Effects of Naltrexone on Lipopolysaccharide-Induced Sepsis in Rats
Content
Journal of Biomedical Science 12: 431–440, 2005. DOI 10.1007/s11373-005-0647-x
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Effects of naltrexone on lipopolysaccharide-induced sepsis in rats Shinn-Long Lin1, Yen-Mei Lee2, Hsin-Yi Chang3, Yu-Wen Cheng4 & Mao-Hsiung Yen2,* 1
Department of Anesthesiology, Tri-Service General Hospital, Taipei, Taiwan; 2Department of Pharmacology, National Defense Medical Center; 3Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan; 4Department of Pharmacy, Taipei Medical University, Taipei, Taiwan
Received 12 September 2004; accepted in revised form 10 December 2004 2005 National Science Council, Taipei
Key words: hepatic dysfunction, naltrexone, nitric oxide, reactive oxygen species, sepsis, TNF-a
Summary Naltrexone, an opioid antagonist, has been reported to possess an anti-inflammatory effect via blockade of opioid receptor. The aim of this study is to evaluate the protective effect of naltrexone on LPS-induced septic shock in rats. Sepsis was induced by administration of LPS (10 mg/kg, i.v.) in anesthetized rats. Results demonstrated that pretreatment with naltrexone (10 mg/kg, i.v.) significantly ameliorated hypotension and bradycardia of rats 6 h after LPS administration. In isolated blood vessel, study showed that pretreatment with naltrexone significantly improved norepinephrine-induced vasoconstriction and ACh-induced vasorelaxation in aorta of endotoxemic animals. Naltrexone significantly reduced the elevation of serum glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase (as index of hepatic function) induced by LPS. The infiltration of polymorphonuclear neutrophils into liver 48 h after LPS treatment in mice was also reduced by naltrexone. On the other hand, naltrexone significantly decreased the levels of plasma TNF-a and inhibited overproduction of superoxide anions in aortic rings. However, naltrexone did not suppress the overproduction of NO (measured by its metabolites nitrite/nitrate in plasma) and iNOS expression in lungs induced by LPS. In in vitro study, naltrexone did not attenuate non-enzymatic ironinduced lipid peroxidation in rat brain homogenates. In conclusion, pretreatment with naltrexone significantly improved circulatory failure and hepatic dysfunction in sepsis. These effects were associated with reduction of TNF-a levels and superoxide anion formation, which may be attributed to antagonism of opioid receptors. Introduction Inflammatory and immune responses are vital features of septic shock and play an essential role in the pathogenesis of sepsis. Pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b), are vital components in the cascade of mediators leading to septic shock and their overproduction is associated with elevated body temperature, hypotension, tachycardia, tachypnea, leukopenia, end-organ dysfunction,
* To whom correspondence should be addressed. Fax: +886-287921704; E-mail: [email protected]
and death [1, 2]. Cytokines also can activate gene expression and protein synthesis for phospholipase A2, cyclooxygenase type II, inducible NO synthase (iNOS), leading to increased PAF, PGE2, and NO level [3–6]. Because of up-regulation of endothelial leukocyte adhesion molecule, adherence of circulating neutrophils to the endothelium, it leads to neutrophils migration to tissues for degranulation and tissue damage [7–9]. Based on existing data it appears that both pro-inflammatory cytokine overexpression and complete blockade are not satisfactory and lead to significant morbidity and mortality [7, 10]. It may be an ideal intervention for partial attenuation on cytokines overexpression without complete blockade.
432 Endogenous opiate substances (enkephalin, endorphin, dynorphin) exist in pituitary gland [11], sympathetic ganglions, adrenal medulla [11, 12]. They play a role as neuromodulator, neurotransmitters in the nerve system [13, 14] and endocrine in blood vessels [15]. Early on, endorphin was focused on pain modulation [16] and mental illness [17]. Then, Holaday and Faden proposed that endorphin is a mediator in shock [18]. They found that the opiate antagonist, naloxone, significantly improved hypotension, respiratory rate, acidosis, end-phase hypoglycemia, increase of leukocyte count, and platelet concentrations, and prolonged survival time in endotoxin-induced shock rats [19]. In endotoxin shock animal, opiate receptor blockade causes an enhanced adrenergic response of arterioles [20]. In addition, in immune aspect, purified mice macrophage treated with LPS and interferon-c, it demonstrated that naltrexone can antagonize morphineenhanced IL-12 mRNA and TNF-a expression [21]. Based on previous evidence, opioid antagonists show promise to be a prophylactic or therapeutic agent in sepsis. Therefore, we investigated the protective effect of naltrexone, an opioid antagonist, on sepsis/septic shock induced by LPS in rats and its possible mechanism of action. Materials and methods Animal preparation Male Wistar-Kyoto rats (220–280 g) were purchased from National Laboratory Animal Breeding and Research Center of National Science Council, Taiwan. All animals were housed at an ambient temperature of 23 ± 1C and moisture of 55 ± 5%. The rats were anesthetized by intraperitoneal injection of urethane (1.2 g/kg) and pentobarbital (5 mg/kg, i.v.). The trachea was cannulated to facilitate respiration. The left femoral artery was cannulated with full 100 units/ml heparin in polyethylene-50 (PE-50) and connected to a pressure transducer (P231D, Statham, Oxnard, CA, USA) for measurement of mean arterial pressure (MAP) and heart rate (HR) which were displayed on a Gould model TA5000 polygraph recorder (Gould, Valley View, OH, USA). The left femoral vein was cannulated for administration of drugs. After the surgical procedure was completed, all cardiovascular parameters were allowed to stabilize for 30–60 min.
