Systemic inflammatory response syndrome (SIRS), culminating in multiple organ failure (MOF), is considered to be a direct cause of death in patients with sepsis and/or endotoxemia (1). Therefore, the prevention of SIRS and subsequent MOF is a critical issue in the management of sepsis or endotoxin shock. A pathophysiological feature of SIRS in humans is the liberation and overexpression of cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1, and IL-6 (2). It has been shown that released cytokines and nitric oxide (NO) play an important role in the development of lipopolysaccharide (LPS)-mediated endotoxin shock (3, 4). Therefore, inhibition of cytokine and NO elevation is important in therapy for endotoxin shock. Some previous studies have shown that heat stress preceding sepsis improves the survival rate and minimizes organ damage (5, 6). This protective effect in sepsis is likely related to the induction of heat shock proteins (HSPs) (7). HSPs are an important family of endogenous cytoprotective proteins that are induced in response to heat and a variety of other stresses (8, 9) such as inflammation (10), reactive oxygen species (ROS) (11), hypoxia, and ischemia (12). The induction of HSPs results in organ protection against stresses (9, 13). HSPs are necessary to counteract the incorrect aggregation of proteins and actively assist the folding process that occurs immediately after new synthesis or under conditions of stress such as heat shock, when native proteins have unfolded (14). Furthermore, it has recently been reported that some HSPs serve as anti-inflammatory factors (10, 15). Hence, pharmacological manipulations that trigger HSP induction may be potential therapeutic tools. However, several agents that increase the level of HSPs are toxic or have harmful side effects and are not recommended for use in a clinical situation (16-18). Geranylgeranylacetone (GGA), an acyclic polyisoprenoid developed in Japan and known as an antiulcer agent, has been used clinically without any serious side effects and is reported to induce HSP70 in several rat organs (18, 19). To date, there are few published reports on the relationship between HSP70 induction in multiple organs with GGA and the amelioration of systemic diseases including endotoxin shock. In the present study, we determined whether oral GGA administration would induce HSP70 in multiple organs of rats and thus protect rats against LPS-mediated endotoxin shock.
MATERIALS AND METHODS
GGA and gum arabic were provided by Eisai Co. Ltd (Tokyo, Japan). Anti-HSP70 polyclonal antibody was purchased from Upstate Corp. (Charlottesville, VA). Rabbit anti-HSP25 polyclonal antibody was obtained from StressGen Biotechnologies (Victoria, BC, Canada). Quercetin (Q) was purchased from Nakarai Tesque (Kyoto, Japan). All other chemicals used were of analytical grade.
Animals and treatments
Male Sprague-Dawley rats (weighing 200-250 g) were purchased from Japan SLC (Shizuoka, Japan) and were housed two to three per plastic cage. The animals were maintained on a 12-h light/dark cycle under controlled temperatures (23°C ± 3°C) and humidity (55% ± 5%) for 1 week before use in experiments. They were allowed unlimited access to standard laboratory chow and water, but were fasted 8 h before the onset of the experiment. GGA as an emulsion with 5% gum arabic and 0.008% α-tocopherol was given orally at a dose of 100, 200, or 400 mg/kg (GGA group). The dose volume was 10 mL/kg. Rats in the control group were given the same dose of vehicle. The Escherichia coli LPS serotype 0111:B4 (Sigma Chemical Co., St. Louis, MO) was dissolved in distilled water and administered intraperitoneally at a dose of 20 mg/kg at 4, 8, 16, and 24 h after GGA (200 mg/kg) administration (group G4, G8, G16, and G24, respectively) or 8 h after administration of the vehicle (group V). The dose volume of LPS was 1 mL/kg. Rats were monitored for survival over 24 h after LPS administration. Rats were anesthetized with pentobarbital intraperitoneally and blood samples were taken from the heart with a heparinized syringe 6 h after LPS administration. After collection of the blood samples, the rats' hearts, lungs, livers, and kidneys were removed, frozen in liquid nitrogen immediately, and stored at −80°C until assayed. In some experiments, rat organs were removed 8, 16, or 24 h after GGA administration.
In experiments using Q, rats were divided into three groups: LPS alone (n = 7), LPS/GGA (n = 7), or LPS/GGA/Q (n = 7). GGA (200 mg/kg) was administered orally 8 h before LPS (20 mg/kg) injection. The dose volume of GGA was 10 and 5 mL/kg when GGA was used alone and together with Q, respectively. Q was suspended in distilled water and administered orally at a dose of 200 mg/kg 1 h before GGA treatment. The dose volume of Q was 5 mL/kg. The survival rate of rats was determined 24 h after LPS injection.
