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ALPHA-LIPOIC ACID PREVENTS ENDOTOXIC SHOCK AND MULTIPLE ORGAN DYSFUNCTION SYNDROME INDUCED BY ENDOTOXEMIA IN RATS Hsin-Hsueh Shen,* Kwok-Keung Lam,†‡ Pao-Yun Cheng,§ Ching-Wen Kung,|| Shu-Ying Chen,¶ Pei-Chiang Lin,* Ming-Ting Chung,** and Yen-Mei Lee* *Department and Institute of Pharmacology, National Defense Medical Center; and † Department of Pharmacology, Taipei Medical University, Taipei; ‡ Department of Anesthesiology, Catholic Mercy Hospital, Hsinchu; §Department of Physiology and Biophysics, National Defense Medical Center, Taipei; ||Department of Nursing, Tzu Chi College of Technology, Hualien; ¶ Department of Biotechnology, HungKuang University, Taichung; and **Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Chi-Mei Medical Center, Tainan, Taiwan Received 28 Aug 2014; first reviewed completed 15 Sep 2014; accepted in final form 30 Oct 2014 ABSTRACT—Alpha-lipoic acid (ALA), a naturally occurring disulfide derivative of octanoic acid, serves as a strong antioxidant and has been reported to possess anti-inflammatory effects. The aim of the present study is to investigate the preventive and therapeutic effects of ALA on multiple organ dysfunction syndrome (MODS) caused by endotoxemia in rats. Male Wistar rats were intravenously infused with lipopolysaccharide (LPS) (10 mg/kg) to induce endotoxemia. Alpha-lipoic acid 10, 20, or 40 mg/kg was administered intravenously 60 min before (pretreatment) LPS challenge, and ALA 40 mg/kg was administered intravenously 30 min after (posttreatment) LPS challenge. Pretreatment and posttreatment with ALA significantly improved the deleterious hemodynamic changes 8 h after LPS challenge, including hypotension and bradycardia. Alpha-lipoic acid reduced the plasma levels of glutamic pyruvic transaminase, blood urea nitrogen, lactate dehydrogenase, tumor necrosis factor-!, nitric oxide metabolites, and thrombin-antithrombin complex, which increased markedly after LPS challenge. The induction of inducible nitric oxide synthase both in the liver and the lung and vascular superoxide anion production were also significantly suppressed by ALA. Moreover, ALA significantly attenuated LPS-induced caspase3 activation in cardiomyocytes and improved survival rate. In conclusion, ALA effectively attenuated LPS-induced acute inflammatory response and improved MODS. The antioxidant and anti-inflammatory effects of ALA may contribute to these beneficial effects. Alpha-lipoic acid might be considered as a novel therapeutic strategy in the prevention of sepsis-induced MODS and inflammatory vascular diseases. KEYWORDS—Alpha-lipoic acid, endotoxemia, reactive oxygen species, nitric oxide, multiple organ dysfunction syndrome
INTRODUCTION
production of NO. Nitric oxide can further react with superoxide anion (O2.-) to form the peroxynitrite anion (ONOOj), which oxidizes sulfhydryl groups and generates the hydroxyl radical (IOH), resulting in vascular hyporeactivity to vasoconstrictors in vitro (3) and vasopressor-resistant hypotension in vivo (4), leading to MODS and septic shock with high mortality (5, 6). Suppressing overproduction of these mediators by antioxidants may prevent the pathophysiology of septic shock. Antioxidants can react with O2.- and prevent tissue oxidation by processes involving free radical scavenging (7). Thus, exogenous supplementation of antioxidants seems to be the therapeutic strategies in attenuating mortality or preventing organ damage in the LPSinduced endotoxemic animal model (8) and helpful in protecting MODS in patients with septic shock (9). Alpha-lipoic acid (ALA), or 1,2-dithiolane-3-pentanoic acid, is a naturally occurring disulfide derivative of octanoic acid and serves as a cofactor of pyruvate dehydrogenase in mitochondria energy metabolism. Alpha-lipoic acid is readily reduced to dihydrolipoic acid, both of which serve as strong antioxidants through several mechanisms, including scavenging ROS, restoring intracellular glutathione, and chelating metals such as copper, iron, and mercury (10Y12). Moreover, it has been implicated as a modulator of various inflammatory signaling pathways, such as inhibiting nuclear translocation of nuclear factor-kappa B (NF-.B) by attenuating phosphorylation/ degradation of I.B-! (13). This dramatic array of cellular and molecular functions has led to considerable interest for the utilization of ALA not only as a nutritive supplement but also
Sepsis is one of the prominent causes of morbidity and mortality in intensive care units, characterized by systemic inflammatory response syndrome and multiple organ dysfunction syndrome (MODS), despite recent advances in medical therapy (1). Lipopolysaccharide (LPS), also known as endotoxin, derived from the outer membrane of gram-negative bacteria provokes similar pathophysiology mimicking sepsis in experimental animal models. The pathophysiology of LPSinduced endotoxemia is characterized by the excessive release of reactive oxygen species (ROS) and proinflammatory cytokines, including interleukins (IL-1, IL-6, IL-10), tumor necrosis factor-! (TNF-!), and prostaglandin E2, suggesting activation of the inflammatory response, eventually leading to severe shock and MODS, and contributing significantly to the lethality of sepsis (2). Among the proinflammatory cytokines, TNF-! seems to be essential for most endotoxemic effects, which induces inducible nitric oxide synthase (iNOS) expression that catalyzes the Address reprint requests to Yen-Mei Lee, PhD, Department and Institute of Pharmacology, National Defense Medical Center, No. 161, Section 6, Min-Chuan East Rd, Taipei 114, Taiwan. E-mail: [email protected]. Co-correspondence: Ming-Ting Chung, MD, Department of Obstetrics and Gynecology, Chi-Mei Medical Center, No. 901 Zhonghua Rd, Yongkang District, Tainan, Taiwan. E-mail: [email protected]. This work was supported by research grants from the Ministry of National Defense (DOD98-9-03) and Chi-Mei Hospital (CMNDMC10308), Republic of China, Taiwan. DOI: 10.1097/SHK.0000000000000295 Copyright Ó 2015 by the Shock Society
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a therapeutic agent. There is a growing number of publications confirming the therapeutic effects of ALA for many oxidative stressYrelated diseases, including diabetic polyneuropathies, neurodegeneration, and cardiovascular diseases (14, 15). In addition, the beneficial effects of ALA on LPS-induced oxidative stress and inflammation are associated with inhibition of TNF-! production and transcriptional induction of iNOS in vitro (16). Recently, the antioxidant properties of ALA have been reported to afford protective effects on the spleen (17) and improve cardiac dysfunction via activation of the phosphoinositide 3-kinase/protein kinase B (Akt) signaling pathway during endotoxemia (18). However, little is known about the therapeutic effect of ALA in LPS-induced coagulopathy and MODS. The aim of the present study is to investigate the potential preventive and therapeutic effects of ALA on MODS caused by endotoxemia in rats and to explore its possible mechanism. MATERIALS AND METHODS
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CaCl2 2.5, and glucose 11. Isometric force was measured using Grass FT03-type transducers (Grass Instruments, Quincy, Mass) and recorded on a PowerLab Recording and Analysis System (ADInstruments). In the segment, 2 g passive tension was applied, and the preparations were equilibrated for 60 min. The Krebs solution in the organ baths was changed every 15 min two to three times. The presence of functional endothelium was confirmed by a minimum of 80% relaxation with acetylcholine (ACh) (1 2M) when the vessels were precontracted with norepinephrine (NE) (0.1 2M). Concentration-response curves for NE were performed by adding varying concentrations of NE (1 nM Y 10 2M) to the organ bath; concentration-response curves for ACh were carried out by adding NE (1 2M) to obtain maximum contraction, then adding ACh cumulatively from 1 nM to 10 2M to evaluate the endothelium-dependent vasorelaxation.
Measurement of caspase-3 activity in cardiac tissue To clarify whether apoptotic pathway was activated after injection of LPS, caspase-3 activity was measured in the left ventricle. Cardiac caspase-3 activity was determined using colorimetric assay kits (Assay Designs, Ann Arbor, Mich) according to the manufacturer’s instructions. Results are expressed as units per microgram protein.