Drug administration Animals were randomly assigned into the following groups: (1) control group: rats were treated with saline; (2) LPS group: rats were treated with E. coli lipopolysaccharide 3129 (LPS) 10 mg/kg (i.v.) [22]; (3) naltrexone-pretreatment group: rats were pretreated with naltrexone (5 or 10 mg/kg, i.v.) at 30 min prior to LPS administration. Blood sample 0.5 ml was withdrawn from venous canulation at 0, 1, 2, 4 and 6 h after the injection of saline or LPS. Blood samples were centrifuged for 3 min at 12,000 · g. Plasma samples were stored at )70 C until analysis. At the end of the experiment for 6 h thoracic aorta of each group were dissected out for vascular response and superoxide analysis. Organ bath experiments The blood vessels were cleared of adhering periadventitial fat and cut 3–4 mm in length. The rings were mounted in 20 ml organ baths filled with 37 C, 95% O2/5% CO2 oxygenated Kreb’s solution (pH 7.4) (consisting of mM: NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, glucose 11). Isometric force was measured with Grass FT03 type transducers (Grass instruments, Quincy, MA, USA) and recorded on a MacLab Recording and Analysis System (ADInstrument, Castle Hill, Australia). In the rings, 2 g tension was applied and equilibrated for 60– 90 min. After rings were stable under setting, to the ring was added 0.1 lM norepinephrine (NE) to contract maximally, then acetylcholine (ACh) 1 lM was added to relax. If (contractile tension – relaxation tension)/contractile tension is >80%, it reveals that the endothelium is intact. Kreb’s solution in organ baths was changed every 15 min for 2–3 times. After the rings with (contractile tension – relaxation tension)/contractile tension >80% were stable toward baseline tension level then the next steps were performed. (1) Concentration–response curves of NE: different concentrations of NE (10 nM to 0.3 lM) were added into the organ bath and the tension change was monitored by force–displacement transducer, and recorded by computer. Kreb’s solution was used to wash two times for every 30 min for the next step; (2) Concentration–response curves of ACh: NE (1 lM) for maximum contraction, then ACh was
433 added accumulatively from 10 nM to 1 lM for relaxation recording. Then Kreb’s solution was changed two times every 15 min for the next step; (3) Concentration–response curves of L -arginine: NE (1 lM) for maximum contraction, then L arginine (0.1–3 lM) was added accumulatively for relaxation recording. Evaluation of hepatic injury At 0 and 6 h after the injection of LPS, blood samples were collected. The blood sample was centrifuged 12,000 · g to prepare serum. All serum was analyzed within 24 h. After adding 10 ll serum to glutamate-oxalacetate transminase paper (GOT; a non-specific marker for hepatic parenchymal injury), and glutamate-pyruvate transaminase paper (GPT; a specific marker for hepatic parenchymal injury), they were put into a machine (DRI-Chem 3000, Colorige Tric Analyzer, FUJIFILM, Tokyo, Japan) to detect biochemical values.
Kit, Genzyme Co., Cambridge, MA, USA), as previously described [22]. Plasma nitrite/nitrate determination A sample of 30 ll thawed plasma was de-proteinated with 100 ll 95% alcohol for 30 min (4 C). Subsequently, these serum samples were centrifuged for 6 min at 12,000 · g. The supernatant (6 ll) was injected into a collection chamber containing 5% VCL3. In this strong reducing environment, both nitrate and nitrite were converted to NO. A constant stream of helium gas carried NO into a NO analyzer (Sievers 280 NOA; Sievers Instruments Inc., Boulder, CO, USA), where the NO reacted with ozone (O3), resulting in the emission of light. Light emission is proportional to the NO formed. Standard amounts of sodium nitrate were used for calibration (Sigma Chemical Co., St. Louis, MO, USA). Western blot analysis of iNOS protein expression in lungs
Histological studies in livers In order to observe markedly histological changes, liver was obtained from surviving ICR mice (30–35 g) in each group as described above after 48-h LPS treatment [22]. Mice were pretreated with naltrexone (10 mg/kg, i.p.) 30 min prior to LPS (60 mg/kg, i.p.) administration, with a second injection of naltrexone (5 mg/kg) 6 h later. These tissues were fixed in Carson-Millonig solution for histopathological examination [23]. The fixed tissues were dehydrated in graded ethanol and embedded in paraffin. Sections of 3 lm were stained with the hematoxylin and eosin reagent for light microscopy. This histological alteration was quantitatively analyzed as an index on the severity of tissue injury. This index was a neutrophil infiltration index that was determined by counting the numbers of polymorphonuclear neutrophil (PMN) in 10 randomly selected high-power fields. The index was expressed as the mean of these 10 numbers ± SE/high-power field. Measurement of plasma TNF-a concentration The blood sample (0.5 ml) was collected at 0, 1, and 2 h after the injection of LPS for measurement of the TNF-a level in plasma by an enzyme-linked immunoadsorbent assay (mouse TNF-a ELISA
At 6 h after the injection of saline or LPS, the experimental animals were sacrificed. The lung tissues were obtained from rats in control, LPS, and naltrexone-pretreatment groups, and frozen at )80 C before assay. Frozen samples were homogenized on ice with a polytron homogenizer (Model PRO 200, PRO Scientific Inc., Oxford, CT, USA) and a ultrasonic convertor (MISONIX Inc., Farmingdale, NY, USA) in a lysis buffer composed of (mM): Tris–HCl 50, EDTA 1, DTT 1, leupeptin 10, aprotinin 10, PMSF 1, and 1% Triton X-100 (pH 7.4). The homogenized tissues were centrifuged at 12,000 · g for 30 min and the supernatant was stored at )80 C until further analysis. Aliquots of tissue homogenates were used for protein assay (Bio-Rad protein assay reagent) and then mixed with the sample buffer (volume 3:1) composed of: 62.5 mM Tris–HCl, 2% SDS, 10% glycerol, 5% b-mercapto-ethanol, 2.5% bromophenol blue (pH 6.8). After heating at 95 C for 5 min, Western blot analysis was performed as described previously [22]. Aortic superoxide anion detection by chemiluminescence Detection of superoxide anions was performed as described previously [24]. Thoracic aorta was cut into 3–4 mm rings and incubated in 95% O2/5%
434 CO2 oxygenated modified Kreb’s/HEPES solution (37 C) for 30 min. Then the aorta sections were put into a 96 well plate in which every well was filled with 200 ll modified Kreb’s/HEPES, and placed in a luminescence measurement system (Microplate Luminometer LB 96 V, Windows Software for MicroLumat Plus LB 96 V, EG & GBERTHOLD, Germany). It can perform autoinjection of 250 lM lucigenin (final volume of 250 ll) into the vessels for interacting with superoxide. Counts were obtained at 15 min intervals at room temperature. After recording was complete, the vessel ring was dried in a 95 C oven for 24 h. The results were expressed as relative units of luminescence (RUL) per 15 min per milligram dry weight vessel (i.e., RUL/15 min/mg).
within 15 min. The MAP then slowly returned to 90 ± 4 mm Hg at 1 h, and gradually decreased to 56 ± 4 mm Hg at the end of the experiment. Results are shown in Figure 1a; the MAP of pretreatment with naltrexone (5 and 10 mg/kg) groups were significantly greater than that of the LPS group (p < 0.05). The mean baseline values of HR in four groups were about 440 ± 8 beats/ min and there was no significant difference among groups. In the LPS group, HR progressively increased and peaked at 2 h, and then decreased until the end of the experiment. However, pretreatment with naltrexone (10 mg/kg) significantly reduced HR at 1 h after LPS administration, and prevented LPS-induced bradycardia at 4–6 h (Figure 1b).
