j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e9
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Protective effect of astaxanthin against multiple organ injury in a rat model of sepsis Liping Zhou, MM,a Min Gao, MD, PhD,b Zhiming Xiao, MD, PhD,c Juan Zhang, MD, PhD,a Xiangmin Li, MD, PhD,a and Aimin Wang, MD, PhDa,* a
Department of Emergency Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China b Department of Critical Care Medicine, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China c Department of Gastroenterology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China
article info
abstract
Article history:
Background: Astaxanthin, a xanthophyll carotenoid, holds exceptional promise as an
Received 27 November 2014
antioxidant, anti-inflammatory, and anticancer agent. No evidence has been published
Received in revised form
whether it has protective effects on sepsis. The study aimed to investigate the potential
10 February 2015
effects of astaxanthin on sepsis and multiple organ dysfunctions.
Accepted 12 February 2015
Materials and methods: Sepsis was induced by cecal ligation and puncture (CLP) in Spraguee
Available online xxx
Dawley rats. Animals subjected to CLP and sham-operated control rats were given vehicle or astaxanthin 100 mg/kg/d by oral gavage for 7 d before the operation. The rats were killed
Keywords:
at the indicated time points, and the specimen was collected. Cytokines and multiorgan
Astaxanthin
injury-associated enzymatic and oxidative stress indicators were investigated. Multiorgan
Sepsis
tissues were assessed histologically, the peritoneal bacterial load and the 72-h survival was
Cecal ligation and puncture
observed too.
Multiple organ dysfunction
Results: Sepsis resulted in a significant increase in serum tumor necrosis factor-a, inter-
syndrome
leukin-1b, and interleukin-6 levels showing systemic inflammatory response; it also caused a remarkable decrease in the superoxide dismutase activity and a significant increase in the malondialdehyde content showing oxidative damage; sepsis caused a great increase in organ injury-associated indicators, including blood urea nitrogen, creatinine, lactate dehydrogenase, creatine kinase isoenzyme-MB isotype, alanine aminotransferase, and aspartate aminotransferase, which was confirmed by histologic examination. And there was a dramatical increase of colony-forming units in the peritoneal cavity in septic rats. Astaxanthin reversed these inflammatory and oxidant response, alleviated the organ injury, reduced the peritoneal bacterial load, and improved the survival of septic rats induced by CLP. Conclusions: Astaxanthin exerts impressively protective effects on CLP-induced multiple organ injury. It might be used as a potential treatment for clinical sepsis. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Emergency Medicine, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, People’s Republic of China. Tel./fax: þ86 731 84327089. E-mail address: [email protected] (A. Wang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.02.026
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1.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e9
Introduction
Sepsis is a devastating and complex syndrome with a high mortality rate and limited therapeutic options. Despite recent advances in surgical techniques and critical care medicine, overall case mortality from septic events is still high, ranging between 30% and 50% [1]. Septic causes are responsible for 200,000 deaths per year in the United States [2], making it a leading cause of death in hospitals of the 21st century. In words, the burden of morbidity, mortality, reduced quality of life, and excessive cost of sepsis on the healthcare system ($14e16 billion/year) [3] are obvious indicators of how much of an unmet medical challenge this condition truly represents [4]. Because of increase in the aging population, immunosuppressive therapies and invasive procedures, the incidence and mortality of sepsis are likely to increase despite the recent medical advances. Thus, new therapeutic perspectives are highly warranted. Sepsis is a complex clinical syndrome that is caused by a harmful host response to infection. Over the past decades, our collective knowledge regarding the pathophysiology of sepsis has grown exponentially. During the development of sepsis, bacterial components, such as lipopolysaccharide (LPS), may activate an inflammatory cascade, which results in the release of inflammatory mediators, including tumor necrosis factor-a (TNF-a), interleukin-1b (IL)-1b, IL-6, and so on [5]. The overproduction of inflammatory mediators induces endothelial and epithelial injury, vascular leakage, edema, and vasodilatation, subsequently causing the development of multiple organ dysfunction syndrome (MODS). Marked oxidative stress as a result of the inflammatory responses inherent with sepsis also initiates changes in mitochondrial function, which may result in organ damage and MODS [6]. Thus, the development of new drugs with an effective anti-inflammatory and antioxidant profile to reduce the incidence and mortality associated with this devastating condition would be valuable. Astaxanthin is a xanthophyll carotenoid, which is found in various microorganisms and marine animals [7]. It plays biological roles and possesses a number of desired features for food applications, such as natural origin, nil toxicity, high versatility, and both hydro and liposolubility [8,9]. The United States Food and Drug Administration has approved the use of astaxanthin as a food colorant in animal and fish feed [10]. The European Commission considers natural astaxanthin as a food dye [11]. In addition to its pigmentation function, astaxanthin has shown a variety of physiologic and pharmacologic important properties such as antioxidant, anti-inflammatory, and anticancer activity [12,13]. It has been reported that astaxanthin is effective for the prevention or treatment of diabetes [14], cardiovascular diseases [15], and neurodegenerative disorders [16], and also stimulates immunization [17]. Moreover, research has so far reported no significant side effects of astaxanthin consumption in animals and humans. Previous studies have shown that astaxanthin can decrease inflammation by inhibiting reactive oxygen species (ROS)-induced nuclear factor kappa-B (NF-kB) activation [18], it also has therapeutic properties protecting U937 cells from LPS-induced inflammatory and oxidative stress [19]. Astaxanthin inhibited LPS-stimulated IL-6 messenger RNA and protein in BV-2
microglial cells by suppressing extracellular signal-regulated kinase (ERK-), mitogen- and stress-activated protein kinase (MSK-), and NF-kBemediated signals [20]. No evidence has been published, however, whether it has protective effects on sepsis mortality and associated organ dysfunction. Therefore, the present study was designed to investigate the potential effect of astaxanthin on sepsis and sepsis-induced MODS.
