THE GREEN TEA POLYPHENOL EPIGALLOCATECHIN-3-GALLATE IMPROVES SYSTEMIC HEMODYNAMICS AND SURVIVAL IN RODENT MODELS OF POLYMICROBIAL SEPSIS

Abstract

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid found in green tea. Recent in vitro studies have suggested that EGCG inhibits activation of the nuclear factor-κB (NF-κB) pathway. The NF-κB is a transcriptional factor required for gene expression of many inflammatory mediators, including the inducible isoform of nitric oxide synthase (NOS2). Excessive NO production by NOS2 is directly linked to the vasoplegia, shock, and mortality associated with sepsis. Accordingly, we hypothesized that EGCG administration would inhibit NOS2 gene expression and thereby improve survival in a rodent model of polymicrobial sepsis. Polymicrobial sepsis was induced in male Sprague-Dawley rats (hemodynamic study) and C57BL6 mice (mortality study) via cecal ligation and double puncture (CL2P). Rodents were treated with either EGCG (10 mg/kg intraperitoneally) or vehicle at 1 and 6 h after CL2P and every 12 h thereafter. In the hemodynamic study, mean arterial blood pressure was monitored for 18 h, and rats were killed at 3, 6, and 18 h after CL2P. In the mortality study, survival was monitored for 72 h after CL2P in mice. In vehicle-treated rodents, CL2P was associated with profound hypotension and greater than 80% mortality rate. Epigallocatechin-3-gallate treatment significantly improved both the hypotension and survival. In vitro experiments further showed that EGCG inhibited activation of NF-κB and subsequent NOS2 gene expression in a primary culture of rat aortic smooth muscle cells. Epigallocatechin-3-gallate may therefore represent a potential nutritional supplement or pharmacologic agent in patients with sepsis.

INTRODUCTION

Tea (Camellia sinensis) is cultivated in more than 30 different countries around the world, and with an average per capita intake of 120 mL/d, tea is now second only to water in worldwide consumption. Recently, there has been growing interest in the beneficial effects of tea on health and in disease prevention. Epidemiological studies have suggested that the regular consumption of green tea reduces the risk of cancer and heart disease through its potent anti-inflammatory and antioxidant effects (¹). Although green tea consists of more than 2,000 components, the polyphenolic flavonoids are the most abundant and perhaps the most studied. These compounds are commonly known as catechins and include epigallocatechin-3-gallate (EGCG), the major flavonoid present in green tea. Apart from their antioxidant properties, the catechins, especially EGCG, have been shown to inhibit several proteins involved in inflammation, including the transcription factors nuclear factor-κB (NF-κB) (^2-6) and activator protein 1 (⁷) both in vitro and in vivo. For example, we have previously shown that EGCG is a potent inhibitor of tumor necrosis factor-α (TNF-α)- and interleukin-1β (IL-1β)-mediated NF-κB activation and subsequent IL-8 gene expression in A549 cells (^{2, 6}). Interleukin 8 is the most important chemotactic cytokine for neutrophils in humans and appears to be regulated in part by NF-κB. Epigallocatechin-3-gallate has previously been shown to inhibit gene expression of the inducible nitric oxide synthase (NOS2) in peritoneal macrophages (^{3, 8, 9}). In addition, green tea polyphenols have recently been shown to inhibit NOS2 expression in the lung and intestine in a zymosan-induced model of shock (¹⁰).

Nuclear factor-κB is a pluripotent transcription factor that functions as a "master switch" or control point for the expression of a large number of proinflammatory genes thought to be involved in the pathophysiology of sepsis, including NOS2. Excessive NO production by NOS2 is directly linked to the vasoplegia, shock, and mortality associated with sepsis (^11-13). Therefore, given the importance of both NF-κB and the NOS2 pathway in the pathophysiology of septic shock, we hypothesized that administration of EGCG would modulate NOS2 gene expression, improve hemodynamics, and increase survival in a model of polymicrobial sepsis induced by cecal ligation and double puncture (CL2P). In addition, septic shock is a common cause of acute lung injury in the intensive care setting. We therefore hypothesized that administration of EGCG would inhibit NF-κB expression in the lung after CL2P.