Anti-oxidant activity in rat brain homogenate (a)
Effects of naltrexone on MAP and HR in rats with endotoxemia The baseline MAPs of four groups were about 100 ± 3.1 to 116 ± 5 mmHg and did not show significant difference among groups (p > 0.05). The MAP of the control group is not significantly changed during the period of the experiment. The injection of LPS resulted in a rapid decrease in MAP from 102 ± 2 mm Hg to 85 ± 5 mm Hg
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Statistical analysis Data are expressed as mean ± SE. One-way ANOVA was performed in the statistical analysis of data. When group comparisons showed a significant difference, the Student–Newman Keuls test was used. A value of p < 0.05 was accepted to indicate statistical significance.
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To evaluate whether naltrexone (50 lM) scavenges free radicals, rat brain homogenate was prepared from the brains of freshly killed Wistar rats and its ferrous-induced peroxidation was measured by the thiobarbituric acid (TBA) method, as described by Hsiao et al. [25]. The results were expressed as nmoles of malondialdehyde (MDA) per mg of protein.
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Figure 1. Effects of pretreatment with naltrexone (Nalt 5: naltrexone 5 mg/kg, i.v., n ¼ 10 and Nalt 10: naltrexone 10 mg/kg, i.v., n ¼ 15) on mean MAP (a) and HR (b) changes after LPS (10 mg/kg, i.v.) administered for 6 h. Data represent means ± SE. *p < 0.05: LPS vs. control (n ¼ 6), #p < 0.05: naltrexone vs. LPS.
435 Effects of naltrexone on NE-induced vasoconstriction, ACh- and L -arginine-induced vasorelaxation, in vitro Results in Figure 2 show that vascular hyporeactivity of NE and ACh was found in the LPS-
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The basal plasma levels of GOT and GPT were not different among three experimental groups. After injection of LPS, GOT and GPT values elevated with time. At the end of the experiment (6 h), pretreatment with naltrexone (10 mg/kg) significantly lowered the GOT and GPT values induced by LPS (p < 0.05; Figure 3a, b)
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treated group. This hyporesponse to NE and to ACh was significantly reversed by pretreatment with naltrexone (10 mg/kg, i.v.) (p < 0.05, Figure 2a, b). However, there was no significant difference in L -arginine-induced vasorelaxation between LPS and naltrexone-pretreatment groups (Figure 2c).
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Time (h) Figure 3. Effects of pretreatment with naltrexone (Nalt) 10 mg/kg on GOT (a) and GPT (b) at 0 and 6 h after LPS treatment. Data represent mean ± SE (n ¼ 10). *p < 0.05: LPS vs. control, #p < 0.05: naltrexone vs. LPS.
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Figure 4. Effects of naltrexone pretreatment on LPS-induced histological changes (a: control; b: naltrexone alone; c: LPS; d: Nalt + LPS) in livers from ICR mice. Mice were pretreated with naltrexone (25 mg/kg, i.p.) 30 min prior to LPS (60 mg/kg, i.p.) administration, with a second injection of naltrexone (10 mg/kg) 15 h later. Liver sections were stained with hematoxylin and eosin and viewed by light microscopy (400 ·). Black arrows indicate marked infiltration of PMN after LPS treatment. e: PMN index in livers. Data are expressed as mean ± SE (n ¼ 6). *p < 0.05 represents significant difference between the LPS group and the control group. # p < 0.05 represents significant difference between the LPS group and the naltrexone group.
Effect of naltrexone on PMN infiltration in liver In the control group, light microscopy did not show any infiltration of PMN in livers (Figure 4a). Naltrexone alone (without LPS) also did not find
infiltration of PMN (Figure 4b). After 48-h LPS treatment, there was an obvious infiltration of PMN in livers (Figure 4c). In addition, all LPStreated mice showed marked interstitial edema and congestion diffusely in livers. In mice treated with
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naltrexone, the pathological change was mild in intensity (Figure 4d). Forty-eight hours after the injection of LPS, an over-infiltration on PMN in liver was observed. In mice treated with naltrexone, the PMN infiltration index was significantly improved in hepatic tissues (Figure 4e) (p < 0.05).
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Effect of naltrexone on superoxide anion formation The content of superoxide anions in thoracic aorta of LPS was significantly higher than that of the control group (38182 ± 8066 vs. 8499 ± 1878 RLU/15 min/mg, p < 0.05). However, pretreatment with naltrexone (10 mg/kg) was significantly different as compared with the LPS group (11866 ± 1195 vs. 38182 ± 8066 RLU/15 min/ mg, p < 0.05) (Figure 6a).
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Time (h) Figure 6. Effects of pretreatment with naltrexone (Nalt; 10 mg/ kg) on superoxide anion formation in aortic tissues (a) and on plasma nitrite/nitrate (b) from rats treated with LPS for 6 h. Data represent means ± SE (n ¼ 10). *p < 0.05: LPS vs. control, #p < 0.05: naltrexone vs. LPS.
Effect of naltrexone on plasma nitrite/nitrate content In LPS-treated groups, plasma nitrite/nitrate content at 4 and 6 h was significantly elevated as compared with the control group. However, pretreatment of naltrexone (10 mg/kg) did not suppress this increase in levels of NO metabolites after LPS administration (Figure 6b). Effect of naltrexone on expression of iNOS protein in lung tissues
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Figure 5. Effects of pretreatment with naltrexone (Nalt) 10 mg/ kg on plasma TNF-a level in LPS-treated rat. Data represent means ± SE (n ¼ 10). *p < 0.05: LPS vs. control, #p < 0.05: naltrexone vs. LPS.
An iNOS protein expression was undetectable in lung homogenates obtained from control rats, whereas a significant induction of iNOS protein (130 kD) was observed in lung homogenates of rats treated with LPS for 6 h (Figure 7). However, the content of iNOS protein expression induced by LPS was not affected by the pretreatment of naltrexone (10 mg/kg) (Figure 7).