These studies were approved by the Institutional Animal Care and Use Committee at Tottori University Faculty of Medicine, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and blood urea nitrogen (BUN), creatinine, and NO2/NO3 levels were determined spectrophotometrically by means of commercially available kits (Wako Pure Chemistry Industries, Osaka, Japan). Plasma levels of TNF-α, IL-6, and macrophage inflammatory protein-2 (MIP-2) were measured with enzyme-linked immunosorbent assay (ELISA) kits (BioSource International, Camarillo, CA).
Western blot analysis
Western blot analysis for HSPs was carried out as described previously (8). Briefly, frozen tissues (about 100 mg) were homogenized in 1.5 mL of sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10 mM dithiothreitol, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride), boiled for 5 min, passed through a 22-gauge needle three times to shear the DNA, and stored at −80°C until use. Each sample (50 μg of protein) was separated by SDS-polyacrylamide gel electrophoresis with a 12.5% polyacrylamide gel and electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking of the nonspecific binding sites, the polyvinylidene difluoride membrane was incubated for 2 h with rabbit anti-HSP70 polyclonal antibody or rabbit anti-HSP25 polyclonal antibody (1:1000 dilution), washed, and incubated for an additional hour with horseradish peroxidase-conjugated sheep anti-rabbit antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK) at 1:2000 dilution. The immunoblot was revealed using an enhanced chemiluminescence (ECL) Western Blotting Detection System or ECL Advance Western Blotting Detection Kit (Amersham Pharmacia Biotech). The membrane was exposed to Hyperfilm-ECL (Amersham Pharmacia Biotech) to localize antibody binding. Western blots were quantified using Scion Image software, which is based on NIH Image (Scion Corp., Frederick, MD).
Protein contents were determined using Bradford's method (20) with bovine serum albumin as a standard.
The results are expressed as means ± SE. The Kaplan-Meier method was used to calculate survival rate. A log-rank test and Welch's t test were used to determine statistically significant differences. Differences at P < 0.05 were considered significant.
Effect of GGA pretreatment on survival rate in rats administered LPS
Rats intraperitoneally injected with LPS (20 mg/kg body weight) were monitored for survival over 24 h (Fig. 1). The survival rate decreased to about 30% 24 h after LPS administration. Surprisingly, the treatment of rats with GGA (200 mg/kg) 8 h before LPS (G8) enabled all of them to survive. GGA treatment 16 h before LPS administration (G16) also increased the survival rate to more than 80%. In contrast, GGA treatment 4 or 24 h before LPS (G4 or G24) failed to improve the survival rate. As deaths were observed in the vehicle-treated group (group V) of rats from 6 h after LPS injection, we carried out biochemical analyses of blood and tissue samples from rats treated with LPS for 6 h in the following experiments to elucidate the mechanisms underlying the protective effect of GGA.
Inhibition by GGA of LPS-mediated proinflammatory cytokine liberation and NO production in rats
Because GGA reportedly suppresses inflammation (21), we evaluated the levels of plasma cytokines (TNF-α, IL-6, and MIP-2) in rats after LPS administration, and the effects of GGA on those levels. Plasma TNF-α, IL-6, and MIP-2 in rats increased 18-, 10-, and 6-fold from control levels, respectively, 6 h after LPS injection (Fig. 2, A-C). GGA pretreatments (G8 and G16) significantly inhibited the increase in plasma TNF-α and IL-6 (Fig. 2, A and B). Plasma MIP-2 elevation was significantly suppressed in G16 (Fig. 2C). GGA treatment 8 h before LPS (G8) tended to inhibit plasma MIP-2 elevation. Administration of GGA 4 or 24 h before LPS (G4 or G24) did not reduce any plasma cytokine levels. It is well known that LPS administration induces NO production in rats, leading to hypotension (2, 4, 22). Therefore, we determined the effect of GGA on plasma levels of NO2/NO3 as an index of NO production in rats treated with LPS (Fig. 2D). Plasma NO2/NO3 increased dramatically approximately 7-fold from the control level 6 h after LPS injection. Treatment of rats with GGA 8 h before LPS injection (G8) decreased plasma NO2/NO3 to an almost normal level. The plasma NO2/NO3 level decreased to double the normal level in group G16. To a lesser extent, treatment with GGA 4 or 24 h before LPS significantly inhibited plasma NO production.