Plasma TNF-! detection Blood samples (0.5 mL) were collected at 1, 2, and 8 h after injection of saline or LPS for measurement of the TNF-! level in plasma by an enzyme-linked immunoadsorbent assay (mouse TNF-! ELISA Kit; Genzyme Co., Cambridge, Mass).
Animal preparation Male Wistar rats (aged 10 Y 12 weeks, weighing 280 Y 300 g) were purchased from the National Laboratory Animal Breeding and Research Center of Ministry of Science and Technology, Taiwan. Handling of the animals was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85Y23, revised in 1985). This study was approved by the Institutional Animal Care and Use Committee of National Defense Medical Center, Taiwan. All animals were housed at an ambient temperature of 22-C T 1-C and humidity of 55% T 5%. Reagents including LPS (Escherichia coli serotype 0127:B8) and antiY"actin were purchased from Sigma-Aldrich (St. Louis, Mo) unless otherwise specified. Rats were anesthetized by intraperitoneal injections of urethane (0.6 g/kg) and pentobarbital (25 mg/kg). The trachea was cannulated to facilitate respiration. The left femoral artery was cannulated with a polyethylene-50 catheter and connected to a pressure transducer (MLT844; ADInstruments Pty. Ltd., Castle Hill, New South Wales, Australia) for the measurement of mean blood pressure (MBP) and heart rate (HR), which were displayed on a polygraph recorder (ML 785; PowerLab, ADInstruments Pty. Ltd.). The left femoral vein was cannulated for the administration of drugs. After the completion of surgery, rats were allowed to stabilize for 30 to 60 min.
Experimental groups Animals were divided into five groups: (I) Sham group: rats were treated with normal saline (4 mL/kg) intravenously (n = 3); (II) ALA 40 group: rats were treated with ALA 40 mg/kg intravenously, not given E. coli LPS (n = 3); (III) LPS group: rats were treated with LPS 10 mg/kg (i.v. infusion for 10 min) (19) (n = 25); (IV) ALA + LPS groups: rats were injected with ALA (10, 20, or 40 mg/kg) intravenously 60 min before LPS administration (pretreatment) (n = 12 in each group); (V) LPS + ALA 40 group: rats were injected with ALA (40 mg/kg) intravenously 30 min after LPS administration (posttreatment) (n = 12). At 0, 1, 2, 4, 6, and 8 h after LPS administration, we examined the changes in hemodynamics (MBP and HR), and 0.5 mL of blood was drawn to measure the levels of TNF-!, NO metabolites, thrombin-antithrombin (TAT) complex, and organ function markers, such as glutamic pyruvic transaminase (GPT) for hepatic function, blood urea nitrogen (BUN) for renal function, and lactate dehydrogenase (LDH) for cell toxicity. All blood drawn was immediately replaced by an injection of an equal volume of saline (i.v.) to maintain the blood volume of the animals. Blood samples were centrifuged for 5 min at 12,000g. Plasma samples were stored at j80-C until analysis. At the end of the experiment (8 h after LPS initiation), animals were sacrificed to obtain organs immediately. Thoracic aortas were dissected out for the measurement of vascular reactivity and superoxide anion formation immediately. Lungs and livers were obtained for iNOS protein expression assays and hearts were isolated for the measurement of caspase-3 activity, which were collected and immediately frozen in liquid nitrogen and stored at j80-C until processed. The survival rate in each group was also evaluated.
Detection of aortic superoxide anion formation by chemiluminescence Thoracic aorta was cut into rings of 3 to 4 mm in length and incubated in modified Krebs/HEPES solution (37-C) oxygenated with 95% O2/5% CO2 for 30 min. Then the aorta sections were put into a 96-well plate in which every well was filled with 200 2L modified Krebs/HEPES solution and placed in a luminescence measurement system (Hidex, Microplate Luminometer, Finland). It can perform autoinjection of 125 2M lucigenin (final volume of 250 2L) into the vessels for interacting with superoxide anions. Counts were obtained at 10-s intervals at room temperature. After recording was complete, the vessel ring was dried in a 95-C oven for 24 h. These results were expressed as counts per second (CPS) per milligram dry weight of aorta.
Determination of plasma nitrite/nitrate levels Thawed plasma (30 2L) was deproteinated with 100 mL 95% alcohol for 30 min (4-C). Serum samples were then centrifuged for 6 min at 12,000g. The supernatant (6 2L) was injected into a collection chamber containing 5% VCl3. In this strong reducing environment, both nitrate and nitrite are converted to NO. A constant stream of helium gas was used to carry the output into an NO analyzer (Sievers 280NOA; Sievers Instruments Inc., Boulder, Colo), where the NO reacts with ozone (O3), resulting in the emission of light. Light emission is proportional to the NO formed. Standard amounts of sodium nitrate (Sigma-Aldrich, St. Louis, Mo) were used for calibration.
Western blot analysis of iNOS protein expression in the liver and the lung Detection of the iNOS protein in the liver and the lung by Western blotting was performed as described previously (20). The primary antibody probes in this experiment were mouse monoclonal anti-iNOS (1:1,000; BD Biosciences, USA) and mouse antiY"-actin (1:2,000 dilution; Sigma-Aldrich). The density of the individual bands was quantified by densitometric scanning of the blots using Image-Pro software as described previously.
TAT complex immunoassay Blood was drawn at 4 and 8 h after the injection of vehicle or LPS. All samples were diluted (1:9, vol/vol) with 0.13 M sodium citrate. A commercial ELISA system (Enzygnost TAT; Behringwerke, Marburg, Germany) was used to determine plasma levels of TAT.
Statistical analysis The data are expressed as mean T SEM. Statistical evaluation was performed with one-factor analysis of variance, followed by the Newman-Keuls post hoc comparison test. Log-rank test was used for comparison of the survival distributions among groups of rats. A value of P G 0.05 was accepted to indicate significance.
RESULTS
Vascular reactivity experiments After clearing adhering periadventitial fat, the thoracic aorta was cut into several sections (3 Y 4 mm in length). Each segment was mounted in 20 mL organ bath filled with 95% O2/5% CO2 oxygenated Krebs solution (pH 7.4) at 37-C, consisting of (in mM) NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2,
Survival rate
As shown in Figure 1, the survival rate decreased to 26.9% at 8 h after LPS administration in the LPS group. However,
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Effects of ALA on vascular reactivity in vitro
In the thoracic aorta of the sham group, NE (10j9 Y 10j5 M) caused cumulative contractions, in which the strength is significantly higher than that of the LPS group. Pretreatment with ALA 40 mg/kg significantly ameliorated the hyporeactivity caused by LPS (Fig. 3A). The ACh-induced vasodilatation of precontracted aortic rings in the LPS group was significantly reduced when compared with that in the sham group. Alpha-lipoic acid significantly attenuated the impairment of endothelium-dependent vasodilatation destroyed by LPS (Fig. 3B). Effects of ALA on cardiac caspase-3 activity
FIG. 1. Effects of treatment with ALA on survival rate of rats after LPS ministration. ALA 10 and 20 mg/kg was given 60 min before LPS 10 mg/kg injection; ALA 40 mg/kg was given 60 min before or 30 min after LPS injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
pretreatment with ALA 20 and ALA 40 (but not 10 mg/kg) and posttreatment with ALA 40 mg/kg significantly increased the survival rate when compared with that in the LPS group. Effects of ALA on hemodynamic changes in endotoxemic rats
The baseline values for MBP and HR did not show a significant difference among groups. Mean blood pressure and HR of the sham and ALA 40 group did not significantly change during the experimental period (ALA 40 group data not shown). After administration of LPS, MBP markedly dropped at 1 h and then gradually returned between 1 and 2 h in each group. However, the MBP of the LPS group was falling increasingly between 2 and 8 h after LPS administration and was significantly lower than the sham group at 8 h. Pretreatment and posttreatment with ALA 40 mg/kg significantly ameliorated the hypotension caused by LPS at 8 h (Fig. 2A). As shown in Figure 2B, after LPS injection, HR progressively increased and reached its peak at 4 h and then decreased by degrees until the end of the experiment. Pretreatment and posttreatment of ALA 40 mg/kg showed profound elevation of HR at 8 h, preventing LPS-induced bradycardia.