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Figure 7. Effects of pretreatment with naltrexone (Nalt) 10 mg/ kg on iNOS expression in the lungs from rats treated with LPS for 6 h. Depicted is a typical display of iNOS protein expression (a) and the statistical analysis of the changes of iNOS protein (b). Data represent mean ± SE. *p < 0.05: LPS vs. control.
Anti-oxidant activity in rat brain homogenate Naltrexone did not exert the inhibition of ironcatalyzed lipid peroxidation in rat brain homogenate, even at a concentration of 50 lM (Resting: 0.24 ± 0.06; Ferrous-induced: 0.85 ± 0.04; Ferrous with naltrexone: 0.98 ± 0.16 nmol MDA/mg protein, n ¼ 3).
Discussion The present study demonstrated that pretreatment with naltrexone significantly ameliorated circulatory failure resulting from LPS injection. This effect is associated with (i) the prevention of vascular hyporeactivity; (ii) inhibition of TNF-a production in plasma, and (iii) reduction of superoxide anion formation in aortae. Besides, naltrexone prevented the elevation of GOT and GPT, and suppressed the infiltration of PMN in liver after 6-h LPS treatment, indicating that naltrexone may be used as a prophylactic and therapeutic agent for sepsis-induced circulatory failure and hepatic dysfunction. It has been reported that b-endorphin can induce hypotension and bradycardia in dogs, which
can be transiently blocked by high doses of the opioid antagonist naloxone [26]. In 1985, Almqvist et al. reported that endotoxin shock caused a marked rise in circulating b-endorphin and suggested that the endorphins may participate in septic shock [27]. In endotoxin shock rats, opiate receptor blockade causes an enhanced adrenergic response of arterioles [20]. Accordingly, results in the present studies demonstrated that pretreatment with naltrexone, an antagonist of opioid receptors, significantly attenuated LPS-induced deleterious hemodynamic changes. This further confirmed the role of endorphins in the pathogenesis of sepsis and septic shock. Molina et al. reported that cytokines such as TNF, interleukin-1 alpha (IL-1a), IL-6, IL-10 are at a higher level than normal in rat especially during lung and spleen of hemorrhage shock in rat. Pretreatment with naltrexone blunted the magnitude of the increases in tissue content of cytokine in response to a given blood loss [28]. Greeneltch et al. reported that naltrexone blocks acute endotoxic shock by inhibiting TNF-a production in mice [29]. In accordance with the above evidence, the results of rats in this study showed that the plasma TNF-a level elevated by LPS was significantly suppressed by pretreatment with naltrexone. However, the mechanism of action is still uncertain and needs to be further elucidated. In sepsis, abundant reactive oxygen species (ROS) has been produced. Several sources of oxygen radical species have been raised to be causes of tissue damage. Following transmigration and activation, the infiltrating neutrophils produce abundant oxygen radicals via the oxidative burst. Other sources of oxygen radical species include activated macrophages and various extracellular molecular processes such as arachidonic acid metabolism and xanthine dehydrogenase oxidation. Many anti-oxidants have been reported to possess beneficial effects on sepsis [30]. Overproducion of superoxide anions can react with NO to form the peroxynitrite anion (ONOO)) [31]. In 1999, Cuzzocrea et al. proved that ONOO) can prompt damage in DNA, which can activate the PARS (polyadenylribosyl synthase) and lead to ATP depletion causing multiple organ failure [32]. Results in this study demonstrated that pretreatment with naltrexone significantly suppressed the superoxide anion production in blood vessels induced by LPS. However, in in vitro study,
439 naltrexone did not attenuate non-enzymatic ironinduced lipid peroxidation in rat brain homogenates, indicating that naltrexone cannot show a free radical scavenging effect to protect cell membrane against oxidative stress. Therefore, the antioxidant effect of naltrexone on blood vessels may be mediated via blockade of opioid receptors or other unknown mechanism(s). Naltrexone improved NE-induced vasocontraction and ACh-induced vasorelaxation (Figure 2), indicating that it prevented LPS-induced vascular hyporeactivity and endothelial dysfunction. However, naltrexone did not reduce the L -arginine-induced relaxation after LPS treatment, implying that it could not suppress the activity of iNOS. This point was further supported by the results in which plasma nitrate/nitrite concentration and iNOS protein expression in lung tissue between LPS and naltrexone-treated groups were not significantly different. These results indicated that the beneficial effect of naltrexone on vascular reactivity was not mediated by suppression of NO overproduction in sepsis. Naltrexone reduced the amount of ROS formation in aorta and may preserve the function and integrity of vascular smooth muscle and endothelium, leading to increased vasoconstrictor-induced responses during sepsis. Although naltrexone significantly improved the vasoreactivity and prevented hypotension induced by LPS, surprisingly the plasma level of nitrite/ nitrate and iNOS protein expression in lungs was not inhibited. On the contrary, Lysle and How have reported that naltrexone significantly reduces LPS-induced iNOS mRNA and protein expression in spleenocytes and suppresses elevation of plasma nitrite/nitrate in rats. These results are mediated via blocking central opioid receptors but not peripheral tissues [33]. The discrepant results may be mainly attributed to the different doses of naltrexone and LPS used. In the report of Lysle et al., naltrexone 10 mg/kg was given twice, at the same time as LPS (100 lg/kg, s.c.) administration, and 4 h later. They used a lower dose of LPS and a higher dose of naltrexone, by which a less severe condition of sepsis by LPS is produced, and a pronounced inhibition of iNOS expression by naltrexone. In contrast, although we used a higher dose of LPS to induce a more severe sepsis, only one dose of naltrexone 10 mg/kg given is sufficient to prevent circulatory failure, maintain vascular response to NE, and improve endothelial and
hepatic function after 6-h LPS treatment. All of these beneficial results may be, at least partly, attributed to blockade of opioid receptors, no matter if this occurs in the central nervous system or peripheral tissues. However, we cannot clarify this point in the present study. Interestingly, we found that pretreatment with naltrexone (10 mg/kg, i.v.) significantly improved hepatic function after 6-h LPS administration in rats. Recently, Greeneltch et al. reported that pretreatment with naltrexone (10 mg/kg, i.p.) cannot reduce the liver damage in mice of acute endotoxic shock [29]. The following reasons may explain this discrepancy. (i) In that earlier study, sepsis is induced by 1 mg/kg LPS + 25 mg D -galactosamine (D -gal, i.p.). Concurrently using two challenging agents may lead to more complicated conditions and severe hepatic damage than LPS alone. (ii) Because the doses of naltrexone for rats with endotoxic shock in several reports are higher than 10 mg/kg [20, 33], the dose of naltrexone 10 mg/kg (i.p.) for mice is too low and not sufficient to protect liver from severe damage. These two factors may result in the failure of naltrexone in hepatic protection in that endotoxic mice study. Therefore, naltrexone may still show promise in hepatoprotective effect. In conclusion, this is the first in vivo experiment to demonstrate beneficial effect of naltrexone on circulatory failure and hepatic dysfunction induced by LPS. The protective effect was associated with reduction of TNF-a level and superoxide anion production. However, the molecular mechanism of action of naltrexone on sepsis needs to be clarified in further experiments.