Effect of GGA on hepatic and renal functions in LPS-injected rats
Because endotoxemia is often accompanied by damage to multiple organs, including the liver and kidney (3, 23), we next determined whether LPS induces hepatic and renal injuries in rats and whether GGA pretreatment prevents such LPS-mediated injuries (Fig. 3). Six hours after LPS injection, plasma ALT and AST in rats increased 6- and 8.5-fold from control level, respectively (Fig. 3, A and B). Treatment of rats with GGA 8, 16, or 24 h before LPS injection significantly inhibited plasma ALT and AST activities (Fig. 3, A and B). In contrast, GGA treatment 4 h before LPS injection failed to suppress the increase in plasma transaminase activities after LPS injection. In terms of renal function, LPS treatment increased the plasma BUN level to double the control level (Fig. 3C). Among groups pretreated with GGA (G4, G8, G16, and G24), the inhibitory effect of GGA on plasma BUN was evident only in G8. LPS administration did not affect plasma creatinine levels (data not shown).
Expression of HSP70 in rat tissues after GGA treatment
We used western blotting to examine whether oral administration of GGA induced HSP70 expression in the hearts, livers, kidneys, and lungs of rats (Fig. 4). An increase in HSP70 levels in rat heart became evident 8 h after 200 mg/kg GGA treatment and almost reached a plateau thereafter (Fig. 4A). HSP70 expression in rat liver showed a similar pattern to that in the heart (Fig. 4B). In the kidney, HSP70 levels increased 8 h after GGA and thereafter gradually decreased (Fig. 4C). In contrast, we were unable to detect any HSP70 accumulation in rat lungs (data not shown). GGA increased HSP70 expression in rat heart in a dose-dependent manner (Table 1). In the liver and kidney, 200 mg/kg of GGA caused the maximum induction of HSP70 (Table 1).
Pretreatment of rats with GGA enhances HSP70 expression after LPS injection
We examined whether pretreatment of rats with GGA (200 mg/kg) increased the HSP70 level in heart, liver, lung, and kidney 6 h after intraperitoneal administration of LPS (Fig. 5). GGA treatment 8 or 16 h before LPS was administered enhanced HSP70 expression to a greater extent in heart and lung 6 h after LPS injection (Fig. 5, A and C), although LPS itself induced HSP70 expression (data not shown). The HSP70 level increased significantly in the liver of rats treated with GGA 8 h before LPS injection (Fig. 5B). GGA administration did not affect the HSP70 level in the kidney (data not shown).
We also examined the protein expression of another HSP, HSP25, in the heart and liver. In rat heart, administration of LPS did not alter the protein level of HSP25 at 6 h after LPS, which remained similar to that of the control, and treatment with GGA in addition to LPS (G8 and G16) did not affect the HSP25 level (data not shown). In contrast, HSP25 was undetectable 6 h after LPS administration in the liver and this was not affected by treatment with GGA (data not shown).
Effect of Q on improvement by GGA of survival rate in LPS-treated rats
Because GGA pretreatment enhanced HSP70 expression in several organs in LPS-injected rats and improved survival rate in G8 and G16 (see Fig. 1), we investigated whether Q, an HSP inhibitor (18), abrogated the protective effect of GGA on death in rats. The survival rates 24 h after LPS administration were 28.5%, 100%, and 42.9% in LPS alone, LPS/GGA, and LPS/GGA/Q, respectively.
GGA has been shown to have HSP-inducing capacity, although it is currently used only as an antiulcer drug in clinical field. Therefore, effects of GGA pretreatment on insults in organs other than stomach have recently been investigated in animal models, but not in clinical studies (18, 19). In the present study, we observed that pretreatment with GGA dramatically improved the survival rate of LPS-treated rats. In particular, the survival rate 24 h after LPS administration was more than 80% when GGA was administered at 8 or 16 h before the LPS treatment. In contrast, treatment with GGA 4 or 24 h before administering LPS did not reduce the death rate of the rats (Fig. 1). These results suggest that there is a critical time point for administration of GGA to rescue rats from endotoxemic death. To our knowledge, there are no reports about the effect of timing of GGA administration at a single dose on survival rate in animal models, although GGA treatment of a fixed time period reportedly improved the survival rate in rat models of massive hepatectomy (21) and liver transplantation (12). LPS is well known to induce proinflammatory cytokine liberation and NO production, leading to death from endotoxin shock in animal models (4, 6, 10). Specifically, hypotension in endotoxin shock is considered to be attributable to a profound reduction of vascular reactivity to vasoconstrictors resulting from the action of NO produced in excess by inducible NO synthase expressed within the vasculature (24, 25). Furthermore, myocardial depression induced by NO might also contribute to hypotension induced by endotoxins (26). Therefore, inhibition of such inflammatory reactions is thought to be a promising approach for the prevention of endotoxin shock. The present study revealed that GGA treatment 8 or 16 h before treatment with LPS reduced blood circulation levels of proinflammatory cytokines such as TNF-α and IL-6, as well as NO, and 6 h after administration of LPS, these levels were less than one-half of those in rats treated with LPS alone (Fig. 2), indicating that GGA exhibits an anti-inflammatory action. Recently, GGA pretreatment was shown to suppress the elevation of serum TNF-α and IL-6 in a rat massive hepatectomy model (21) and a rat liver transplantation model (12).