MATERIALS AND METHODS

The CL2P model of polymicrobial sepsis

All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Use of Laboratory Animals (National Institutes of Health Publication 85-23, revised 1996) and with approval of the Institutional Animal Care and Use Committee, Cincinnati Children's Research Foundation. Animals were acclimatized for 7 days before surgical manipulation and maintained on 12-h light/dark cycles with access to food and water ad libitum. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass) weighing 150 to 200 g were used in the hemodynamic studies, and male C57BL/6 mice (Charles River Laboratories) weighing 25 to 30 g were used in the mortality studies. Cecal ligation and double puncture was performed as previously described (¹⁴). Briefly, all animals were anesthetized with an intraperitoneal (i.p.) administration of thiopentone sodium (60 mg/kg rat and 400 μg/10 g body weight mouse, respectively). Once adequate anesthesia was achieved, the rodents were placed in a supine position, and the lower abdomen was shaved and prepared with alcohol. The cecum was exteriorized via a small abdominal incision and then ligated with a 3-0 silk suture placed approximately 5 mm distal to the ileocecal valve. The ligated cecum was punctured twice with an 18-gauge needle (rat) or 19-gauge needle (mouse) and gently squeezed to extrude a small amount of fecal contents. The cecum was replaced in the abdomen, and the incision was closed in 2 layers with 4-0 silk sutures and cyanoacrylate adhesive. Five milliliters (rat) or 1 mL (mouse) of 0.9% isotonic sodium chloride solution was injected s.c. to compensate for third-space fluid loss that occurred during the procedure. Animals were placed on a heating pad and allowed to recover from anesthesia.

Two treatment groups were used for the hemodynamic study performed in the rats. The first group received an equal volume of vehicle (0.9% sterile saline) instead of EGCG (vehicle group); a second group received EGCG (10 mg/kg i.p.; EGCG group). Epigallocatechin-3-gallate or vehicle was administered at 1 and 6 h after CL2P. Mean arterial blood pressure was measured in both the EGCG group and vehicle group. After induction of anesthesia, the trachea and carotid artery were cannulated to facilitate respiration and to measure mean arterial blood pressure (MAP), respectively. Mean arterial blood pressure was measured via a pressure transducer connected to a Maclab A/D converter (AD Instruments, Milford, Mass) for 3 h (n = 5 rats in each treatment group) or 6 h (n = 5 rats in each treatment group) after CL2P. A second set of rats underwent CL2P (n = 5 rats in each treatment group) and were allowed to recover. Fourteen hours later, the rats were anesthetized, the trachea and carotid artery were cannulated, and MAP was measured for 4 h (i.e., up to 18 h after CL2P). Animals that died before the end of the experiment were excluded from the study. These animals were then euthanized at 3, 6, and 18 h after CL2P, and plasma samples and lung tissue were obtained for biochemical analysis.

Two groups of mice were used for the mortality study. The first group (n = 36) received an equal volume vehicle (0.9% sterile saline) instead of EGCG (vehicle group), whereas the second group (n = 36) received EGCG (10 mg/kg i.p.; EGCG group). Epigallocatechin-3-gallate or vehicle was administered at 1 and 6 h after CL2P and every 12 h thereafter until the end of the experimental period. Survival was monitored for 72 h, at which time any remaining animals were euthanized.

Measurement of plasma concentrations of cytokines

Plasma concentrations of TNF-α, IL-6, and IL-10 were measured using a commercially available enzyme-linked immunosorbent assay kit (Biosource International, Camarillo, Calif) using the protocol recommended by the manufacturer.

