^high, CD204^high, IL-10^high, TGF-β^high, IL-6^low, IL-12^low, VEGF^high, and MMP-9^high and promotion of tumor cell invasion were more pronounced in M-2-polarized THP-1 macrophages generated by differentiating THP-1 cells with PMA and subsequently polarizing them with Th2 cytokines (IL-4/IL-13). Upon IFN-γ exposure, THP-1-derived TAM changed their phenotypes to the M-1-like morphology and intracellular granular pattern with an expression of an increased level of proinflammatory and immunostimulatory cytokines and a reduced level of immunosuppressive and tumor progressive mediators. These results explain the underlying mechanism of the anti-tumor activity of HS-1793. The elevated level of IFN-γ production after HS-1793 treatment evoked reprogramming of M-2 phenotype TAM, which efficiently countered the immunosuppressive and tumor progressive influences of TAM.     

Pioglitazone can downregulate bone morphogenetic protein-2 expression induced by high glucose in human umbilical vein endothelial cells

Bone morphogenetic protein-2 (BMP-2) plays a key role in both vascular development and pathophysiological processes. However, the effect of high glucose on BMP-2 expression remains unclear. Here we show in human umbilical vein endothelial cells that high glucose increased BMP-2 expression, associated with NF-kappaB activation, whereas pioglitazone suppressed BMP-2 expression and NF-kappaB65 activation induced by high glucose. Furthermore, the increase in MDA levels and decrease in activities of total superoxide dismutase of cell culture stimulated by high glucose were reversed by pioglitazone treatment. Our findings indicate that BMP-2 expression induced by high glucose might be closely related to NF-kappaB activation, and both effects can be suppressed by pioglitazone.