Acknowledgements This work was supported by grants from the National Science Council (NSC 91-2320-B-016-040) and from the Ministry of National Defense (DOD92-08), Taiwan and Jenken Bioscience, Inc., USA.
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431
Effects of naltrexone on lipopolysaccharide-induced sepsis in rats Shinn-Long Lin1, Yen-Mei Lee2, Hsin-Yi Chang3, Yu-Wen Cheng4 & Mao-Hsiung Yen2,* 1
Department of Anesthesiology, Tri-Service General Hospital, Taipei, Taiwan; 2Department of Pharmacology, National Defense Medical Center; 3Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan; 4Department of Pharmacy, Taipei Medical University, Taipei, Taiwan
Received 12 September 2004; accepted in revised form 10 December 2004 2005 National Science Council, Taipei
Key words: hepatic dysfunction, naltrexone, nitric oxide, reactive oxygen species, sepsis, TNF-a
Summary Naltrexone, an opioid antagonist, has been reported to possess an anti-inflammatory effect via blockade of opioid receptor. The aim of this study is to evaluate the protective effect of naltrexone on LPS-induced septic shock in rats. Sepsis was induced by administration of LPS (10 mg/kg, i.v.) in anesthetized rats. Results demonstrated that pretreatment with naltrexone (10 mg/kg, i.v.) significantly ameliorated hypotension and bradycardia of rats 6 h after LPS administration. In isolated blood vessel, study showed that pretreatment with naltrexone significantly improved norepinephrine-induced vasoconstriction and ACh-induced vasorelaxation in aorta of endotoxemic animals. Naltrexone significantly reduced the elevation of serum glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase (as index of hepatic function) induced by LPS. The infiltration of polymorphonuclear neutrophils into liver 48 h after LPS treatment in mice was also reduced by naltrexone. On the other hand, naltrexone significantly decreased the levels of plasma TNF-a and inhibited overproduction of superoxide anions in aortic rings. However, naltrexone did not suppress the overproduction of NO (measured by its metabolites nitrite/nitrate in plasma) and iNOS expression in lungs induced by LPS. In in vitro study, naltrexone did not attenuate non-enzymatic ironinduced lipid peroxidation in rat brain homogenates. In conclusion, pretreatment with naltrexone significantly improved circulatory failure and hepatic dysfunction in sepsis. These effects were associated with reduction of TNF-a levels and superoxide anion formation, which may be attributed to antagonism of opioid receptors. Introduction Inflammatory and immune responses are vital features of septic shock and play an essential role in the pathogenesis of sepsis. Pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b), are vital components in the cascade of mediators leading to septic shock and their overproduction is associated with elevated body temperature, hypotension, tachycardia, tachypnea, leukopenia, end-organ dysfunction,
* To whom correspondence should be addressed. Fax: +886-287921704; E-mail: [email protected]
and death [1, 2]. Cytokines also can activate gene expression and protein synthesis for phospholipase A2, cyclooxygenase type II, inducible NO synthase (iNOS), leading to increased PAF, PGE2, and NO level [3–6]. Because of up-regulation of endothelial leukocyte adhesion molecule, adherence of circulating neutrophils to the endothelium, it leads to neutrophils migration to tissues for degranulation and tissue damage [7–9]. Based on existing data it appears that both pro-inflammatory cytokine overexpression and complete blockade are not satisfactory and lead to significant morbidity and mortality [7, 10]. It may be an ideal intervention for partial attenuation on cytokines overexpression without complete blockade.
432 Endogenous opiate substances (enkephalin, endorphin, dynorphin) exist in pituitary gland [11], sympathetic ganglions, adrenal medulla [11, 12]. They play a role as neuromodulator, neurotransmitters in the nerve system [13, 14] and endocrine in blood vessels [15]. Early on, endorphin was focused on pain modulation [16] and mental illness [17]. Then, Holaday and Faden proposed that endorphin is a mediator in shock [18]. They found that the opiate antagonist, naloxone, significantly improved hypotension, respiratory rate, acidosis, end-phase hypoglycemia, increase of leukocyte count, and platelet concentrations, and prolonged survival time in endotoxin-induced shock rats [19]. In endotoxin shock animal, opiate receptor blockade causes an enhanced adrenergic response of arterioles [20]. In addition, in immune aspect, purified mice macrophage treated with LPS and interferon-c, it demonstrated that naltrexone can antagonize morphineenhanced IL-12 mRNA and TNF-a expression [21]. Based on previous evidence, opioid antagonists show promise to be a prophylactic or therapeutic agent in sepsis. Therefore, we investigated the protective effect of naltrexone, an opioid antagonist, on sepsis/septic shock induced by LPS in rats and its possible mechanism of action. Materials and methods Animal preparation Male Wistar-Kyoto rats (220–280 g) were purchased from National Laboratory Animal Breeding and Research Center of National Science Council, Taiwan. All animals were housed at an ambient temperature of 23 ± 1C and moisture of 55 ± 5%. The rats were anesthetized by intraperitoneal injection of urethane (1.2 g/kg) and pentobarbital (5 mg/kg, i.v.). The trachea was cannulated to facilitate respiration. The left femoral artery was cannulated with full 100 units/ml heparin in polyethylene-50 (PE-50) and connected to a pressure transducer (P231D, Statham, Oxnard, CA, USA) for measurement of mean arterial pressure (MAP) and heart rate (HR) which were displayed on a Gould model TA5000 polygraph recorder (Gould, Valley View, OH, USA). The left femoral vein was cannulated for administration of drugs. After the surgical procedure was completed, all cardiovascular parameters were allowed to stabilize for 30–60 min.