Subcellular fractionation and nuclear protein extraction

Lung samples were homogenized with a Polytron homogenizer (Brinkmann Instruments, West Orange, NY) in a buffer containing 0.32 M sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM ethyleneglycol bis-2-aminoethyl ether-N,N,N&,n-tetraacetic acid, 2 mM EDTA, 5 mM NaN₃, 10 mM 2-ME, 20 μM leupeptin, 0.15 μM pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM sodium fluoride, 1 mM sodium orthovanadate, and 0.4 nM microcystin. The homogenates were centrifuged (1,000g, 10 min), and the pellets were solubilized in Triton buffer (1% Triton X-100, 150 mM sodium chloride, 10 mM Tris-HCl (pH 7.4), 1 mM ethyleneglycol bis-2-aminoethyl ether-N,N,N,n-tetraacetic acid, 1 mM EDTA, 0.2 mM sodium orthovanadate, 20 μM leupeptin A, and 0.2 mM PMSF). The lysates were centrifuged (15,000g, 30 min, 4°C), and the supernatant (nuclear extract) was collected for evaluation of DNA binding of NF-κB as described previously.

Cell culture

Rat aortic vascular smooth muscle cells (RASMCs) were isolated and maintained as previously described (¹⁵) for in vitro studies of the effect of EGCG on NOS2 gene expression. Briefly, RASMCs were harvested from Wistar rats weighing 200 to 225 g (Harlan, Indianapolis, Ind) by enzymatic dissociation. The cells were positively identified as smooth muscle by indirect immunofluorescent staining for α-actin with the use of mouse anti-actin antibody and antimouse immunoglobulin G (IgG)-fluorescein isothiocyanate conjugate. Cells were grown in 50% Dulbecco modified Eagle medium and 50% F-12 nutrient medium supplemented with 10% fetal bovine serum, glutamine (0.2 g/L), penicillin (100 units/mL), and streptomycin (100 units/mL). All cultures were grown in a humidified incubator at 37°C under 5% carbon dioxide in air. Cells at passages 2-4 were used in all studies. The RASMCs were treated with 1 ng/mL of human IL-1β (Roche Applied Science, Indianapolis, Ind) and 100 ng/mL of rat recombinant interferon-γ (IFN-γ; R&D Systems, Minneapolis, Minn) for 24 h. Epigallocatechin-3-gallate (Sigma Chemical Co, St Louis, Mo) was diluted in filtered phosphate-buffered saline to a stock concentration of 10 mmol/L and further diluted to experimental concentrations ranging from 1 to 10 μmol/L in culture media. Cells were pretreated with EGCG or vehicle for 1 h before incubation with IL-1β and IFN-γ. Control cells were treated with culture media alone. In separate experiments, cells were pretreated with EGCG or vehicle before treatment with lipopolysaccharide (LPS) from Escherichia coli serotype O127:B8 (Sigma Chemical Co) 10 ng/mL and IFN-γ 100 ng/mL for 24 h. Cell viability after 24-h continuous exposure to EGCG was measured by Trypan blue exclusion and by a colorimetric assay based on the ability of mitochondria in viable cells to reduce the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as previously described (¹⁶).

Determination of nitrite concentration

To determine NO production, nitrite (a stable oxidative end product of NO) accumulation in the media of cells was measured as previously described (¹⁶). Cells were plated in 12-well tissue culture plates at a density of 2 × 10⁵ cells/well. For each experiment, conditions were carried out in triplicate. Twenty-four hours after exposure to experimental conditions, nitrite accumulation (μM) was determined via a colorimetric reaction based on the Griess reagent.

Western blot

After the experimental protocol, RASMCs were rinsed twice with ice-cold phosphate-buffered saline and lysed with 100 μL of a lysis buffer consisting of 20mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 1% Triton, 10% glycerol, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM sodium fluoride, 10 mM Na₄P₂O₇, and 1 mM PMSF. Cytosol extracts were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol), and 50 μg of protein was loaded per lane on an 8% to 16% Tris-glycine gradient gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked in 5% nonfat dried milk in Tris-buffered saline for 1 h and then incubated with primary antibodies for NOS2 (rabbit polyclonal IgG, BD Transduction Laboratories, Lexington, Ky) at 1:5,000 dilution, IκBα (rabbit polyclonal IgG) at 1:1,000 dilution (Santa Cruz Biotechnology, Santa Cruz, Calif), NF-κB-inducing kinase (NIK) (rabbit polyclonal IgG, Santa CruzBiotechnology) at 1:500 dilution, phospho-NIK (rabbit polyclonal IgG, Santa Cruz Biotechnology) at 1:500 dilution, and α-actin (rabbit polyclonal IgG, Santa Cruz Biotechnology) at 1:1,000 dilution for 1 h. The membranes were washed in Tris-buffered saline with 0.1% Tween 20, incubated with secondary peroxidase-conjugated antibody, and developed with enhanced chemiluminescence detection system (Amersham, Piscataway, NJ) and exposed on film.