Overexpression of HSP70 has been reported to reduce the production of proinflammatory mediators (10, 15, 27) and to protect against organ damage (8, 21, 28), apoptosis (29), and ischemia-reperfusion injury (30). It is known that the expression of HSP70 inhibits the production of cytokines in macrophages activated by LPS (11). It has been demonstrated that whole-body heat shock (HS) pretreatment prevents TNF-α-induced NO and IL-6 production and consequently death in mice, and that such HS-induced protection is absent in HSP70 knockout mice (31).
These results suggest that the induction of HSP70 confers protection against LPS-induced death at least in part by inhibiting the induction of cytokine production. Given that GGA is a potent HSP70 inducer, the anti-inflammatory action of GGA is thought to be possibly linked to HSP70 induction. In fact, HSP70 induction by GGA pretreatment was accompanied by inhibition of serum TNF-α and IL-6 levels in a rat massive hepatectomy model (21). Therefore, we investigated the induction of HSP70 by GGA and the effect of GGA pretreatment on HSP70 expression after LPS injection in several organs. We observed that HSP70 was most strongly induced 8 h after GGA administration in the heart, liver, and kidney of the rats (Fig. 4). The fact that there was no difference in the chronological pattern of HSP70 expression among organs shows that, in this study, orally administered GGA was absorbed and delivered to organs equally. Whereas GGA reportedly seems to exhibit a priming effect by enhancing HS factor-1 activation rather than directly inducing HSP70 in rat liver (30) and rat cultured hepatocytes (32), in rat heart, GGA can induce substantial HSP70 (19). Further research is required to clarify the precise mechanisms underlying HSP induction by GGA. Regarding HSP70 expression after LPS administration, treatment with GGA at 8 or 16 h before LPS enhanced HSP70 expression in tissues tested more effectively than GGA treatment 4 or 24 h before LPS (Fig. 5), consistent with the previous observations of cytokine inhibition and protection against death.
As for another protective effect of GGA, it has been reported that GGA enhanced expression of thioredoxin and suppressed ethanol-induced death of hepatocytes (33). However, the result that GGA enhanced nuclear factor (NF)-κB activity in that study is inconsistent with the inhibitory effect of GGA on production of NO and proinflammatory cytokines in the present study because their production is NF-κB dependent. The effects of GGA on LPS itself, LPS receptor such as Toll-like receptor 4, and CD14 remain to be elucidated. Further studies are required. Administration of LPS causes tissue damage in rat liver (34) and heart (35), and also in mouse kidney (36). When we orally treated rats in the present study with GGA 8, 16, or 24 h before the LPS challenge, we observed a clear reduction of LPS-induced hepatic damage as shown by a decrease in plasma ALT and AST activities. Regarding renal damage, treatment of rats with GGA 8 h before LPS only suppressed plasma BUN elevation.
In addition, treatment with Q, known as an inhibitor of HSP70 induction (18), decreased the survival rate of rats treated with LPS and GGA from 100% to 42.9%, strongly supporting the idea that protection by GGA of rats against LPS-mediated lethality is related to HSP70 induction by GGA, at least in part. Taken together, it has been suggested that oral administration of GGA at an optimal time before LPS injection induces and enhances HSP70 expression in several organs, inhibits proinflammatory cytokine and NO production, and prevents organ damage, resulting in improvement of survival rates.
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Geranylgeranylacetone; heat shock protein 70; lipopolysaccharide; cytokine; nitric oxide; endotoxin shock; rat