6]-Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo

[6]-Gingerol, a naturally occurring plant phenol, is one of the major components of fresh ginger (Zingiber officinale Roscoe, Zingiberaceae) and has diverse pharmacologic effects. Here, we describe its novel anti-oxidant, anti-apoptotic, and anti-inflammatory activities in vitro and in vivo. In vitro, pre-treatment with [6]-gingerol reduced UVB-induced intracellular reactive oxygen species levels, activation of caspase-3, -8, -9, and Fas expression. It also reduced UVB-induced expression and transactivation of COX-2. Translocation of NF-kappaB from cytosol to nucleus in HaCaT cells was inhibited by [6]-gingerol via suppression of IkappaBalpha phosphorylation (ser-32). Examination by EMSAs and immunohistochemistry showed that topical application of [6]-gingerol (30 microM) prior to UVB irradiation (5 kJ/m(2)) of hairless mice, also inhibited the induction of COX-2 mRNA and protein, as well as NF-kappaB translocation. These results suggest that [6]-gingerol could be an effective therapeutic agent providing protection against UVB-induced skin disorders.     

Metabolism of [6]-gingerol in rats

The metabolic fate of [6]-gingerol, one of the active constituents of Zingiber officinale Roscoe, was investigated using rats. The bile of rats orally administered [6]-gingerol was shown to contain a major metabolite (1) by HPLC analysis. Although the metabolites derived from [6]-gingerol were not detected in the urine, the ethyl acetate extract of the urine after enzymatic hydrolysis was shown to contain six minor metabolites (2-7). Their structures were determined to be (S)-[6]-gingerol-4'-O-beta-glucuronide (1), vanillic acid (2), ferulic acid (3), (S)-(+)-4-hydroxy-6-oxo-8-(4-hydroxy-3-methoxyphenyl) octanoic acid (4), 4-(4-hydroxy-3-methoxyphenyl)butanoic acid (5), 9-hydroxy [6]-gingerol (6) and (S)-(+)-[6]-gingerol (7) based on spectroscopic and chemical data. The total cumulative amount of 1 excreted in the bile and 2-7 in the urine during 60 h after the oral administration of [6]-gingerol were approximately 48% and 16% of the dose, respectively. The excretion of 2-7 in the urine decreased after gut sterilization. On the other hand, the incubations of [6]-gingerol with rat liver showed the presence of 9-hydroxy [6]-gingerol, gingerdiol (8), and (S)-[6]-gingerol-4'-O-beta-glucuronide (1). These findings suggest that the gut flora and enzymes in the liver play an important part in the metabolism of [6]-gingerol.     

6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

干扰FSCN1基因表达对前列腺癌细胞凋亡、活性氧水平影响的研究

目的探讨干扰FSCN1基因表达对前列腺癌细胞凋亡、活性氧(ROS)水平影响及机制。方法以正常前列腺上皮细胞RWPE-1为对照细胞,通过RT-PCR及Western blot检测前列腺癌LNCaP、DU145和PC-3细胞中FSCN1 mRNA及蛋白表达;以LipofectamineTM 2000为载体,DU145细胞分为si-FSCN1组(靶向抑制FSCN1的小干扰RNAs转染DU145细胞)、阴性对照组(随机序列转染DU145细胞)及空白对照组(未转染的细胞),siRNA转染48 h,Western blot检测FSCN1、PCNA、NF-κB p65、p-NF-κB p65、IKKα和p-IKKα的蛋白表达。CCK8检测细胞活力,流式细胞术检测细胞凋亡率及ROS水平。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果与RWPE-1细胞比较,LNCaP、DU145和PC-3细胞中FSCN1 mRNA(1比2.561±0.189、7.183±0.882、4.796±0.567、4.796±0.567)及蛋白表达(0.053±0.007比0.217±0.013、0.654±0.058、0.316±0.035)均升高,差异具有统计学意义(P均<0.05)。与阴性对照组比较,si-FSCN1组FSCN1表达(0.473±0.052比0.086±0.010)降低,差异具有统计学意义(P均<0.05)。与si-FSCN1组比较,空白对照组、阴性对照组细胞活力(0.302±0.033比0.787±0.069、0.764±0.063)均升高,凋亡率(24.54﹪±1.47﹪比3.04﹪±0.36﹪、3.28﹪±0.40﹪)和ROS相对荧光强度(90.04±5.73比47.88±3.62、49.62±4.11)均降低,差异具有统计学意义(P均<0.05)。与si-FSCN1组比较,空白对照组、阴性对照组PCNA(0.255±0.032比0.654±0.062、0.631±0.058)、NF-κB p65(0.092±0.011比0.296±0.032、0.318±0.037)、p-NF-κB p65(0.042±0.008比0.155±0.018、0.151±0.016)、IKKα(0.112±0.01比0.172±0.020、0.192±0.023)和p-IKKα的蛋白表达(0.051±0.005比0.102±0.011、0.091±0.009)均升高,Cleaved caspase3蛋白表达(0.206±0.018比0.074±0.009、0.085±0.010)均降低,差异具有统计学意义(P均<0.05)。阴性对照组与空白对照组细胞活力、凋亡率、ROS水平及FSCN1、PCNA、Cleaved caspase3、NF-κB p65、p-NF-κB p65、IKKα和p-IKKα的蛋白表达差异均无统计学意义(P>0.05)。结论干扰FSCN1基因表达可降低前列腺癌细胞活力及诱导凋亡,机制可能与ROS水平升高及NF-κB信号下调有关。     