Drug administration Animals were randomly assigned into the following groups: (1) control group: rats were treated with saline; (2) LPS group: rats were treated with E. coli lipopolysaccharide 3129 (LPS) 10 mg/kg (i.v.) [22]; (3) naltrexone-pretreatment group: rats were pretreated with naltrexone (5 or 10 mg/kg, i.v.) at 30 min prior to LPS administration. Blood sample 0.5 ml was withdrawn from venous canulation at 0, 1, 2, 4 and 6 h after the injection of saline or LPS. Blood samples were centrifuged for 3 min at 12,000 · g. Plasma samples were stored at )70 C until analysis. At the end of the experiment for 6 h thoracic aorta of each group were dissected out for vascular response and superoxide analysis. Organ bath experiments The blood vessels were cleared of adhering periadventitial fat and cut 3–4 mm in length. The rings were mounted in 20 ml organ baths filled with 37 C, 95% O2/5% CO2 oxygenated Kreb’s solution (pH 7.4) (consisting of mM: NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, glucose 11). Isometric force was measured with Grass FT03 type transducers (Grass instruments, Quincy, MA, USA) and recorded on a MacLab Recording and Analysis System (ADInstrument, Castle Hill, Australia). In the rings, 2 g tension was applied and equilibrated for 60– 90 min. After rings were stable under setting, to the ring was added 0.1 lM norepinephrine (NE) to contract maximally, then acetylcholine (ACh) 1 lM was added to relax. If (contractile tension – relaxation tension)/contractile tension is >80%, it reveals that the endothelium is intact. Kreb’s solution in organ baths was changed every 15 min for 2–3 times. After the rings with (contractile tension – relaxation tension)/contractile tension >80% were stable toward baseline tension level then the next steps were performed. (1) Concentration–response curves of NE: different concentrations of NE (10 nM to 0.3 lM) were added into the organ bath and the tension change was monitored by force–displacement transducer, and recorded by computer. Kreb’s solution was used to wash two times for every 30 min for the next step; (2) Concentration–response curves of ACh: NE (1 lM) for maximum contraction, then ACh was
433 added accumulatively from 10 nM to 1 lM for relaxation recording. Then Kreb’s solution was changed two times every 15 min for the next step; (3) Concentration–response curves of L -arginine: NE (1 lM) for maximum contraction, then L arginine (0.1–3 lM) was added accumulatively for relaxation recording. Evaluation of hepatic injury At 0 and 6 h after the injection of LPS, blood samples were collected. The blood sample was centrifuged 12,000 · g to prepare serum. All serum was analyzed within 24 h. After adding 10 ll serum to glutamate-oxalacetate transminase paper (GOT; a non-specific marker for hepatic parenchymal injury), and glutamate-pyruvate transaminase paper (GPT; a specific marker for hepatic parenchymal injury), they were put into a machine (DRI-Chem 3000, Colorige Tric Analyzer, FUJIFILM, Tokyo, Japan) to detect biochemical values.
Kit, Genzyme Co., Cambridge, MA, USA), as previously described [22]. Plasma nitrite/nitrate determination A sample of 30 ll thawed plasma was de-proteinated with 100 ll 95% alcohol for 30 min (4 C). Subsequently, these serum samples were centrifuged for 6 min at 12,000 · g. The supernatant (6 ll) was injected into a collection chamber containing 5% VCL3. In this strong reducing environment, both nitrate and nitrite were converted to NO. A constant stream of helium gas carried NO into a NO analyzer (Sievers 280 NOA; Sievers Instruments Inc., Boulder, CO, USA), where the NO reacted with ozone (O3), resulting in the emission of light. Light emission is proportional to the NO formed. Standard amounts of sodium nitrate were used for calibration (Sigma Chemical Co., St. Louis, MO, USA). Western blot analysis of iNOS protein expression in lungs
Histological studies in livers In order to observe markedly histological changes, liver was obtained from surviving ICR mice (30–35 g) in each group as described above after 48-h LPS treatment [22]. Mice were pretreated with naltrexone (10 mg/kg, i.p.) 30 min prior to LPS (60 mg/kg, i.p.) administration, with a second injection of naltrexone (5 mg/kg) 6 h later. These tissues were fixed in Carson-Millonig solution for histopathological examination [23]. The fixed tissues were dehydrated in graded ethanol and embedded in paraffin. Sections of 3 lm were stained with the hematoxylin and eosin reagent for light microscopy. This histological alteration was quantitatively analyzed as an index on the severity of tissue injury. This index was a neutrophil infiltration index that was determined by counting the numbers of polymorphonuclear neutrophil (PMN) in 10 randomly selected high-power fields. The index was expressed as the mean of these 10 numbers ± SE/high-power field. Measurement of plasma TNF-a concentration The blood sample (0.5 ml) was collected at 0, 1, and 2 h after the injection of LPS for measurement of the TNF-a level in plasma by an enzyme-linked immunoadsorbent assay (mouse TNF-a ELISA
At 6 h after the injection of saline or LPS, the experimental animals were sacrificed. The lung tissues were obtained from rats in control, LPS, and naltrexone-pretreatment groups, and frozen at )80 C before assay. Frozen samples were homogenized on ice with a polytron homogenizer (Model PRO 200, PRO Scientific Inc., Oxford, CT, USA) and a ultrasonic convertor (MISONIX Inc., Farmingdale, NY, USA) in a lysis buffer composed of (mM): Tris–HCl 50, EDTA 1, DTT 1, leupeptin 10, aprotinin 10, PMSF 1, and 1% Triton X-100 (pH 7.4). The homogenized tissues were centrifuged at 12,000 · g for 30 min and the supernatant was stored at )80 C until further analysis. Aliquots of tissue homogenates were used for protein assay (Bio-Rad protein assay reagent) and then mixed with the sample buffer (volume 3:1) composed of: 62.5 mM Tris–HCl, 2% SDS, 10% glycerol, 5% b-mercapto-ethanol, 2.5% bromophenol blue (pH 6.8). After heating at 95 C for 5 min, Western blot analysis was performed as described previously [22]. Aortic superoxide anion detection by chemiluminescence Detection of superoxide anions was performed as described previously [24]. Thoracic aorta was cut into 3–4 mm rings and incubated in 95% O2/5%
434 CO2 oxygenated modified Kreb’s/HEPES solution (37 C) for 30 min. Then the aorta sections were put into a 96 well plate in which every well was filled with 200 ll modified Kreb’s/HEPES, and placed in a luminescence measurement system (Microplate Luminometer LB 96 V, Windows Software for MicroLumat Plus LB 96 V, EG & GBERTHOLD, Germany). It can perform autoinjection of 250 lM lucigenin (final volume of 250 ll) into the vessels for interacting with superoxide. Counts were obtained at 15 min intervals at room temperature. After recording was complete, the vessel ring was dried in a 95 C oven for 24 h. The results were expressed as relative units of luminescence (RUL) per 15 min per milligram dry weight vessel (i.e., RUL/15 min/mg).