Eight hours after administration of LPS, the caspase-3 activity of the left ventricle of rats in the LPS group was significantly higher than that of the sham group (sham, 326.3 T 17.2 units/2g protein; LPS, 546.5 T 16.0 units/2g protein) (P G 0.05) (Fig. 3C). Pretreatment and posttreatment with ALA 40 mg/kg significantly reduced the elevation of caspase-3 activity caused by LPS (ALA 40 + LPS, 485.1 T 12.5 units/2g protein; LPS + ALA 40, 512.3 T 18.3 units/2g protein) (P G 0.05), but it remained significantly higher than that of the sham group (P G 0.05). The level of caspase-3 activity in the ALA group (345.4 T 20.6 units/2g protein) did not significantly differ from that of the sham group. These results indicate that pretreatment and posttreatment with a high dose of ALA (40 mg/kg) can attenuate myocardial apoptosis induced by endotoxemia. Effects of ALA on organ dysfunction and cell toxicity
To elucidate the protective effects of ALA on LPS-induced organ dysfunction, we measured the plasma levels of GPT, BUN, and LDH. Injection of LPS significantly increased plasma levels of GPT, BUN, and LDH than those of the sham group at 8 h. Pretreatment and posttreatment of ALA 40 mg/kg significantly attenuated the elevation of GPT, BUN, and LDH levels caused by LPS, which were still significantly higher than that of the sham group (Fig. 4, A Y C). Effects of ALA on plasma levels of TNF-! and nitrite/nitrate
As shown in Figure 5A, the plasma level of TNF-! significantly increased 1 and 2 h after LPS injection and dramatically decreased at 8 h. Pretreatment of rats with ALA 10, 20, and 40 mg/kg significantly attenuated the elevation of plasma TNF-! level caused by LPS.
FIG. 2. Effects of treatment with ALA (10, 20, 40 mg/kg) on mean blood pressure (A) and heart rate (B) in endotoxemic rats induced by injection of LPS 10 mg/kg. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
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FIG. 3. Effects of treatment with ALA on the concentration-response curves of NE (A) and ACh (B) in aortic rings and on caspase-3 activity in the left ventricular myocardium (C) 8 h after LPS 10 mg/kg injection. ALA 40 mg/kg was given 1 h before LPS injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
Lipopolysaccharide challenge significantly induced the elevation of plasma nitrite/nitrate content at 4 and 8 h as compared with that in the sham group (P G 0.05). Pretreatment and posttreatment with ALA 40 mg/kg significantly inhibited the LPS-induced increase of plasma nitrite/nitrate content (Fig. 5B). The effect of ALA on superoxide anion formation in the thoracic aorta
The formation of superoxide anions in the thoracic aorta at 8 h after LPS administration was significantly higher than that in the sham group (sham, 11.0 T 1.5 CPS/mg tissue; LPS, 39.0 T 2.0 CPS/mg tissue). Pretreatment and posttreatment with ALA 40 mg/kg significantly reduced the superoxide anion formation compared with that in the LPS group (ALA 40 + LPS, 22.0 T 1.0 CPS/mg tissue; LPS + ALA 40, 25.0 T 2.3 CPS/mg tissue) (Fig. 5C). Effects of ALA on the expression of iNOS protein in the livers and the lungs
As shown in Figure 6, iNOS protein expression in the livers and the lungs was low in the sham and ALA 40 groups. Eight hours after LPS administration, the levels of hepatic and pulmonary expression of iNOS protein were significantly increased compared with those of the sham groups. Pretreatment and posttreatment with ALA 40 mg/kg significantly reduced the induction of iNOS protein in rats with LPS challenge. However, these levels were significantly higher than those of the sham group (Fig. 6, B and D).
Effects of ALA on plasma TAT complex levels
To evaluate the activation of coagulation in ALA-treated rats after LPS challenge, we assessed the plasma TAT complex levels. Lipopolysaccharide challenge resulted in profound elevation of TAT complex levels at 4 and 8 h, which were significantly higher than those of the sham group (P G 0.05). Pretreatment and posttreatment with ALA 40 mg/kg significantly attenuated the LPS-induced elevation of plasma TAT complex levels. However, the TAT complex levels in the ALA-treated groups were still significantly higher than that of the sham group (Fig. 7). DISCUSSION In the present study, we demonstrated that pretreatment and posttreatment with ALA, a potent antioxidant, possessed the preventive and therapeutic effects on severe endotoxemia and endotoxic shock in rats. Alpha-lipoic acid prevented hypotension and bradycardia and maintained vascular reactivity and endothelial function in late-stage endotoxemia to improve the circulatory function. It also ameliorated hepatic and renal functions and reduced coagulopathy caused by endotoxemia. These data suggest that the antioxidant and anti-inflammatory effects of ALA accounted for reducing proinflammatory cytokines and increasing the survival rate of endotoxemic rats. Antioxidants have been suggested to attenuate mortality or prevent organ damage in LPS-induced endotoxemic animal models (8, 21) and are helpful in protecting against MODS in
FIG. 4. Effects of treatment with ALA on plasma levels of GPT (A), BUN (B), and LDH (C) in endotoxemic rats 8 h after LPS (10 mg/kg) injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
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FIG. 5. Effects of treatment with ALA on plasma TNF-! level (A), plasma nitrite/nitrate content (B), and aortic superoxide anion formation (C) in endotoxemic rats induced by injection of LPS 10 mg/kg. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
patients with septic shock (9). Evidences have shown that N-acetylcysteine, vitamins, and statins exert antioxidant capacity to demonstrate beneficial effects in sepsis (22, 23). Clinical trials of antioxidant supplementation (eicosapentaenoic acid and +-linolenic acid) reduced the incidence of septic shock and played a beneficial role in the management of patients with late sepsis-associated respiratory failure (24). The potent antioxidant ALA has been reported to suppress mild portal endotoxemiaYinduced steatohepatitis in fructose-fed rats (25) and to improve the survival rate of endotoxemic mice (26). Alpha-lipoic acid also attenuates oxidative stress caused by endotoxin in the heart (27) and brain (28). Alpha-lipoic acid possesses the thiol groups that make it capable of scavenging ROS. Alpha-lipoic acid also acts indirectly to maintain the cellular antioxidant state by enhancing the synthesis of hepatic ascorbic acid levels (29) and by induction of glutathione against ischemic damage (30). In our experimental model, pretreatment or posttreatment with ALA effectively attenuated the superoxide anion formation in the thoracic aorta (Fig. 5C), which may contribute to the in vivo protective effects of ALA in endotoxemia. Based on this evidence, ALA may attenuate the oxidative stress in endotoxemia, leading to improvement of organ function and increase in survival rate. Lipopolysaccharide elicited the release of proinflammatory cytokines (e.g., ILs, TNF-!) from neutrophil or phagocytic cells, leading to endothelial cell damage and excessive generation of ROS. During endotoxemia, ROS had been shown to be involved in activating the transcription factor NF-.B, which is a central regulator of immunity, inflammation, and cell survival both in vivo and in vitro (31, 32). It also served as a second messenger in different signaling events, comprising induction of nitric oxide synthase (iNOS) (33). Overproduction of NO by iNOS protein might contribute to hypotension and vascular hyporeactivity in septic shock (8, 34). Nitric oxide reacts with superoxide anion (O2.-) to produce the more potent reactive oxygen metabolite, the peroxynitrite anion (ONOOj), which oxidizes sulfhydryl groups and generates the hydroxyl radical (IOH). Both ONOOj and IOH are capable of cellular lipid peroxidation, protein oxidation, and mitochondria devastation, which cause further impairments to tissues and induce cell death, leading to the progression of circulatory failure and MODS (35). In the present study, we demonstrated that ALA suppressed iNOS expression induced by LPS in the liver and the lung (Fig. 6, A and B), which may contribute to the improvement of MODS. A previous report also showed that ALA inhibited the induction of iNOS gene expression at a posttranscriptional level via destabilization of iNOS mRNA in
an in vitro liver injury model (36). Collectively, these findings have shown clinical importance because treatment with ALA may delay the initiation of the inflammatory diseases.
FIG. 6. Effects of treatment with ALA on iNOS protein expression in the liver (A) and lung (C) in endotoxemic rats 8 h after injection of LPS 10 mg/kg. ALA 40 mg/kg was given 1 h before or 30 min after LPS 10 mg/kg injection. Depicted is a typical display of iNOS protein expression (upper panel), and the statistical analysis of the changes of iNOS protein is in the lower panel. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; # P G 0.05 vs. the LPS group, n = 3 Y 12.