Imidazo[4,5-d]thiazolo[5,4-b]pyridine based inhibitors of IKK2: synthesis, SAR, PK/PD and activity in a preclinical model of rheumatoid arthritis

The synthesis, structure-activity relationships (SAR) and biological evaluation of thiazole based tricyclic inhibitors of IKK2 are described. Compound 9 was determined to be orally efficacious in a murine model of rheumatoid arthritis.     

Scutellarein inhibits RANKL-induced osteoclast formation in vitro and prevents LPS-induced bone loss in vivo

Osteoporosis, arthritis, Peget's disease, bone tumor, periprosthetic joint infection, and periprosthetic loosening have a common characteristic of osteolysis, which is characterized by the enhanced osteoclastic bone resorptive function. At present, the treatment target of these diseases is to interfere with osteoclastic formation and function. Scutellarein (Scu), a flavonoids compound, can inhibit the progress of tumor and inflammation. However, the role of Scu in inflammatory osteolysis isn’t elucidated clearly. Our study showed that Scu inhibited bone destruction induced by LPS in vivo and OC morphology and function induced by RANKL in vitro. Mechanistic studies revealed that Scu suppressed osteoclastic marker gene expression by RANKL-induced, such as Ctsk9, Mmp9, Acp5, and Atp6v0d2. In addition, we found that the inhibition effects of osteoclastogenesis and bone resorption function of Scu were mediated via attenuating NF-κB and NFAT signaling pathways. In conclusion, the results showed that Scu may become a potential new drug for the treatment of inflammatory osteolysis.     

[B2-Lys]-胰岛素的制备与性质

本文报道了用化学半合成途径从天然猪胰岛素制备[B2-Lys]-胰岛素的过程。人胎盘细胞膜胰岛素受体结合试验表明:[B2-Lys]-胰岛素的受体结合能力只有天然胰岛素的80％,降兔血糖作用与时间关系的结果表明它没有长效作用。本文还对这些结果进行了讨论。     

Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis

A novel α-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. α-iso-cubebenol inhibited LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production. Consistent with these findings, α-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. α-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-κB p65 subunit. Furthermore, α-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel α-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPS in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.     

Carboplatin induces apoptotic cell death through downregulation of constitutively active nuclear factor-kappaB in human HPV-18 E6-positive HEp-2 cells