within 15 min. The MAP then slowly returned to 90 ± 4 mm Hg at 1 h, and gradually decreased to 56 ± 4 mm Hg at the end of the experiment. Results are shown in Figure 1a; the MAP of pretreatment with naltrexone (5 and 10 mg/kg) groups were significantly greater than that of the LPS group (p < 0.05). The mean baseline values of HR in four groups were about 440 ± 8 beats/ min and there was no significant difference among groups. In the LPS group, HR progressively increased and peaked at 2 h, and then decreased until the end of the experiment. However, pretreatment with naltrexone (10 mg/kg) significantly reduced HR at 1 h after LPS administration, and prevented LPS-induced bradycardia at 4–6 h (Figure 1b).
Anti-oxidant activity in rat brain homogenate (a)
Effects of naltrexone on MAP and HR in rats with endotoxemia The baseline MAPs of four groups were about 100 ± 3.1 to 116 ± 5 mmHg and did not show significant difference among groups (p > 0.05). The MAP of the control group is not significantly changed during the period of the experiment. The injection of LPS resulted in a rapid decrease in MAP from 102 ± 2 mm Hg to 85 ± 5 mm Hg
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To evaluate whether naltrexone (50 lM) scavenges free radicals, rat brain homogenate was prepared from the brains of freshly killed Wistar rats and its ferrous-induced peroxidation was measured by the thiobarbituric acid (TBA) method, as described by Hsiao et al. [25]. The results were expressed as nmoles of malondialdehyde (MDA) per mg of protein.
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Figure 1. Effects of pretreatment with naltrexone (Nalt 5: naltrexone 5 mg/kg, i.v., n ¼ 10 and Nalt 10: naltrexone 10 mg/kg, i.v., n ¼ 15) on mean MAP (a) and HR (b) changes after LPS (10 mg/kg, i.v.) administered for 6 h. Data represent means ± SE. *p < 0.05: LPS vs. control (n ¼ 6), #p < 0.05: naltrexone vs. LPS.
435 Effects of naltrexone on NE-induced vasoconstriction, ACh- and L -arginine-induced vasorelaxation, in vitro Results in Figure 2 show that vascular hyporeactivity of NE and ACh was found in the LPS-
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treated group. This hyporesponse to NE and to ACh was significantly reversed by pretreatment with naltrexone (10 mg/kg, i.v.) (p < 0.05, Figure 2a, b). However, there was no significant difference in L -arginine-induced vasorelaxation between LPS and naltrexone-pretreatment groups (Figure 2c).
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Figure 4. Effects of naltrexone pretreatment on LPS-induced histological changes (a: control; b: naltrexone alone; c: LPS; d: Nalt + LPS) in livers from ICR mice. Mice were pretreated with naltrexone (25 mg/kg, i.p.) 30 min prior to LPS (60 mg/kg, i.p.) administration, with a second injection of naltrexone (10 mg/kg) 15 h later. Liver sections were stained with hematoxylin and eosin and viewed by light microscopy (400 ·). Black arrows indicate marked infiltration of PMN after LPS treatment. e: PMN index in livers. Data are expressed as mean ± SE (n ¼ 6). *p < 0.05 represents significant difference between the LPS group and the control group. # p < 0.05 represents significant difference between the LPS group and the naltrexone group.
Effect of naltrexone on PMN infiltration in liver In the control group, light microscopy did not show any infiltration of PMN in livers (Figure 4a). Naltrexone alone (without LPS) also did not find
infiltration of PMN (Figure 4b). After 48-h LPS treatment, there was an obvious infiltration of PMN in livers (Figure 4c). In addition, all LPStreated mice showed marked interstitial edema and congestion diffusely in livers. In mice treated with
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naltrexone, the pathological change was mild in intensity (Figure 4d). Forty-eight hours after the injection of LPS, an over-infiltration on PMN in liver was observed. In mice treated with naltrexone, the PMN infiltration index was significantly improved in hepatic tissues (Figure 4e) (p < 0.05).
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Effect of naltrexone on superoxide anion formation The content of superoxide anions in thoracic aorta of LPS was significantly higher than that of the control group (38182 ± 8066 vs. 8499 ± 1878 RLU/15 min/mg, p < 0.05). However, pretreatment with naltrexone (10 mg/kg) was significantly different as compared with the LPS group (11866 ± 1195 vs. 38182 ± 8066 RLU/15 min/ mg, p < 0.05) (Figure 6a).
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Effect of naltrexone on plasma nitrite/nitrate content In LPS-treated groups, plasma nitrite/nitrate content at 4 and 6 h was significantly elevated as compared with the control group. However, pretreatment of naltrexone (10 mg/kg) did not suppress this increase in levels of NO metabolites after LPS administration (Figure 6b). Effect of naltrexone on expression of iNOS protein in lung tissues
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An iNOS protein expression was undetectable in lung homogenates obtained from control rats, whereas a significant induction of iNOS protein (130 kD) was observed in lung homogenates of rats treated with LPS for 6 h (Figure 7). However, the content of iNOS protein expression induced by LPS was not affected by the pretreatment of naltrexone (10 mg/kg) (Figure 7).
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Figure 7. Effects of pretreatment with naltrexone (Nalt) 10 mg/ kg on iNOS expression in the lungs from rats treated with LPS for 6 h. Depicted is a typical display of iNOS protein expression (a) and the statistical analysis of the changes of iNOS protein (b). Data represent mean ± SE. *p < 0.05: LPS vs. control.
Anti-oxidant activity in rat brain homogenate Naltrexone did not exert the inhibition of ironcatalyzed lipid peroxidation in rat brain homogenate, even at a concentration of 50 lM (Resting: 0.24 ± 0.06; Ferrous-induced: 0.85 ± 0.04; Ferrous with naltrexone: 0.98 ± 0.16 nmol MDA/mg protein, n ¼ 3).