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FIG. 7. Effects of treatment with ALA on TAT complex in endotoxemic rats 4 and 8 h after LPS administration. ALA 40 mg/kg was given 60 min before LPS 10 mg/kg injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
Nuclear transcription of NF-.B and proinflammatory cytokines, including TNF-! and IL-1", play a pivotal role in iNOS expression and NO production (37). Previous studies have demonstrated that ALA attenuated LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway (26). This pathway plays a crucial role in promoting cellular proliferation and acts as a negative regulator of LPS-induced NF-.B activation in vivo (26, 38). Consistent evidence had demonstrated that the mechanism underlying the cytoprotection by ALA may be mediated by inhibition of translocation of NF-.B into the nucleus in vitro (39). Alpha-lipoic acid has been shown to inhibit NF-.B activation in lung tissue in a cecal ligation and punctureYinduced sepsis model and to reduce serum proinflammatory cytokines TNF-! and IL-6 levels, which are associated with its antioxidant capacity (40). In this study, we revealed that treatment with ALA can suppress LPS-induced iNOS protein expression and TNF-! release (Fig. 5A), which may be mediated via suppression of NF-.B signaling pathway activation. Caspase signaling, especially caspase-3, has been extensively involved in cardiac myocyte apoptotic pathways (41). In addition, the caspase-3 activity markedly increased after LPS injection and had been correlated with the apoptotic death of cardiac myocytes (42, 43). In this experiment, activation of apoptotic pathways in the myocardium was evaluated by measuring the caspase-3 enzymatic activity. Apoptosis was shown 8 h after LPS injection (Fig. 3C). Both pretreatment and posttreatment with ALA effectively ameliorated LPS-induced increase in caspase-3 activity. A previous study reported that the blockade of caspase-3 can reduce subsequent proinflammatory cytokine levels (44), implying that the antiapoptotic effect of ALA may contribute to reducing the inflammatory response. Thus, suppression of LPS-induced caspase-3 activation in the myocardium may contribute to the cardioprotection of ALA during sepsis. By contrast, ALA can increase ROS generation, leading to DNA damage and caspase-3 activity upregulation in hepatoma cells and eventually induce apoptosis (45, 46). These effects may be dose dependent and/or in different pathophysiological circumstances to present the opposite results.
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Disseminated intravascular coagulation is characterized by a distinct thromboembolic event or may manifest fibrin deposition in the microvasculature (47). Excessive thrombin generation and subsequent fibrin deposition exacerbate inflammation and ischemia, contributing to the development of MODS, and seem to be the leading cause of lethality in patients with sepsis (48). The proinflammatory cytokines induced by LPS resulted in activation of the coagulation cascade, with concurrent inhibition of anticoagulant mechanisms and fibrinolysis (49). The TAT complex is one of the most sensitive markers of the coagulation cascade activity and reflects the amount of thrombin generated in the circulating bloodstream (50). Here we showed that LPS increased plasma TAT levels in endotoxemic rats (Fig. 7), and pretreatment with ALA significantly reduced the LPS-induced elevation of plasma TAT complex (Fig. 7), indicating that ALA prevented the development of LPS-induced disseminated intravascular coagulation. The antioxidant and anti-inflammatory properties of ALA are likely attributed to its anticoagulant effects, by which ALA can improve microcirculation of organs and prevent organ dysfunction. Although ROS plays a crucial role in sepsis progression (7) and numerous studies reported the beneficial effects of antioxidants in sepsis patients (22, 24), it is still a debate whether antioxidants can be used as therapeutic agents for sepsis. However, treatment of sepsis is currently restricted to mainly supportive and reactive treatments. Therefore, to explore any novel therapy that reduces the incidence and impact of organ failure would have extensive assistance. The results of this study also showed the promising potential of antioxidants to prevent MODS in sepsis. The detailed mechanism of action of ALA needs to be further investigated. In conclusion, ALA effectively attenuated LPS-induced acute inflammatory response and improved multiple organ function in endotoxemia. It might be considered as a novel therapeutic strategy in the prevention of endotoxemia-induced MODS and inflammatory vascular diseases in the future. REFERENCES 1. Bone RC, Grodzin CJ, Balk RA: Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 112(1):235Y243, 1997. 2. Macdonald J, Galley HF, Webster NR: Oxidative stress and gene expression in sepsis. Br J Anaesth 90(2):221Y232, 2003. 3. Macarthur H, Westfall TC, Riley DP, Misko TP, Salvemini D: Inactivation of catecholamines by superoxide gives new insights on the pathogenesis of septic shock. Proc Natl Acad Sci USA 97(17):9753Y9758, 2000. 4. Thiemermann C, Vane J: Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. Eur J Pharmacol 182(3):591Y595, 1990. 5. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA: Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87(4): 1620Y1624, 1990. 6. Deitch EA: Multiple organ failure. Pathophysiology and potential future therapy. Ann Surg 216(2):117Y134, 1992. 7. Victor VM, Rocha M, Esplugues JV, De la Fuente M: Role of free radicals in sepsis: antioxidant therapy. Curr Pharm Des 11(24):3141Y3158, 2005. 8. Wu CC, Chiao CW, Hsiao G, Chen A, Yen MH: Melatonin prevents endotoxininduced circulatory failure in rats. J Pineal Res 30(3):147Y156, 2001. 9. Galley H, Howdle P, Walker B, Webster N: The effects of intravenous antioxidants in patients with septic shock. Free Radic Biol Med 23(5): 768Y774, 1997. 10. Matsugo S, Yan LJ, Han D, Trischler HJ, Packer L: Elucidation of antioxidant activity of alpha-lipoic acid toward hydroxyl radical. Biochem Biophys Res Commun 208(1):161Y167, 1995.
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11. Coombes JS, Powers SK, Hamilton KL, Demirel HA, Shanely RA, Zergeroglu MA, Sen CK, Packer L, Ji LL: Improved cardiac performance after ischemia in aged rats supplemented with vitamin E and alpha-lipoic acid. Am J Physiol Regul Integr Comp Physiol 279(6):R2149YR2155, 2000. 12. Bilska A, Wlodek L: Lipoic acidVthe drug of the future? Pharmacol Rep 57(5):570Y577, 2005. 13. Zhang WJ, Frei B: Alpha-lipoic acid inhibits TNF-alpha-induced NF-kappaB activation and adhesion molecule expression in human aortic endothelial cells. FASEB J 15(13):2423Y2432, 2001. 14. Smith AR, Shenvi SV, Widlansky M, Suh JH, Hagen TM: Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress. Curr Med Chem 11(9):1135Y1146, 2004. 15. Shay KP, Moreau RF, Smith EJ, Smith AR, Hagen TM: Alpha-lipoic acid as a dietary supplement: molecular mechanisms and therapeutic potential. Biochim Biophys Acta 1790(10):1149Y1160, 2009. 16. Kiemer AK, Muller C, Vollmar AM: Inhibition of LPS-induced nitric oxide and TNF-alpha production by alpha-lipoic acid in rat Kupffer cells and in RAW 264.7 murine macrophages. Immunol Cell Biol 80(6):550Y557, 2002. 17. Gor&ca A, Huk-Kolega H, Kleniewska P, Piechota-Polacczyk A, Skibska B: Effects of lipoic acid on spleen oxidative stress after LPS administration. Pharmacological reports : PR 65(1):179Y186, 2013. 18. Jiang S, Zhu W, Li C, Zhang X, Lu T, Ding Z, Cao K, Liu L: Alpha-lipoic acid attenuates LPS-induced cardiac dysfunction through a PI3K/Akt-dependent mechanism. Int Immunopharmacol 16(1):100Y107, 2013. 19. Thiemermann C, Wu CC, Szabo C, Perretti M, Vane JR: Role of tumour necrosis factor in the induction of nitric oxide synthase in a rat model of endotoxin shock. Br J Pharmacol 110(1):177Y182, 1993. 20. Cheng PY, Lee YM, Wu YS, Chang TW, Jin JS, Yen MH: Protective effect of baicalein against endotoxic shock in rats in vivo and in vitro. Biochem Pharmacol 73(6):793Y804, 2007. 21. Hsu DZ, Li YH, Chu PY, Chien SP, Chuang YC, Liu MY: Attenuation of endotoxin-induced oxidative stress and multiple organ injury by 3,4methylenedioxyphenol in rats. Shock 25(3):300Y305, 2006. 22. Steckert AV, de Castro AA, Quevedo J, Dal-Pizzol F: Sepsis in the central nervous system and antioxidant strategies with N-acetylcysteine, vitamins and statins. Curr Neurovasc Res 11(1):83Y90, 2014. 23. Ritter C, Andrades ME, Reinke A, Menna-Barreto S, Moreira JC, Dal-Pizzol F: Treatment with N-acetylcysteine plus deferoxamine protects rats against oxidative stress and improves survival in sepsis. Crit Care Med 32(2):342Y349, 2004. 24. Pontes-Arruda A, Martins LF, de Lima SM, Isola AM, Toledo D, Rezende E, Maia M, Magnan GB: Enteral nutrition with eicosapentaenoic acid, gammalinolenic acid and antioxidants in the early treatment of sepsis: results from a multicenter, prospective, randomized, double-blinded, controlled study: the INTERSEPT study. Crit Care 15(3):R144, 2011. 25. Tian YF, He CT, Chen YT, Hsieh PS: Lipoic acid suppresses portal endotoxemia-induced steatohepatitis and pancreatic inflammation in rats. World J Gastroenterol 19(18):2761Y2771, 2013. 26. Zhang WJ, Wei H, Hagen T, Frei B: Alpha-lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway. Proc Natl Acad Sci USA 104(10):4077Y4082, 2007. 27. Goraca A, Piechota A, Huk-Kolega H: Effect of alpha-lipoic acid on LPSinduced oxidative stress in the heart. J Physiol Pharmacol 60(1):61Y68, 2009. 28. Goraca A, Aslanowicz-Antkowiak K: Prophylaxis with alpha-lipoic acid against lipopolysaccharide-induced brain injury in rats. Arch Immunol Ther Exp (Warsz) 57(2):141Y146, 2009. 29. Lykkesfeldt J, Hagen TM, Vinarsky V, Ames BN: Age-associated decline in ascorbic acid concentration, recycling, and biosynthesis in rat hepatocytesVreversal with (R)-alpha-lipoic acid supplementation. FASEB J 12(12):1183Y1189, 1998. 30. Dulundu E, Ozel Y, Topaloglu U, Sehirli O, Ercan F, Gedik N, Sener G: Alphalipoic acid protects against hepatic ischemia-reperfusion injury in rats. Pharmacology 79(3):163Y170, 2007.