Electromobility Gel Shift Assay

All nuclear extraction procedures were performed on ice with ice-cold reagents as previously described (^{2, 6}). Nuclear proteins were stored at -70°C until used for electromobility gel shift assay (EMSA). Oligonucleotide probes corresponding to the NF-κB consensus sequence (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) were labeled with [γ-³²P]ATP using T4 polynucleotide kinase and were purified in Bio-Spin chromatography columns (Bio-Rad, Hercules, Calif). Ten micrograms of nuclear protein was preincubated with EMSA buffer (12 mM HEPES [pH 7.9], 4 mM Tris-HCl [pH 7.9], 25 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, 50 ng/mL polydeoxy[inosinate-cytidylate], 12% glycerol [vol/vol], and 0.2 mM PMSF) on ice for 10 min before addition of the radiolabeled oligonucleotide for an additional 10 min. Protein-nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29/1 ratio of acrylamide/bisacrylamide) and were run in 0.5× tetrabromoethane (45 mM Tris-HCl, 45 mM boric acid, and 1 mM EDTA) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3M paper (Clifton, NJ), dried under a vacuum at 80°C for 1 h, and exposed to photographic film at −70°C with an intensifying screen. Densitometric analysis was performed using ImageQuant (Molecular Dynamics).

Data analysis

Data was analyzed using SigmaStat version 3.11 (Systat Software, Inc). All values in the figures and text are expressed as mean ± SEM of n observations. The results were examined by analysis of variance followed by Bonferroni correction post hoc t test. Survival after CL2P was compared using the log-rank method. A P < 0.05 was considered significant.

RESULTS

Effect of EGCG on survival after CL2P

After CL2P, vehicle-treated mice exhibited signs of septicemia (ruffled fur, tachypnea, decreased activity, and diarrhea) at 24 h, whereas the EGCG-treated mice did not exhibit these signs of septicemia until 48 h. Epigallocatechin-3-gallate administration at 1 h after CL2P resulted in a significant improvement in survival compared with the vehicle group (P = 0.004; Fig. 1).

Fig. 1: Epigallocatechin-3-gallate administration improves survival after CL2P. Cecal ligation and double puncture was performed using a 19-gauge needle in C57BL/6 mice. The first group of mice (n = 36) received an equal volume vehicle (0.9% sterile saline) instead of EGCG (vehicle group), whereas the second group (n = 36) received EGCG (10 mg/kg i.p.; EGCG group). Epigallocatechin-3-gallate or vehicle was administered at 1 and 6 h after CL2P and every 12 h thereafter, until the end of the experimental period. Survival was monitored for 72 h, at which time any remaining animals were euthanized. Epigallocatechin-3-gallate administration beginning at 1 h after CL2P resulted in a significant improvement in survival compared with the vehicle group (P = 0.004).

Effect of EGCG on hypotension after CL2P

In vehicle-treated rats, CL2P resulted in a profound and sustained decrease in MAP. Treatment with EGCG after CL2P, however, resulted in a significant improvement in MAP (Fig. 2).

Fig. 2: Epigallocatechin-3-gallate administration improves systemic hemodynamics after CL2P. Cecal ligation and double puncture was performed using an 18-gauge needle in Sprague-Dawley rats (150-200 g) after instrumentation for blood pressure monitoring via the left common carotid artery (see "Materials and Methods"). The first group of rats (n = 10) received an equal volume of vehicle (0.9% sterile saline) instead of EGCG (vehicle group); a second group of rats received EGCG (10 mg/kg i.p.; EGCG group). Epigallocatechin-3-gallate or vehicle was administered at 1 and 6 h after CL2P. In vehicle-treated rats, CL2P resulted in a profound and sustained decrease in MAP. Treatment with EGCG beginning at 1 h after CL2P, however, resulted in a significant improvement in MAP (*P < 0.05).