Discussion The present study demonstrated that pretreatment with naltrexone significantly ameliorated circulatory failure resulting from LPS injection. This effect is associated with (i) the prevention of vascular hyporeactivity; (ii) inhibition of TNF-a production in plasma, and (iii) reduction of superoxide anion formation in aortae. Besides, naltrexone prevented the elevation of GOT and GPT, and suppressed the infiltration of PMN in liver after 6-h LPS treatment, indicating that naltrexone may be used as a prophylactic and therapeutic agent for sepsis-induced circulatory failure and hepatic dysfunction. It has been reported that b-endorphin can induce hypotension and bradycardia in dogs, which
can be transiently blocked by high doses of the opioid antagonist naloxone [26]. In 1985, Almqvist et al. reported that endotoxin shock caused a marked rise in circulating b-endorphin and suggested that the endorphins may participate in septic shock [27]. In endotoxin shock rats, opiate receptor blockade causes an enhanced adrenergic response of arterioles [20]. Accordingly, results in the present studies demonstrated that pretreatment with naltrexone, an antagonist of opioid receptors, significantly attenuated LPS-induced deleterious hemodynamic changes. This further confirmed the role of endorphins in the pathogenesis of sepsis and septic shock. Molina et al. reported that cytokines such as TNF, interleukin-1 alpha (IL-1a), IL-6, IL-10 are at a higher level than normal in rat especially during lung and spleen of hemorrhage shock in rat. Pretreatment with naltrexone blunted the magnitude of the increases in tissue content of cytokine in response to a given blood loss [28]. Greeneltch et al. reported that naltrexone blocks acute endotoxic shock by inhibiting TNF-a production in mice [29]. In accordance with the above evidence, the results of rats in this study showed that the plasma TNF-a level elevated by LPS was significantly suppressed by pretreatment with naltrexone. However, the mechanism of action is still uncertain and needs to be further elucidated. In sepsis, abundant reactive oxygen species (ROS) has been produced. Several sources of oxygen radical species have been raised to be causes of tissue damage. Following transmigration and activation, the infiltrating neutrophils produce abundant oxygen radicals via the oxidative burst. Other sources of oxygen radical species include activated macrophages and various extracellular molecular processes such as arachidonic acid metabolism and xanthine dehydrogenase oxidation. Many anti-oxidants have been reported to possess beneficial effects on sepsis [30]. Overproducion of superoxide anions can react with NO to form the peroxynitrite anion (ONOO)) [31]. In 1999, Cuzzocrea et al. proved that ONOO) can prompt damage in DNA, which can activate the PARS (polyadenylribosyl synthase) and lead to ATP depletion causing multiple organ failure [32]. Results in this study demonstrated that pretreatment with naltrexone significantly suppressed the superoxide anion production in blood vessels induced by LPS. However, in in vitro study,
439 naltrexone did not attenuate non-enzymatic ironinduced lipid peroxidation in rat brain homogenates, indicating that naltrexone cannot show a free radical scavenging effect to protect cell membrane against oxidative stress. Therefore, the antioxidant effect of naltrexone on blood vessels may be mediated via blockade of opioid receptors or other unknown mechanism(s). Naltrexone improved NE-induced vasocontraction and ACh-induced vasorelaxation (Figure 2), indicating that it prevented LPS-induced vascular hyporeactivity and endothelial dysfunction. However, naltrexone did not reduce the L -arginine-induced relaxation after LPS treatment, implying that it could not suppress the activity of iNOS. This point was further supported by the results in which plasma nitrate/nitrite concentration and iNOS protein expression in lung tissue between LPS and naltrexone-treated groups were not significantly different. These results indicated that the beneficial effect of naltrexone on vascular reactivity was not mediated by suppression of NO overproduction in sepsis. Naltrexone reduced the amount of ROS formation in aorta and may preserve the function and integrity of vascular smooth muscle and endothelium, leading to increased vasoconstrictor-induced responses during sepsis. Although naltrexone significantly improved the vasoreactivity and prevented hypotension induced by LPS, surprisingly the plasma level of nitrite/ nitrate and iNOS protein expression in lungs was not inhibited. On the contrary, Lysle and How have reported that naltrexone significantly reduces LPS-induced iNOS mRNA and protein expression in spleenocytes and suppresses elevation of plasma nitrite/nitrate in rats. These results are mediated via blocking central opioid receptors but not peripheral tissues [33]. The discrepant results may be mainly attributed to the different doses of naltrexone and LPS used. In the report of Lysle et al., naltrexone 10 mg/kg was given twice, at the same time as LPS (100 lg/kg, s.c.) administration, and 4 h later. They used a lower dose of LPS and a higher dose of naltrexone, by which a less severe condition of sepsis by LPS is produced, and a pronounced inhibition of iNOS expression by naltrexone. In contrast, although we used a higher dose of LPS to induce a more severe sepsis, only one dose of naltrexone 10 mg/kg given is sufficient to prevent circulatory failure, maintain vascular response to NE, and improve endothelial and
hepatic function after 6-h LPS treatment. All of these beneficial results may be, at least partly, attributed to blockade of opioid receptors, no matter if this occurs in the central nervous system or peripheral tissues. However, we cannot clarify this point in the present study. Interestingly, we found that pretreatment with naltrexone (10 mg/kg, i.v.) significantly improved hepatic function after 6-h LPS administration in rats. Recently, Greeneltch et al. reported that pretreatment with naltrexone (10 mg/kg, i.p.) cannot reduce the liver damage in mice of acute endotoxic shock [29]. The following reasons may explain this discrepancy. (i) In that earlier study, sepsis is induced by 1 mg/kg LPS + 25 mg D -galactosamine (D -gal, i.p.). Concurrently using two challenging agents may lead to more complicated conditions and severe hepatic damage than LPS alone. (ii) Because the doses of naltrexone for rats with endotoxic shock in several reports are higher than 10 mg/kg [20, 33], the dose of naltrexone 10 mg/kg (i.p.) for mice is too low and not sufficient to protect liver from severe damage. These two factors may result in the failure of naltrexone in hepatic protection in that endotoxic mice study. Therefore, naltrexone may still show promise in hepatoprotective effect. In conclusion, this is the first in vivo experiment to demonstrate beneficial effect of naltrexone on circulatory failure and hepatic dysfunction induced by LPS. The protective effect was associated with reduction of TNF-a level and superoxide anion production. However, the molecular mechanism of action of naltrexone on sepsis needs to be clarified in further experiments.
Acknowledgements This work was supported by grants from the National Science Council (NSC 91-2320-B-016-040) and from the Ministry of National Defense (DOD92-08), Taiwan and Jenken Bioscience, Inc., USA.
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