Because the role of nuclear factor kappaB (NF-kappaB) is in cellular growth control and neoplasia, we explored the status of NF-kappaB and investigated its role in survival of human HPV-18 E6-positive HEp-2 cells. We observed accumulation of p65 in the nucleus. Moreover, without any external stimulus constitutive NF-kappaB DNA binding and transactivation activity were detected in HEp-2 cells. Treatment with NF-kappaB inhibitor curcumin (diferuloylmethane) and transient transfection of the mutant form of IkappaBalpha, IkappaBalpha super repressor (IkappaBalpha-SR), suppressed constitutive NF-kappaB activity as well as proliferation, suggesting that constitutive NF-kappaB activity is required for the survival of HEp-2 cells. Carboplatin treatment downregulated constitutive NF-kappaB activity and prevented nuclear retention of p65. Further, carboplatin also suppressed the constitutive IkappaBalpha phosphorylation leading to stabilization of IkappaBalpha protein in the cells. Carboplatin inhibited NF-kappaB binding to its response element present in Bcl-2 promoter resulting in downregulation of antiapoptotic Bcl-2 protein. Thus, our results for the first time indicate that constitutive NF-kappaB has a significant role in the survival of HPV-18 E6-positive HEp-2 cells. Moreover, inactivation of NF-kappaB is one of the mechanisms underlying the induction of carboplatin-mediated apoptosis in HPV-18 E6-positive cancer cells.     

ST2 suppresses IL-6 production via the inhibition of IkappaB degradation induced by the LPS signal in THP-1 cells

LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-kappaB to the IL-6 promoter. Furthermore, the degradation of IkappaB in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated that ST2 negatively regulates LPS-induced IL-6 production via the inhibition of IkappaB degradation in THP-1 cells.     

Novel tricyclic inhibitors of IKK2: discovery and SAR leading to the identification of 2-methoxy-N-((6-(1-methyl-4-(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-7-yl)pyridin-2-yl)methyl)acetamide (BMS-066)

The synthesis, structure-activity relationships (SAR), and biological results of pyridyl-substituted azaindole based tricyclic inhibitors of IKK2 are described. Compound 4m demonstrated potent in vitro potency, acceptable pharmacokinetic and physicochemical properties, and efficacy when dosed orally in a mouse model of inflammatory bowel disease.     

Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells

The anti-inflammatory activity of the predominant procyanidin dimer in cocoa, dimer B2, was investigated in this study. Pretreatment of the procyanidin dimer B2 reduced COX-2 expression induced by the endotoxin lipopolysaccharide (LPS) in differentiated human monocytic cells (THP-1) in culture. To further elucidate the underlying mechanism of COX-2 inhibition by procyanidin, we examined their effects on the activation of extracellular signal-regulated protein kinase (ERK), Jun-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), which are upstream enzymes known to regulate COX-2 expression in many cell types. Pretreatment with procyanidin dimer B2 decreased the activation of ERK, JNK, and p38 MAPK. In addition, procyanidin dimer B2 suppressed the NF-kappaB activation through stabilization of IkappaB proteins, suggesting that these signal-transducing enzymes could be potential targets for procyanidin dimer B2. By affecting the expression rather than the activity of COX-2, these in vitro data reported herein give further evidence on the anti-inflammatory protection by procyanidins.     

Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor-calcium signaling pathways

Abstract

Homocysteine (Hcy) at elevated levels is a putative risk factor for many cardiovascular disorders including atherosclerosis. In the present study, we investigated the effect of Hcy on the expression of cyclooxygenase (COX)-2 in murine macrophages and the mechanisms involved. Hcy increased the expression of COX-2 mRNA and protein in dose- and time-dependent manners, but did not affect COX-1 expression. Hcy-induced COX-2 expression was attenuated not only by the calcium chelators, EGTA and BAPTA-AM, but also by an antioxidant, N-acetylcysteine. Calcium chelators also attenuated Hcy-induced reactive oxygen species (ROS) production in macrophages, indicating that Hcy-induced COX-2 expression might be mediated through ROS generated by calcium-dependent signaling pathways. In another series of experiments, Hcy increased the intracellular concentration of calcium in a dose-dependent manner, which was attenuated by MK-801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, but not by bicuculline, a gamma-aminobutyric acid receptor inhibitor. Molecular inhibition of NMDA receptor using small interfering RNA also attenuated Hcy-induced increases in intracellular calcium. Furthermore, both ROS production and Hcy-induced COX-2 expression were also inhibited by MK-801 as well as by molecular inhibition of NMDA receptor. Taken together, these findings suggest that Hcy enhances COX-2 expression in murine macrophages by ROS generated via NMDA receptor-mediated calcium signaling pathways.     