Effect of EGCG on lung NF-κB activation

To investigate the cellular mechanisms by which EGCG may attenuate the inflammatory response during sepsis, we evaluated the nuclear activation of NF-κB, a major transcription factor involved in acute lung injury and septic shock. Polymicrobial sepsis resulted in the early activation of NF-κB in the lungs of vehicle-treated rats, with activity reaching a maximum within 3 h after CL2P. In contrast, treatment with EGCG significantly reduced the DNA binding activity of NF-κB at 3 h after CL2P (Fig. 3). Furthermore, although EGCG reduced DNA binding activity of NF-κB at 6 and 18 h after CL2P compared with vehicle, the difference was not statistically significant.

Fig. 3: Epigallocatechin-3-gallate administration abrogates NF-κB activation in the lung after CL2P. Cecal ligation and puncture was performed using an 18-gauge needle in Sprague-Dawley rats (150-200 g). The first group of rats received an equal volume of vehicle (0.9% sterile saline) instead of EGCG (vehicle group); a second group of rats received EGCG (10 mg/kg i.p.; EGCG group). Epigallocatechin-3-gallate or vehicle was administered at 1 and 6 h after CL2P. Rats were euthanized at 3, 6, and 18 h after CL2P. Lung samples were homogenized as described in the Materials and Methods section, and EMSA was performed using an oligonucleotide probe corresponding to the NF-κB consensus sequence. A, Representative EMSA showing that EGCG inhibits NF-κB activation in the lung after CL2P. Lanes 1, 3, and 5 correspond to vehicle-treated rats at 3, 6, and 18 h after CL2P, respectively. Lanes 2, 4, and 6 correspond to EGCG-treated rats at 3, 6, and 18 h after CL2P, respectively. Lanes 7 and 8 represent cold competitor (vehicle and EGCG, respectively); lanes 9 and 10 represent supershift antibody to p65 subunit (vehicle and EGCG, respectively); and lanes 11 and 12 represent supershift antibody to p50 subunit (vehicle and EGCG, respectively). B, Densitometric analysis of EMSA results (n = 3-5 rats per time point), showing that EGCG inhibits NF-κB activation in the lung after CL2P (*P < 0.05 compared with control).

Effect of EGCG on plasma cytokine concentrations

A substantial increase in TNF-α, IL-6, and IL-10 production was observed at 18 h after CL2P in vehicle-treated and EGCG-treated rats. There were no significant differences in plasma cytokine concentrations between the 2 groups at any of the time points studied (Table 1).

Table 1: Serum cytokine concentrations after CL2P in rats

Effect of EGCG on plasma nitrite and nitrate concentrations

A substantial increase in circulating nitrites and nitrates was noted at 18 h after CL2P in vehicle-treated rats, although the circulating nitrites and nitrates was significantly decreased in the EGCG-treated rats (88.5 ± 13 μmol/L vs 56.6 ± 1, P < 0.05).

Effect of EGCG on NOS2 gene expression and activity in RASMCs in vitro

Given the improvement in hemodynamics and survival after treatment with EGCG in the in vivo studies above, we next determined the effect of EGCG on the NOS2 pathway in RASMCs. We first determined the effect of EGCG on IL-1β/IFN-γ-mediated production of nitrite, a stable oxidative end product of NO via the Griess assay. Treatment with IL-1β/IFN-γ for 24 h increased production of nitrite in RASMCs compared with control cells treated with basal growth media alone (Fig. 4). However, in RASMCs pretreated with EGCG for 1 h before exposure to IL-1β/IFN-γ, nitrite production was significantly inhibited. Epigallocatechin-3-gallate had no adverse effects on cell survival at any of the doses used (data not shown).