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ALPHA-LIPOIC ACID PREVENTS ENDOTOXIC SHOCK AND MULTIPLE ORGAN DYSFUNCTION SYNDROME INDUCED BY ENDOTOXEMIA IN RATS Hsin-Hsueh Shen,* Kwok-Keung Lam,†‡ Pao-Yun Cheng,§ Ching-Wen Kung,|| Shu-Ying Chen,¶ Pei-Chiang Lin,* Ming-Ting Chung,** and Yen-Mei Lee* *Department and Institute of Pharmacology, National Defense Medical Center; and † Department of Pharmacology, Taipei Medical University, Taipei; ‡ Department of Anesthesiology, Catholic Mercy Hospital, Hsinchu; §Department of Physiology and Biophysics, National Defense Medical Center, Taipei; ||Department of Nursing, Tzu Chi College of Technology, Hualien; ¶ Department of Biotechnology, HungKuang University, Taichung; and **Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Chi-Mei Medical Center, Tainan, Taiwan Received 28 Aug 2014; first reviewed completed 15 Sep 2014; accepted in final form 30 Oct 2014 ABSTRACT—Alpha-lipoic acid (ALA), a naturally occurring disulfide derivative of octanoic acid, serves as a strong antioxidant and has been reported to possess anti-inflammatory effects. The aim of the present study is to investigate the preventive and therapeutic effects of ALA on multiple organ dysfunction syndrome (MODS) caused by endotoxemia in rats. Male Wistar rats were intravenously infused with lipopolysaccharide (LPS) (10 mg/kg) to induce endotoxemia. Alpha-lipoic acid 10, 20, or 40 mg/kg was administered intravenously 60 min before (pretreatment) LPS challenge, and ALA 40 mg/kg was administered intravenously 30 min after (posttreatment) LPS challenge. Pretreatment and posttreatment with ALA significantly improved the deleterious hemodynamic changes 8 h after LPS challenge, including hypotension and bradycardia. Alpha-lipoic acid reduced the plasma levels of glutamic pyruvic transaminase, blood urea nitrogen, lactate dehydrogenase, tumor necrosis factor-!, nitric oxide metabolites, and thrombin-antithrombin complex, which increased markedly after LPS challenge. The induction of inducible nitric oxide synthase both in the liver and the lung and vascular superoxide anion production were also significantly suppressed by ALA. Moreover, ALA significantly attenuated LPS-induced caspase3 activation in cardiomyocytes and improved survival rate. In conclusion, ALA effectively attenuated LPS-induced acute inflammatory response and improved MODS. The antioxidant and anti-inflammatory effects of ALA may contribute to these beneficial effects. Alpha-lipoic acid might be considered as a novel therapeutic strategy in the prevention of sepsis-induced MODS and inflammatory vascular diseases. KEYWORDS—Alpha-lipoic acid, endotoxemia, reactive oxygen species, nitric oxide, multiple organ dysfunction syndrome
INTRODUCTION
production of NO. Nitric oxide can further react with superoxide anion (O2.-) to form the peroxynitrite anion (ONOOj), which oxidizes sulfhydryl groups and generates the hydroxyl radical (IOH), resulting in vascular hyporeactivity to vasoconstrictors in vitro (3) and vasopressor-resistant hypotension in vivo (4), leading to MODS and septic shock with high mortality (5, 6). Suppressing overproduction of these mediators by antioxidants may prevent the pathophysiology of septic shock. Antioxidants can react with O2.- and prevent tissue oxidation by processes involving free radical scavenging (7). Thus, exogenous supplementation of antioxidants seems to be the therapeutic strategies in attenuating mortality or preventing organ damage in the LPSinduced endotoxemic animal model (8) and helpful in protecting MODS in patients with septic shock (9). Alpha-lipoic acid (ALA), or 1,2-dithiolane-3-pentanoic acid, is a naturally occurring disulfide derivative of octanoic acid and serves as a cofactor of pyruvate dehydrogenase in mitochondria energy metabolism. Alpha-lipoic acid is readily reduced to dihydrolipoic acid, both of which serve as strong antioxidants through several mechanisms, including scavenging ROS, restoring intracellular glutathione, and chelating metals such as copper, iron, and mercury (10Y12). Moreover, it has been implicated as a modulator of various inflammatory signaling pathways, such as inhibiting nuclear translocation of nuclear factor-kappa B (NF-.B) by attenuating phosphorylation/ degradation of I.B-! (13). This dramatic array of cellular and molecular functions has led to considerable interest for the utilization of ALA not only as a nutritive supplement but also
Sepsis is one of the prominent causes of morbidity and mortality in intensive care units, characterized by systemic inflammatory response syndrome and multiple organ dysfunction syndrome (MODS), despite recent advances in medical therapy (1). Lipopolysaccharide (LPS), also known as endotoxin, derived from the outer membrane of gram-negative bacteria provokes similar pathophysiology mimicking sepsis in experimental animal models. The pathophysiology of LPSinduced endotoxemia is characterized by the excessive release of reactive oxygen species (ROS) and proinflammatory cytokines, including interleukins (IL-1, IL-6, IL-10), tumor necrosis factor-! (TNF-!), and prostaglandin E2, suggesting activation of the inflammatory response, eventually leading to severe shock and MODS, and contributing significantly to the lethality of sepsis (2). Among the proinflammatory cytokines, TNF-! seems to be essential for most endotoxemic effects, which induces inducible nitric oxide synthase (iNOS) expression that catalyzes the Address reprint requests to Yen-Mei Lee, PhD, Department and Institute of Pharmacology, National Defense Medical Center, No. 161, Section 6, Min-Chuan East Rd, Taipei 114, Taiwan. E-mail: [email protected]. Co-correspondence: Ming-Ting Chung, MD, Department of Obstetrics and Gynecology, Chi-Mei Medical Center, No. 901 Zhonghua Rd, Yongkang District, Tainan, Taiwan. E-mail: [email protected]. This work was supported by research grants from the Ministry of National Defense (DOD98-9-03) and Chi-Mei Hospital (CMNDMC10308), Republic of China, Taiwan. DOI: 10.1097/SHK.0000000000000295 Copyright Ó 2015 by the Shock Society
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a therapeutic agent. There is a growing number of publications confirming the therapeutic effects of ALA for many oxidative stressYrelated diseases, including diabetic polyneuropathies, neurodegeneration, and cardiovascular diseases (14, 15). In addition, the beneficial effects of ALA on LPS-induced oxidative stress and inflammation are associated with inhibition of TNF-! production and transcriptional induction of iNOS in vitro (16). Recently, the antioxidant properties of ALA have been reported to afford protective effects on the spleen (17) and improve cardiac dysfunction via activation of the phosphoinositide 3-kinase/protein kinase B (Akt) signaling pathway during endotoxemia (18). However, little is known about the therapeutic effect of ALA in LPS-induced coagulopathy and MODS. The aim of the present study is to investigate the potential preventive and therapeutic effects of ALA on MODS caused by endotoxemia in rats and to explore its possible mechanism. MATERIALS AND METHODS
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CaCl2 2.5, and glucose 11. Isometric force was measured using Grass FT03-type transducers (Grass Instruments, Quincy, Mass) and recorded on a PowerLab Recording and Analysis System (ADInstruments). In the segment, 2 g passive tension was applied, and the preparations were equilibrated for 60 min. The Krebs solution in the organ baths was changed every 15 min two to three times. The presence of functional endothelium was confirmed by a minimum of 80% relaxation with acetylcholine (ACh) (1 2M) when the vessels were precontracted with norepinephrine (NE) (0.1 2M). Concentration-response curves for NE were performed by adding varying concentrations of NE (1 nM Y 10 2M) to the organ bath; concentration-response curves for ACh were carried out by adding NE (1 2M) to obtain maximum contraction, then adding ACh cumulatively from 1 nM to 10 2M to evaluate the endothelium-dependent vasorelaxation.