2.
Materials and methods
2.1.
Animal preparation
All experimental protocols used for animals were approved by the Animal Care and Use Committee of Xiangya School of Medicine, Central South University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male 8-wk-old specific pathogen-free SpragueeDawley rats, each weighing 220e250 g, were purchased from the Laboratory Animals Center of Central South University (Changsha, China). They were acclimated in a humidified room and maintained on a standard pellet diet before the experiment. The temperature in both the feeding room and the operation room was maintained at about 25 C.
2.2.
Experimental design
2.2.1.
Animal groups
Rats were randomly divided into three groups as follows: rats undergoing sham cecal ligation and puncture (CLP) operation (sham group); rats undergoing CLP and treated with vehicle (CLP þ V group), and rats undergoing CLP and treated with astaxanthin (CLP þ Asta group). Astaxanthin was purchased from SigmaeAldrich, St. Louis, MO. It was diluted in olive oil (100 mg/mL) immediately before use. Either astaxanthin (100 mg/kg) or an equal volume of olive oil was administered by oral gavage for 7 d before the operation. Rats in the sham group received olive oil in a volume equivalent to that used to dissolve astaxanthin. The dose of astaxanthin used in this study was based on previous experiments [21].
2.2.2.
Sepsis model: CLP
Animals were anesthetized with ketamine (60 mg/kg, intramuscular) plus xylazine (10 mg/kg, intramuscular). And then a 2-cm midline abdominal incision was performed. The cecum was then exposed, ligated just distal to the ileocecal valve to avoid intestinal obstruction, punctured twice with an 18-gauge needle, and returned to the abdominal cavity. The incision was then closed in layers. In sham group rats, the cecum was exposed and the bowel was massaged as described previously, but it was not ligated or punctured. The rats were resuscitated with 30 mL/kg body weight normal saline subcutaneously immediately after surgery. The animals were then returned to their cages.
2.2.3.
Specimen collection
Under anesthesia, blood samples were collected from the external carotid vein at 1, 3, 6, 12, 24, and 48 h after CLP. The
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e9
serum was separated by centrifugation at 3000g for 15 min at 4 C and then stored at 80 C before cytokines and multiorgan injury-associated enzymatic indicators analysis. Twenty-four hours after CLP, rats were anesthetized and peritoneal fluid was collected. And tissue samples of the heart, kidney, liver, and lung were removed for indicators of oxidative stress determination and histologic analysis.
2.3. Measurement of systemic cytokines by enzyme-linked immunosorbent assay The concentrations of TNF-a, IL-1b, and IL-6 were determined using enzyme-linked immunosorbent assay kits (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. All samples were measured in triplicate.
2.4. Measurement of multiorgan injury-associated enzymatic indicators The serum levels of multiorgan injury-associated enzymatic indicators, including blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase isoenzyme-MB isotype (CK-MB), were evaluated by the Olympus AU5400 Automatic Biochemical Analyzer (Olympus, Tokyo, Japan) using commercially available clinical assay kits.
2.5.
Measurement of oxidative stress in multiple organs
2.5.1.
Measurement of malondialdehyde
The multiorgan tissue samples were weighed and homogenized (1:10, wt/vol) in 0.1 M phosphate buffer (pH 7.4) in an ice bath. The homogenate was centrifuged at 3000g for 20 min at 4 C. Subsequently, malondialdehyde (MDA) content in the supernatants was measured using a commercially available MDA assay kit (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instruction. Absorbance was measured at 532 nm with a 96-well plate reader (Bio-Tek Instruments Inc, Winooski, VT), and MDA concentration was determined and expressed as nanomoles per gram tissue.
2.5.2.
Measurement of superoxide dismutase
The superoxide dismutase (SOD) activity was estimated using a commercially available assay kit (Jiancheng Bioengineering Institute) following the manufacturer’s instruction. In brief, epinephrine undergoes autoxidation rapidly at pH 10.0 to produce adrenochrome, a pink-colored product that was detected at 480 nm in kinetic mode using ultraviolet-visible spectrophotometer. The amount of enzyme required to produce 50% inhibition was defined as one unit of enzyme activity. The SOD activity was expressed as units per milligram protein.