Fig. 4: Epigallocatechin-3-gallate pretreatment inhibits NOS2 gene expression in RASMCs via inhibition of NF-κB activation. A, The RASMCs were pretreated with increasing concentrations of EGCG for 1 h before treatment with IL-1β (1 ng/mL) and IFN-γ (100 ng/mL) for 24 h. Epigallocatechin-3-gallate treatment (5 μmol/L) inhibited cytokine-induced nitrite production, as measured by the Griess assay. Data shown are results of three separate experiments with similar results (*P < 0.05 compared with control; †P < 0.05 compared with IL-1β/IFN-γ alone). B, The RASMCs were pretreated with increasing concentrations of EGCG for 1 h before treatment with IL-1β (1 ng/mL) and IFN-γ (100 ng/mL) for 24 h. Epigallocatechin-3-gallate treatment inhibited cytokine-induced NOS2 expression as measured by Western blot in a dose-dependent fashion, with maximal inhibition at doses of 5 μmol/L. The immunoblot is representative of three separate experiments that showed similar results. C, The RASMCs were pretreated with EGCG for 1 h before treatment with LPS (10 ng/mL) and IFN-γ (100 ng/mL) for 24 h. Epigallocatechin-3-gallate treatment inhibited cytokine-induced NOS2 expression as measured by Western blot in a dose-dependent fashion, with maximal inhibition at doses of 5 μmol/L. The immunoblot is representative of three separate experiments that showed similar results.

Epigallocatechin-3-gallate pretreatment resulted in a dose-dependent decrease in NOS2 expression as determined by Western blot, with maximal inhibition observed at 5 μmol/L, a dose that is readily achievable in vivo (¹). Similarly, pretreatment with 5 μmol/L inhibited NOS2 expression after treatment with LPS and IFN-γ, demonstrating that these effects were not stimulus specific (Fig. 4). As NOS2 gene expression in RASMCs is dependent on activation of the transcription factor, NF-κB (^{15, 17-19}), we next determined the effect of EGCG pretreatment on activation of the NF-κB pathway. Pretreatment with EGCG at 5 μmol/L resulted in complete inhibition of IL-1β/IFN-γ-induced IκBα degradation, phosphorylation of NIK, and NF-κB DNA binding (Fig. 5). Collectively, these data suggest that EGCG inhibits NOS2 gene expression and activity in RASMCs, at least partially via inhibition of the activation of the transcription factor NF-κB.

Fig. 5: Epigallocatechin-3-gallate pretreatment inhibits IL-1β/IFN-γ-mediated NF-κB activation in RASMCs. A, The RASMCs were pretreated with increasing concentrations of EGCG for 1 h before treatment with IL-1β (1 ng/mL) and IFN-γ (100 ng/mL) for 30 min. Epigallocatechin-3-gallate inhibited IκBα degradation as measured by Western blot in a dose-dependent manner. The immunoblot is representative of three separate experiments that showed similar results. B, The RASMCs were pretreated with increasing concentrations of EGCG for 1 h before treatment with IL-1β (1 ng/mL) and IFN-γ (100 ng/mL) for 30 min. Epigallocatechin-3-gallate inhibited phosphorylation of NIK as measured by Western blot in a dose-dependent manner. The immunoblot is representative of three separate experiments that showed similar results. C, Interleukin-1β and IFN-γ (lane 2) increased NF-κB activation as measured by EMSA when compared with control (lane 1), whereas EGCG pretreatment (lane 3) inhibited cytokine-induced NF-κB activation.

DISCUSSION

Sepsis and its related syndromes account for significant morbidity and mortality in critically ill patients. Sepsis is the 10th leading cause of death overall and accounts for nearly $17 billion in annual health care expenditures in the United States alone (²⁰). Recent epidemiologic data suggest that there has been a dramatic increase in the incidence of sepsis within the last 20 years (²¹), and despite the use of newer, potent antimicrobial agents, immunomodulating drugs, and modern intensive care, the overall mortality of patients with sepsis remains unacceptably high. The major pathophysiological derangement in sepsis stems, in large part, from a dysregulated inflammatory cascade, which begins as a normal response to an infection or other inciting event, but ultimately causes significant autoinjury to the host. A large body of indirect and direct evidence links the transcription factor NF-κB to the dysregulated inflammation that is central to the pathogenesis of sepsis. The NF-κB is a pluripotent transcription factor that functions as a master switch or control point for the expression of a large number of proinflammatory genes thought to be involved in the pathophysiology of sepsis, for example, TNF-α, IL-1β, and NOS2 (^{1, 15, 17-19}). The NF-κB, a member of the Rel family of transcription factors, is usually present in the cytoplasm of the cell bound to a related inhibitory protein known as IκB, which physically masks the nuclear translocation sequence of NF-κB, thereby retaining it in the cytoplasm in an inactive state. A common pathway for the activation of NF-κB occurs when IκB is phosphorylated by a serine-threonine kinase known as IκB kinase. Phosphorylated IκB is targeted for rapid ubiquitination, which results in its subsequent degradation by the 26S proteasome, thereby unmasking the nuclear translocation sequence of NF-κB, allowing NF-κB to enter the nucleus and direct transcription of target genes (²²).