Measurement of caspase-3 activity in cardiac tissue To clarify whether apoptotic pathway was activated after injection of LPS, caspase-3 activity was measured in the left ventricle. Cardiac caspase-3 activity was determined using colorimetric assay kits (Assay Designs, Ann Arbor, Mich) according to the manufacturer’s instructions. Results are expressed as units per microgram protein.
Plasma TNF-! detection Blood samples (0.5 mL) were collected at 1, 2, and 8 h after injection of saline or LPS for measurement of the TNF-! level in plasma by an enzyme-linked immunoadsorbent assay (mouse TNF-! ELISA Kit; Genzyme Co., Cambridge, Mass).
Animal preparation Male Wistar rats (aged 10 Y 12 weeks, weighing 280 Y 300 g) were purchased from the National Laboratory Animal Breeding and Research Center of Ministry of Science and Technology, Taiwan. Handling of the animals was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85Y23, revised in 1985). This study was approved by the Institutional Animal Care and Use Committee of National Defense Medical Center, Taiwan. All animals were housed at an ambient temperature of 22-C T 1-C and humidity of 55% T 5%. Reagents including LPS (Escherichia coli serotype 0127:B8) and antiY"actin were purchased from Sigma-Aldrich (St. Louis, Mo) unless otherwise specified. Rats were anesthetized by intraperitoneal injections of urethane (0.6 g/kg) and pentobarbital (25 mg/kg). The trachea was cannulated to facilitate respiration. The left femoral artery was cannulated with a polyethylene-50 catheter and connected to a pressure transducer (MLT844; ADInstruments Pty. Ltd., Castle Hill, New South Wales, Australia) for the measurement of mean blood pressure (MBP) and heart rate (HR), which were displayed on a polygraph recorder (ML 785; PowerLab, ADInstruments Pty. Ltd.). The left femoral vein was cannulated for the administration of drugs. After the completion of surgery, rats were allowed to stabilize for 30 to 60 min.
Experimental groups Animals were divided into five groups: (I) Sham group: rats were treated with normal saline (4 mL/kg) intravenously (n = 3); (II) ALA 40 group: rats were treated with ALA 40 mg/kg intravenously, not given E. coli LPS (n = 3); (III) LPS group: rats were treated with LPS 10 mg/kg (i.v. infusion for 10 min) (19) (n = 25); (IV) ALA + LPS groups: rats were injected with ALA (10, 20, or 40 mg/kg) intravenously 60 min before LPS administration (pretreatment) (n = 12 in each group); (V) LPS + ALA 40 group: rats were injected with ALA (40 mg/kg) intravenously 30 min after LPS administration (posttreatment) (n = 12). At 0, 1, 2, 4, 6, and 8 h after LPS administration, we examined the changes in hemodynamics (MBP and HR), and 0.5 mL of blood was drawn to measure the levels of TNF-!, NO metabolites, thrombin-antithrombin (TAT) complex, and organ function markers, such as glutamic pyruvic transaminase (GPT) for hepatic function, blood urea nitrogen (BUN) for renal function, and lactate dehydrogenase (LDH) for cell toxicity. All blood drawn was immediately replaced by an injection of an equal volume of saline (i.v.) to maintain the blood volume of the animals. Blood samples were centrifuged for 5 min at 12,000g. Plasma samples were stored at j80-C until analysis. At the end of the experiment (8 h after LPS initiation), animals were sacrificed to obtain organs immediately. Thoracic aortas were dissected out for the measurement of vascular reactivity and superoxide anion formation immediately. Lungs and livers were obtained for iNOS protein expression assays and hearts were isolated for the measurement of caspase-3 activity, which were collected and immediately frozen in liquid nitrogen and stored at j80-C until processed. The survival rate in each group was also evaluated.
Detection of aortic superoxide anion formation by chemiluminescence Thoracic aorta was cut into rings of 3 to 4 mm in length and incubated in modified Krebs/HEPES solution (37-C) oxygenated with 95% O2/5% CO2 for 30 min. Then the aorta sections were put into a 96-well plate in which every well was filled with 200 2L modified Krebs/HEPES solution and placed in a luminescence measurement system (Hidex, Microplate Luminometer, Finland). It can perform autoinjection of 125 2M lucigenin (final volume of 250 2L) into the vessels for interacting with superoxide anions. Counts were obtained at 10-s intervals at room temperature. After recording was complete, the vessel ring was dried in a 95-C oven for 24 h. These results were expressed as counts per second (CPS) per milligram dry weight of aorta.
Determination of plasma nitrite/nitrate levels Thawed plasma (30 2L) was deproteinated with 100 mL 95% alcohol for 30 min (4-C). Serum samples were then centrifuged for 6 min at 12,000g. The supernatant (6 2L) was injected into a collection chamber containing 5% VCl3. In this strong reducing environment, both nitrate and nitrite are converted to NO. A constant stream of helium gas was used to carry the output into an NO analyzer (Sievers 280NOA; Sievers Instruments Inc., Boulder, Colo), where the NO reacts with ozone (O3), resulting in the emission of light. Light emission is proportional to the NO formed. Standard amounts of sodium nitrate (Sigma-Aldrich, St. Louis, Mo) were used for calibration.
Western blot analysis of iNOS protein expression in the liver and the lung Detection of the iNOS protein in the liver and the lung by Western blotting was performed as described previously (20). The primary antibody probes in this experiment were mouse monoclonal anti-iNOS (1:1,000; BD Biosciences, USA) and mouse antiY"-actin (1:2,000 dilution; Sigma-Aldrich). The density of the individual bands was quantified by densitometric scanning of the blots using Image-Pro software as described previously.
TAT complex immunoassay Blood was drawn at 4 and 8 h after the injection of vehicle or LPS. All samples were diluted (1:9, vol/vol) with 0.13 M sodium citrate. A commercial ELISA system (Enzygnost TAT; Behringwerke, Marburg, Germany) was used to determine plasma levels of TAT.
Statistical analysis The data are expressed as mean T SEM. Statistical evaluation was performed with one-factor analysis of variance, followed by the Newman-Keuls post hoc comparison test. Log-rank test was used for comparison of the survival distributions among groups of rats. A value of P G 0.05 was accepted to indicate significance.
