We have previously demonstrated that STAT-1 plays a critical role in promoting apoptotic cell death in cardiac myocytes following ischemia/reperfusion (I/R) injury. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has recently been reported to inhibit STAT-1 activity in noncardiac cells. In the present study, we have assessed the protective effects of EGCG and green tea extract (GTE) infusion on both cultures of cardiac myocytes and the isolated rat heart. EGCG reduced STAT-1 phosphorylation and protected cardiac myocytes against I/R-induced apoptotic cell death. Moreover, EGCG reduced the expression of a known STAT-1 pro-apoptotic target gene, Fas receptor. More interestingly, oral administration of GTE as well as EGCG infusion limited the extent of infarct size and attenuated the magnitude of myocyte apoptosis in the isolated rat heart exposed to I/R injury. This reduction cell death was associated with improved hemodynamic recovery and ventricular function in the ischemic/reperfused rat heart. This is the first report to show that consumption of green tea is able to mediate cardioprotection and enhance cardiac function during I/R injury. Because GTE-mediated cardioprotection is achieved, at least in part, through inhibition of STAT-1 activity, we may postulate that a similar action can be implemented in the clinical setting to minimize STAT-1 activation levels in patients with acute coronary artery disease (CAD).

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor ␣ (TNF␣)-inducible NO synthesis, because by affecting NF-B activation it inhibits inducible Ca 2؉ -independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/ TNF␣-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF␣ induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/ TNF␣-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF␣-elicited NF-B activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-B availability. Nitric oxide (NO)1 is a major messenger molecule playing key roles in many physiological and pathological processes (1). NO production is catalyzed by at least two major forms of the NO synthase (NOS) enzyme: a constitutive Ca 2ϩ -dependent NOS isoform (cNOS) and an inducible Ca 2ϩ -independent NOS isoform (iNOS), which is expressed after stimulation with Escherichia coli lipopolysaccharide (LPS) and cytokines. Recently, we have demonstrated that LPS and/or TNF␣ are able to induce iNOS in human ramified microglia leading to a high NO output (2). On the other hand, NO release from mouse microglia is thought to play an important role in neuronal cell death (3-5). In a recent work, Meda et al. (6) suggested a possible involvement of NO produced by rat microglia after activation with ␤-amyloid protein and IFN-␥ in the pathogenesis of neuronal degradation occurring with age and in Alzheimer's disease.Preserving iNOS gene from its undesirable induction may be important for neuronal survival. Down-regulation of iNOS expression was reported to be achieved by some factors such as dexamethasone, interleukin-4, transforming growth factor-␤, and basic fibroblast growth factor (7-9). Recently, Griscavage et al. However, little is known about the regulatory effects on the mechanism by the variable low concentrations of the available NO before iNOS induction. Recently, we have observed that sodium nitroprusside (SNP), a well known NO donor, elicited inhibition of LPS-induced iNOS expression in rat neutrophils, suggesting a possible suppressive effect on iNOS gene expression by exogenous NO (12). The promoter region of human iNOS gene in vascular smooth muscle cells was shown to contain the consensus sequences for the binding of NFB, a nuclear transcriptional factor (13), and iNOS transc...

The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors.

Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP-ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 microM) showed a rapid and transient increase of polyADP-ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non-toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate-mediated neurotoxicity, structurally different inhibitors of pADPRP (3-aminobenzamide, benzamide,3-aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60-100%, assessed 24 hr later. Glutamate-induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca(2+)-mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death.

We previously observed that Gadd45␤/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate-early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45␤ is known to be stimulated by nuclear factor (NF) B, which is activated by tumor necrosis factor alpha (TNF␣) in the early response to partial hepatectomy. We therefore investigated whether TNF␣ and NF B also stimulated Gadd45␤ as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild-type, TNFR1 ؊/؊ , and TNFR1 ؊/؊ TNFR2 ؊/؊ . The results showed that TCPOBOP did not activate NF B in any of the mice, but a strong induction of Gadd45␤ messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NF B did not impair CAR-mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1 I n recent years, the tools of molecular biology have revealed important mechanisms controlling liver regeneration, including critical growth factors, transcription factors, and signal transduction regulators. [1][2][3][4] Within minutes after partial hepatectomy (PH), hepatocytes in the remnant liver undergo a transition from the quiescent G0 to the G1 phase of an active cell cycle. Although the precise mechanisms responsible for triggering this transition are not known, enhanced expression of immediate-early genes, occurring 15 minutes to 2 hours after PH, is believed to be critical. 5 Immediate-early gene induction does not require protein synthesis and is activated by transcription factors that preexist in a latent form. In particular, increased binding of nuclear factor (NF) B, AP1, and C/EBP occurs within minutes after PH, 6-8 whereas STAT3 activates shortly thereafter. Activation of AP1 and NF B, as well as DNA synthesis, can be inhibited by pretreatment of the animals with antibodies against tumor necrosis factor alpha (TNF␣), 9 suggesting that this cytokine plays a central role in the initiation of liver regeneration, by activating transcription factors.

No abstract

Green tea has been reported to show anti-inflammatory properties because of its inhibitory effects on the expression of several pro-inflammatory genes. Because the inducible nitricoxide synthase (iNOS) plays an important role in chronic inflammatory diseases, we have focused our attention on the regulation of iNOS expression by green tea in two different human epithelial cell lines, alveolar A549/8 and colon DLD-1 cells. With the use of electrophoretic mobility shift assays, we found a green tea-mediated down-regulation of the DNA binding activity of the transcription factor signal transducer and activator of transcription-1␣ (STAT-1␣), but not of nuclear factor-B. This down-regulation of the STAT-1␣ DNA binding was shown to result from reduced tyrosine phosphorylation of the STAT-1␣ protein and not from antioxidative effects of the green tea extract. Green tea extract inhibited human iNOS expression in a concentration-dependent manner, quantified in terms of iNOS mRNA, iNOS protein, and nitric oxide production in both cell lines. This inhibitory effect of green tea resulted from transcriptional inhibition as shown in reporter gene experiments. These data suggest that green tea extracts may be promising at least as an auxiliary anti-inflammatory principle in chronic inflammatory diseases.

Inflammatory bowel disease (IBD) is characterised by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. The aim of the present study was to examine the effects of green tea extract in rats subjected to experimental colitis induced by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). At 4 days after DNBS administration the rats were sacrificed. Treatment with green tea extract significantly attenuated diarrhoea and loss of body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture, significant reduction of colonic myeloperoxidase (MPO) and tumor necrosis factor-alpha (TNF-alpha) production. Green tea extract also reduced the appearance of nitrotyrosine immunoreactivity in the colon and reduced the up-regulation of ICAM-1.