NF-κB activation has been demonstrated in vivo in several animal models of sepsis, and in many of these models, inhibition of NF-κB activation by a variety of strategies conferred protection against sepsis (²²). In addition, several clinical studies have demonstrated increased activation of NF-κB in vivo, and more importantly, increased activation of NF-κB in these patients correlated with increased mortality (^22-24). These data support the general hypothesis that increased NF-κB-dependent inflammation directly contributes to the outcome of sepsis and suggests a rational treatment strategy. A potential novel strategy for inhibiting NF-κB activation may involve the polyphenolic flavonoids found in tea. Data from our laboratory and others demonstrate that the green tea-derived flavonoid, EGCG, inhibits activation of the NF-κB pathway in vitro (^2-6). In addition, our laboratory has also previously shown that EGCG inhibits NF-κB activation after myocardial ischemia-reperfusion injury in vivo (⁷).

We show that EGCG treatment administered via i.p. injection at 1 and 6 h and continued every 12 h after CL2P improved survival in mice. Yang et al. (⁴) previously showed that green tea polyphenols administered by oral gavage (0.5 g/kg body weight) 2 h before LPS administration inhibited systemic TNF-α production and improved survival, at least partially via the inhibition of NF-κB activation. Importantly, our results show that EGCG improved survival in a more clinically relevant model of septic shock, that is, polymicrobial sepsis induced by CL2P. In further contrast to Yang et al. (⁴), our results demonstrate that treatment with EGCG initiated after the inciting stimulus is still effective in improving survival in a murine model of intraperitoneal sepsis. In addition, EGCG treatment inhibited NF-κB activation in the lung, consistent with previous data, suggesting that inhibition of NF-κB activation improves survival in various in vivo models of sepsis (^{22, 25, 26}). However, consistent with previous in vitro and in vivo studies (^{27, 28}), EGCG treatment did not seem to affect the serum concentrations of the proinflammatory cytokines TNF-α or IL-6, suggesting that other mechanisms may be involved as well. For example, the tea flavonoids, especially EGCG, have previously been shown to exhibit some antimicrobial effects (^{1, 29}) that could have accounted for some of the protective effects of EGCG treatment in this model of polymicrobial sepsis. The tea flavonoids are potent antioxidants as well (¹). Previous investigators have shown that inhibiting the production of or scavenging the superoxide anion improves outcome in a rat model of polymicrobial sepsis (^{30, 31}). Epigallocatechin-3-gallate and green tea flavonoids have also been shown to chelate iron (^{1, 32}), and recent studies have shown an association between iron and survival in the CL2P model (³³). Epigallocatechin-3-gallate may also abrogate organ injury and improve outcome via similar mechanisms, although further studies are necessary to prove this assertion.