RESULTS
Vascular reactivity experiments After clearing adhering periadventitial fat, the thoracic aorta was cut into several sections (3 Y 4 mm in length). Each segment was mounted in 20 mL organ bath filled with 95% O2/5% CO2 oxygenated Krebs solution (pH 7.4) at 37-C, consisting of (in mM) NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2,
Survival rate
As shown in Figure 1, the survival rate decreased to 26.9% at 8 h after LPS administration in the LPS group. However,
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Effects of ALA on vascular reactivity in vitro
In the thoracic aorta of the sham group, NE (10j9 Y 10j5 M) caused cumulative contractions, in which the strength is significantly higher than that of the LPS group. Pretreatment with ALA 40 mg/kg significantly ameliorated the hyporeactivity caused by LPS (Fig. 3A). The ACh-induced vasodilatation of precontracted aortic rings in the LPS group was significantly reduced when compared with that in the sham group. Alpha-lipoic acid significantly attenuated the impairment of endothelium-dependent vasodilatation destroyed by LPS (Fig. 3B). Effects of ALA on cardiac caspase-3 activity
FIG. 1. Effects of treatment with ALA on survival rate of rats after LPS ministration. ALA 10 and 20 mg/kg was given 60 min before LPS 10 mg/kg injection; ALA 40 mg/kg was given 60 min before or 30 min after LPS injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
pretreatment with ALA 20 and ALA 40 (but not 10 mg/kg) and posttreatment with ALA 40 mg/kg significantly increased the survival rate when compared with that in the LPS group. Effects of ALA on hemodynamic changes in endotoxemic rats
The baseline values for MBP and HR did not show a significant difference among groups. Mean blood pressure and HR of the sham and ALA 40 group did not significantly change during the experimental period (ALA 40 group data not shown). After administration of LPS, MBP markedly dropped at 1 h and then gradually returned between 1 and 2 h in each group. However, the MBP of the LPS group was falling increasingly between 2 and 8 h after LPS administration and was significantly lower than the sham group at 8 h. Pretreatment and posttreatment with ALA 40 mg/kg significantly ameliorated the hypotension caused by LPS at 8 h (Fig. 2A). As shown in Figure 2B, after LPS injection, HR progressively increased and reached its peak at 4 h and then decreased by degrees until the end of the experiment. Pretreatment and posttreatment of ALA 40 mg/kg showed profound elevation of HR at 8 h, preventing LPS-induced bradycardia.
Eight hours after administration of LPS, the caspase-3 activity of the left ventricle of rats in the LPS group was significantly higher than that of the sham group (sham, 326.3 T 17.2 units/2g protein; LPS, 546.5 T 16.0 units/2g protein) (P G 0.05) (Fig. 3C). Pretreatment and posttreatment with ALA 40 mg/kg significantly reduced the elevation of caspase-3 activity caused by LPS (ALA 40 + LPS, 485.1 T 12.5 units/2g protein; LPS + ALA 40, 512.3 T 18.3 units/2g protein) (P G 0.05), but it remained significantly higher than that of the sham group (P G 0.05). The level of caspase-3 activity in the ALA group (345.4 T 20.6 units/2g protein) did not significantly differ from that of the sham group. These results indicate that pretreatment and posttreatment with a high dose of ALA (40 mg/kg) can attenuate myocardial apoptosis induced by endotoxemia. Effects of ALA on organ dysfunction and cell toxicity
To elucidate the protective effects of ALA on LPS-induced organ dysfunction, we measured the plasma levels of GPT, BUN, and LDH. Injection of LPS significantly increased plasma levels of GPT, BUN, and LDH than those of the sham group at 8 h. Pretreatment and posttreatment of ALA 40 mg/kg significantly attenuated the elevation of GPT, BUN, and LDH levels caused by LPS, which were still significantly higher than that of the sham group (Fig. 4, A Y C). Effects of ALA on plasma levels of TNF-! and nitrite/nitrate
As shown in Figure 5A, the plasma level of TNF-! significantly increased 1 and 2 h after LPS injection and dramatically decreased at 8 h. Pretreatment of rats with ALA 10, 20, and 40 mg/kg significantly attenuated the elevation of plasma TNF-! level caused by LPS.
FIG. 2. Effects of treatment with ALA (10, 20, 40 mg/kg) on mean blood pressure (A) and heart rate (B) in endotoxemic rats induced by injection of LPS 10 mg/kg. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
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FIG. 3. Effects of treatment with ALA on the concentration-response curves of NE (A) and ACh (B) in aortic rings and on caspase-3 activity in the left ventricular myocardium (C) 8 h after LPS 10 mg/kg injection. ALA 40 mg/kg was given 1 h before LPS injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
Lipopolysaccharide challenge significantly induced the elevation of plasma nitrite/nitrate content at 4 and 8 h as compared with that in the sham group (P G 0.05). Pretreatment and posttreatment with ALA 40 mg/kg significantly inhibited the LPS-induced increase of plasma nitrite/nitrate content (Fig. 5B). The effect of ALA on superoxide anion formation in the thoracic aorta
The formation of superoxide anions in the thoracic aorta at 8 h after LPS administration was significantly higher than that in the sham group (sham, 11.0 T 1.5 CPS/mg tissue; LPS, 39.0 T 2.0 CPS/mg tissue). Pretreatment and posttreatment with ALA 40 mg/kg significantly reduced the superoxide anion formation compared with that in the LPS group (ALA 40 + LPS, 22.0 T 1.0 CPS/mg tissue; LPS + ALA 40, 25.0 T 2.3 CPS/mg tissue) (Fig. 5C). Effects of ALA on the expression of iNOS protein in the livers and the lungs
As shown in Figure 6, iNOS protein expression in the livers and the lungs was low in the sham and ALA 40 groups. Eight hours after LPS administration, the levels of hepatic and pulmonary expression of iNOS protein were significantly increased compared with those of the sham groups. Pretreatment and posttreatment with ALA 40 mg/kg significantly reduced the induction of iNOS protein in rats with LPS challenge. However, these levels were significantly higher than those of the sham group (Fig. 6, B and D).
Effects of ALA on plasma TAT complex levels
To evaluate the activation of coagulation in ALA-treated rats after LPS challenge, we assessed the plasma TAT complex levels. Lipopolysaccharide challenge resulted in profound elevation of TAT complex levels at 4 and 8 h, which were significantly higher than those of the sham group (P G 0.05). Pretreatment and posttreatment with ALA 40 mg/kg significantly attenuated the LPS-induced elevation of plasma TAT complex levels. However, the TAT complex levels in the ALA-treated groups were still significantly higher than that of the sham group (Fig. 7). DISCUSSION In the present study, we demonstrated that pretreatment and posttreatment with ALA, a potent antioxidant, possessed the preventive and therapeutic effects on severe endotoxemia and endotoxic shock in rats. Alpha-lipoic acid prevented hypotension and bradycardia and maintained vascular reactivity and endothelial function in late-stage endotoxemia to improve the circulatory function. It also ameliorated hepatic and renal functions and reduced coagulopathy caused by endotoxemia. These data suggest that the antioxidant and anti-inflammatory effects of ALA accounted for reducing proinflammatory cytokines and increasing the survival rate of endotoxemic rats. Antioxidants have been suggested to attenuate mortality or prevent organ damage in LPS-induced endotoxemic animal models (8, 21) and are helpful in protecting against MODS in
FIG. 4. Effects of treatment with ALA on plasma levels of GPT (A), BUN (B), and LDH (C) in endotoxemic rats 8 h after LPS (10 mg/kg) injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
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FIG. 5. Effects of treatment with ALA on plasma TNF-! level (A), plasma nitrite/nitrate content (B), and aortic superoxide anion formation (C) in endotoxemic rats induced by injection of LPS 10 mg/kg. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
patients with septic shock (9). Evidences have shown that N-acetylcysteine, vitamins, and statins exert antioxidant capacity to demonstrate beneficial effects in sepsis (22, 23). Clinical trials of antioxidant supplementation (eicosapentaenoic acid and +-linolenic acid) reduced the incidence of septic shock and played a beneficial role in the management of patients with late sepsis-associated respiratory failure (24). The potent antioxidant ALA has been reported to suppress mild portal endotoxemiaYinduced steatohepatitis in fructose-fed rats (25) and to improve the survival rate of endotoxemic mice (26). Alpha-lipoic acid also attenuates oxidative stress caused by endotoxin in the heart (27) and brain (28). Alpha-lipoic acid possesses the thiol groups that make it capable of scavenging ROS. Alpha-lipoic acid also acts indirectly to maintain the cellular antioxidant state by enhancing the synthesis of hepatic ascorbic acid levels (29) and by induction of glutathione against ischemic damage (30). In our experimental model, pretreatment or posttreatment with ALA effectively attenuated the superoxide anion formation in the thoracic aorta (Fig. 5C), which may contribute to the in vivo protective effects of ALA in endotoxemia. Based on this evidence, ALA may attenuate the oxidative stress in endotoxemia, leading to improvement of organ function and increase in survival rate. Lipopolysaccharide elicited the release of proinflammatory cytokines (e.g., ILs, TNF-!) from neutrophil or phagocytic cells, leading to endothelial cell damage and excessive generation of ROS. During endotoxemia, ROS had been shown to be involved in activating the transcription factor NF-.B, which is a central regulator of immunity, inflammation, and cell survival both in vivo and in vitro (31, 32). It also served as a second messenger in different signaling events, comprising induction of nitric oxide synthase (iNOS) (33). Overproduction of NO by iNOS protein might contribute to hypotension and vascular hyporeactivity in septic shock (8, 34). Nitric oxide reacts with superoxide anion (O2.-) to produce the more potent reactive oxygen metabolite, the peroxynitrite anion (ONOOj), which oxidizes sulfhydryl groups and generates the hydroxyl radical (IOH). Both ONOOj and IOH are capable of cellular lipid peroxidation, protein oxidation, and mitochondria devastation, which cause further impairments to tissues and induce cell death, leading to the progression of circulatory failure and MODS (35). In the present study, we demonstrated that ALA suppressed iNOS expression induced by LPS in the liver and the lung (Fig. 6, A and B), which may contribute to the improvement of MODS. A previous report also showed that ALA inhibited the induction of iNOS gene expression at a posttranscriptional level via destabilization of iNOS mRNA in
an in vitro liver injury model (36). Collectively, these findings have shown clinical importance because treatment with ALA may delay the initiation of the inflammatory diseases.