Hypertonic sodium chloride and mannitol induces COX-2 via different signaling pathways in mouse cortical collecting duct M-1 cells

The kidney cortical collecting duct is an important site for the maintenance of sodium balance. Previous studies have shown that, in renal medullary cells, hypertonic stress induces expression of cyclooxygenase-2 (COX-2) via NF-kappaB activation, but little is known about COX-2 expression in response to hypertonicity in the cortical collecting duct. Therefore, we examined the mechanism of hypertonic induction of COX-2 in M-1 cells derived from mouse cortical collecting duct. Induction of COX-2 protein was detected within 6 h of treatment with hypertonic sodium chloride. The treatment also increased COX-2 mRNA accumulation in a cycloheximide-independent manner, suggesting that ongoing protein synthesis is not required for COX-2 induction. Using reporter plasmids containing 0.2-, 0.3-, and 1.5-kb fragments of the COX-2 promoter, we found that hypertonic induction of COX-2 was due to an increase in promoter activity. The COX-2-inductive effect of hypertonicity was inhibited by SB203580, indicating that the effect is mediated by p38 MAPK. Since p38 MAPK can activate NF-kappaB, we made point mutations in the NF-kappaB binding site within the COX-2 promoter. The mutations did not block the induction of COX-2 promoter activity by hypertonic sodium chloride, and hypertonic sodium chloride failed to activate NF-kappaB binding site-driven reporter gene constructs. In contrast, hypertonic mannitol activated NF-kappaB, indicating that hypertonic mannitol and hypertonic sodium chloride activate COX-2 by different mechanisms. Thus, induction of COX-2 expression in M-1 cells by hypertonic sodium chloride does not involve activation of NF-kappaB. Furthermore, the signal transduction pathways that respond to hypertonic stress vary for different osmolytes in cortical collecting duct cells.     

The anti-inflammatory potential of neuropeptide FF in vitro and in vivo

Neuropeptide FF (NPFF) has many functions in regulating various biological processes. However, little attention has been focused on the anti-inflammatory effect of this peptide. In the present study, the in vitro anti-inflammatory activity of NPFF in both primary peritoneal macrophages and RAW 264.7 macrophages was investigated. Our data showed that NPFF suppressed the nitric oxide (NO) production of macrophages in the inflammation process. RF9, a reported antagonist of NPFF receptors, completely blocked the NPFF-induced NO suppression, suggesting a NPFF receptors-mediated pathway is mainly involved. Down-regulation of the nitric oxide synthases significantly inhibited the NPFF-induced NO reduction, indicating the involvement of nitric oxide synthases. However, the nitric oxide synthases were not the only route by which NPFF modulated the NO levels of macrophages. Pharmacological antagonists of the NF-κB signal pathway also completely suppressed the NPFF-induced NO decline. Moreover, we also observed that NPFF is capable of blocking the LPS-induced nuclear translocation of p65 in macrophages, implying the involvement of the NF-κB signal pathway. Finally, we observed that NPFF markedly attenuated the carrageenan-induced mouse paw edema, indicating that NPFF is capable of exerting anti-inflammatory potency in vivo. Collectively, our findings reveal the potential role of NPFF in the anti-inflammatory field both in vitro and in vivo, which will be helpful for the further exploitation of NPFF utility therapeutically.     

TRAF2 expression in differentiated muscle

Recent data involving traf2 knockout mice have suggested a necessity of the protein in viability of skeletal muscle tissue. traf2−/− mice are born with decreased muscle mass that is hypothesized to be due to the increased circulating tumor necrosis factor in these mice. We show that TRAF2 protein is present at high levels in terminally differentiated skeletal muscle in the developing mouse. In vitro differentiation of mouse myoblasts displays a dramatic increase in TRAF2 protein levels. Although basal NF-κB activity decreases during myogenesis, TNF-induced NF-κB activity is 10 times greater in myotubes compared with myoblasts, presumably because of the stockpiling of TRAF2 protein in these cells. This may represent a strong anti-apoptotic TRAF2-mediated response specifically tailored to myotubes. These data help explain why muscle integrity is at risk in traf2−/− mice. J. Cell. Biochem. 71:461–466, 1998. © 1998 Wiley-Liss, Inc.