A large body of experimental and clinical evidence has linked the enhanced formation of endogenous NO with the vasoplegia and myocardial depression characterizing septic shock (³⁴). Nitric oxide in mammalian tissues is synthesized by three distinct isoforms of NOS that catalyze the conversion of l-arginine to l-citrulline and NO. The NOS1 and NOS3 are constitutively expressed and are activated by reversible binding of Ca²⁺/calmodulin after elevations in intracellular Ca²⁺ in response to exposure of cells to various extracellular stimuli. In contrast, NOS2 is expressed in vascular smooth muscle and other cells after induction by cytokines or LPS. When the NOS2 gene is induced, large quantities of NO are produced, resulting in the vasoplegia and hypotension associated with septic shock (^{34, 35}). Peroxynitrite, an oxidant species produced by NO, has been shown to play an important role in the cellular energetic failure and contractile dysfunction of vascular SMCs in septic shock (^{36, 37}). A number of transcription factors, including NF-κB, seem to modulate the expression of NOS2 in RASMCs in response to proinflammatory stimuli (^{15, 17-19, 38}). Previous investigators (^{30, 39}) have shown that inhibition of NOS2 gene expression via inhibition of NF-κB activation ameliorates the vasoplegia and systemic hypotension associated with septic shock, resulting in improved outcome. Epigallocatechin-3-gallate seems to potentiate the contractile response to phenylephrine in an ex vivo thoracic ring preparation, partially through inhibition of NO production (⁴⁰). Consistent with these results, the current study suggests that treatment with the NF-κB inhibitor, EGCG, ameliorates the CL2P-mediated systemic hypotension in a rat model of polymicrobial sepsis.

Epigallocatechin-3-gallate has been shown to inhibit both NOS2 gene expression and activity in macrophages (^{3, 9}) and chondrocytes (⁴¹) in vitro. In addition, green tea extract was recently shown to inhibit NOS2 in the lung and intestine in a zymosan-induced model of nonseptic shock (¹⁰). In the current study, we show that EGCG inhibits IL-1β/IFN-γ-mediated NOS2 gene expression and activity in RASMCs, at least partially via a mechanism involving the inhibition of NF-κB activation. More importantly, the concentration of EGCG necessary for inhibition of NOS2 gene expression (5 μmol/L) in vitro is readily achievable in vivo through the consumption of green tea. For example, 1 cup (approximately 240 mL) of green tea contains 200 mg of EGCG, and a single 200-mg dose of EGCG produces a plasma EGCG concentration of approximately 0.1 μmol/L. However, the consumption of pharmaceutically prepared formulations of green tea polyphenols produces plasma EGCG concentrations approaching 2 μmol/L (¹). Collectively, these data suggest that the improvement in systemic hypotension and survival after CL2P in rats treated with EGCG may have been caused by inhibition of NOS2 gene expression and activity in vivo, although further studies are necessary to prove this assertion.

Our study has a few limitations that deserve mention. First, it is not known whether the improvement in systemic hemodynamics after CL2P results in a reduction in mortality in the rat model. However, although there may be some species-specific differences in the models used in the current study, these differences are likely to be minimal, and it is likely that the reduction in mortality observed in the EGCG-treated mice after CL2P is relevant to the rat model of CL2P as well (^{42, 43}). Second, we chose to study the direct effects of EGCG administration in the absence of antibiotics and surgical source control (i.e., laparotomy with removal of dead necrotic tissue and irrigation of abscess). Importantly, fluid resuscitation and antibiotic administration significantly improved survival after CL2P in NOS2-deficient transgenic mice compared with wild-type mice (⁴⁴), so it is possible that survival in our model will be even greater when EGCG and antibiotics are coadministered. Finally, our studies suggest that EGCG inhibits NOS2 expression and activity in the CL2P model of sepsis, although the source of NO and peroxynitrite was not assessed. For example, Annane et al. (⁴⁵) showed that NO activity is compartmentalized and localized to the site of infection in humans with sepsis. In this study, peripheral blood monocytes and tissue macrophages were an important source of NO as well. Notably, EGCG inhibits NOS2 expression and activity in peritoneal macrophages in vitro (^{3, 8, 9}).

In conclusion, we show that the green tea flavonoid, EGCG, improved hemodynamics and subsequently improved survival in a rodent model of polymicrobial sepsis. Consistent with previous work by our laboratory (^{2, 6, 7}), EGCG inhibited NF-κB activation in the lung, although there were no significant differences in serum cytokine concentrations. The improvement in hemodynamics may occur through a mechanism involving inhibition of NOS2 gene expression and activity. Therefore, EGCG may represent a potential nutritional supplement or pharmacologic agent in patients with sepsis.