FIG. 6. Effects of treatment with ALA on iNOS protein expression in the liver (A) and lung (C) in endotoxemic rats 8 h after injection of LPS 10 mg/kg. ALA 40 mg/kg was given 1 h before or 30 min after LPS 10 mg/kg injection. Depicted is a typical display of iNOS protein expression (upper panel), and the statistical analysis of the changes of iNOS protein is in the lower panel. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; # P G 0.05 vs. the LPS group, n = 3 Y 12.
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FIG. 7. Effects of treatment with ALA on TAT complex in endotoxemic rats 4 and 8 h after LPS administration. ALA 40 mg/kg was given 60 min before LPS 10 mg/kg injection. Data are shown as mean T SEM. *P G 0.05 vs. the sham group; #P G 0.05 vs. the LPS group, n = 3 Y 12.
Nuclear transcription of NF-.B and proinflammatory cytokines, including TNF-! and IL-1", play a pivotal role in iNOS expression and NO production (37). Previous studies have demonstrated that ALA attenuated LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway (26). This pathway plays a crucial role in promoting cellular proliferation and acts as a negative regulator of LPS-induced NF-.B activation in vivo (26, 38). Consistent evidence had demonstrated that the mechanism underlying the cytoprotection by ALA may be mediated by inhibition of translocation of NF-.B into the nucleus in vitro (39). Alpha-lipoic acid has been shown to inhibit NF-.B activation in lung tissue in a cecal ligation and punctureYinduced sepsis model and to reduce serum proinflammatory cytokines TNF-! and IL-6 levels, which are associated with its antioxidant capacity (40). In this study, we revealed that treatment with ALA can suppress LPS-induced iNOS protein expression and TNF-! release (Fig. 5A), which may be mediated via suppression of NF-.B signaling pathway activation. Caspase signaling, especially caspase-3, has been extensively involved in cardiac myocyte apoptotic pathways (41). In addition, the caspase-3 activity markedly increased after LPS injection and had been correlated with the apoptotic death of cardiac myocytes (42, 43). In this experiment, activation of apoptotic pathways in the myocardium was evaluated by measuring the caspase-3 enzymatic activity. Apoptosis was shown 8 h after LPS injection (Fig. 3C). Both pretreatment and posttreatment with ALA effectively ameliorated LPS-induced increase in caspase-3 activity. A previous study reported that the blockade of caspase-3 can reduce subsequent proinflammatory cytokine levels (44), implying that the antiapoptotic effect of ALA may contribute to reducing the inflammatory response. Thus, suppression of LPS-induced caspase-3 activation in the myocardium may contribute to the cardioprotection of ALA during sepsis. By contrast, ALA can increase ROS generation, leading to DNA damage and caspase-3 activity upregulation in hepatoma cells and eventually induce apoptosis (45, 46). These effects may be dose dependent and/or in different pathophysiological circumstances to present the opposite results.
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Disseminated intravascular coagulation is characterized by a distinct thromboembolic event or may manifest fibrin deposition in the microvasculature (47). Excessive thrombin generation and subsequent fibrin deposition exacerbate inflammation and ischemia, contributing to the development of MODS, and seem to be the leading cause of lethality in patients with sepsis (48). The proinflammatory cytokines induced by LPS resulted in activation of the coagulation cascade, with concurrent inhibition of anticoagulant mechanisms and fibrinolysis (49). The TAT complex is one of the most sensitive markers of the coagulation cascade activity and reflects the amount of thrombin generated in the circulating bloodstream (50). Here we showed that LPS increased plasma TAT levels in endotoxemic rats (Fig. 7), and pretreatment with ALA significantly reduced the LPS-induced elevation of plasma TAT complex (Fig. 7), indicating that ALA prevented the development of LPS-induced disseminated intravascular coagulation. The antioxidant and anti-inflammatory properties of ALA are likely attributed to its anticoagulant effects, by which ALA can improve microcirculation of organs and prevent organ dysfunction. Although ROS plays a crucial role in sepsis progression (7) and numerous studies reported the beneficial effects of antioxidants in sepsis patients (22, 24), it is still a debate whether antioxidants can be used as therapeutic agents for sepsis. However, treatment of sepsis is currently restricted to mainly supportive and reactive treatments. Therefore, to explore any novel therapy that reduces the incidence and impact of organ failure would have extensive assistance. The results of this study also showed the promising potential of antioxidants to prevent MODS in sepsis. The detailed mechanism of action of ALA needs to be further investigated. In conclusion, ALA effectively attenuated LPS-induced acute inflammatory response and improved multiple organ function in endotoxemia. It might be considered as a novel therapeutic strategy in the prevention of endotoxemia-induced MODS and inflammatory vascular diseases in the future. REFERENCES 1. Bone RC, Grodzin CJ, Balk RA: Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 112(1):235Y243, 1997. 2. Macdonald J, Galley HF, Webster NR: Oxidative stress and gene expression in sepsis. Br J Anaesth 90(2):221Y232, 2003. 3. Macarthur H, Westfall TC, Riley DP, Misko TP, Salvemini D: Inactivation of catecholamines by superoxide gives new insights on the pathogenesis of septic shock. Proc Natl Acad Sci USA 97(17):9753Y9758, 2000. 4. Thiemermann C, Vane J: Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. Eur J Pharmacol 182(3):591Y595, 1990. 5. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA: Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87(4): 1620Y1624, 1990. 6. Deitch EA: Multiple organ failure. Pathophysiology and potential future therapy. Ann Surg 216(2):117Y134, 1992. 7. Victor VM, Rocha M, Esplugues JV, De la Fuente M: Role of free radicals in sepsis: antioxidant therapy. Curr Pharm Des 11(24):3141Y3158, 2005. 8. Wu CC, Chiao CW, Hsiao G, Chen A, Yen MH: Melatonin prevents endotoxininduced circulatory failure in rats. J Pineal Res 30(3):147Y156, 2001. 9. Galley H, Howdle P, Walker B, Webster N: The effects of intravenous antioxidants in patients with septic shock. Free Radic Biol Med 23(5): 768Y774, 1997. 10. Matsugo S, Yan LJ, Han D, Trischler HJ, Packer L: Elucidation of antioxidant activity of alpha-lipoic acid toward hydroxyl radical. Biochem Biophys Res Commun 208(1):161Y167, 1995.
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