2.6.
Histologic analysis
Twenty-four hours after CLP, tissue samples of the heart, kidney, liver, and lung were removed for histologic analysis. Each sample was immersed in 10% neutral-buffered formalin and processed routinely by embedding in paraffin. Sections of tissues were cut at 4e5 mm, mounted on slides, and stained with hematoxylin-eosin. Slides were examined by an
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experienced pathologist blinded to the treatment, and photographs were representatives of five to six rats per group. Semiquantitative analysis about the histopathologic injury of lungs was evaluated as previously described [22]. Briefly, lung injury was graded from 0 (normal) to 4 (severe) in the following categories: neutrophil infiltration, interstitial edema, congestion, hemorrhage, hyaline membrane formation, and necrosis. Semiquantitative analysis about the histopathologic injury of livers was evaluated as previously described [23]. Briefly, liver injury was graded from 0 (normal) to 4 (severe) in four categories as follows: hepatocellular necrosis, hemorrhage, hepatic parenchymal inflammatory infiltrate, and sinusoidal inflammatory infiltrate. Semiquantitative analysis about the histopathologic injury of kidneys was evaluated as previously described [24]. The score reflected the grading of tubular necrosis, loss of brush border, cast formation, and tubular dilatation, which was judged as follows: 0 (none), 1 (10%), 2 (11%e25%), 3 (26%e45%), 4 (46%e75%), and 5 (76%). Semiquantitative analysis about the histopathologic injury of the heart was evaluated as previously described [25]. Briefly, heart injury was graded from 0 (normal) to 3 (severe) in two criterions as follows: inflammatory cell infiltration and degeneration of muscle fibers. The histologic score of the organ was calculated as the sum of the scores given for each criterion. The histologic changes were evaluated in random, nonconsecutive 200 histologic fields (Olympus BX51/Olympus DP71; Olympus).
2.7.
Measurement of blood and peritoneal bacterial load
Twenty-four hours after CLP, rats were anesthetized and peritoneal fluid was collected. After serial dilutions with phosphate-buffered saline, the peritoneal fluid was cultured overnight on blood-agar base plates (Trypticase Soy Agar Deeps; Becton Dickinson, NJ) at 37 C, and colony-forming units (CFUs) were counted.
2.8.
Survival studies
To determine the effect of astaxanthin pretreatment on mortality from CLP-induced sepsis, survival studies were performed. Rats were randomly divided into three experimental groups (n ¼ 10 per group) as mentioned previously. All rats had free access to water and food and were frequently monitored by dedicated research personnel to determine the 72-h survival statistics.
2.9.
Statistical analysis
Data are presented as mean and standard deviation. Multiple comparisons were analyzed for significant differences using the one-way analysis of variance with Tukey post hoc test for multiple comparisons. KaplaneMeier plots were used to illustrate survival between treatment groups, and statistical assessment was performed by the log-rank test. Animals still alive at 72 h after CLP were censored at 72 h. All tests were two-sided, and significance was accepted at P < 0.05. GraphPad Prism version 5.02 (GraphPad Prism Software Inc, San Diego, CA) was used for data analysis and figure preparation.
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Fig. 1 e Effect of astaxanthin on serum levels of TNF-a (A), IL-1b (B), and IL-6 (C). Astaxanthin was administered by oral gavage for 7 d (100 mg/kg/d) before the operation. The results are presented as mean ± standard deviation. Of 5 to 6 animals per group. * or ** compared with the sham group P < 0.05 or P < 0.01; # or ## compared with the CLP D V group P < 0.05 or P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
3.
Results
3.1.
Effect of astaxanthin on systemic cytokine levels
TNF-a, IL-1b, and IL-6 concentrations were very low in shamoperated rats. In contrast, serum TNF-a and IL-1b concentrations in rats undergoing CLP were increased at 1 h and were maximal at 3 h. The increase in TNF-a and IL-1b concentrations at 1, 3, 6, and 12 h after CLP was attenuated by astaxanthin treatment (P < 0.01; Fig. 1A and B). Serum IL-6 concentrations in rats undergoing CLP were increased at 1 h and were maximal at 6 h, the increased serum level of IL-6 at 1, 3, 6, 12, and 24 h after CLP was significantly suppressed by astaxanthin treatment (P < 0.01 or P < 0.05; Fig. 1C).
48 h after CLP. Treatment with astaxanthin markedly attenuated the increase at 3, 6, 12, and 24 h after CLP (Fig. 2C). In the CLP group, the serum level of CK-MB was not significantly higher than that of the sham group at 1 and 3 h after CLP. However, the CK-MB level began to increase at 6 h after CLP and reached a peak of (765.39 77.38 U/L) at 24 h after CLP. Astaxanthin markedly suppressed the increase of CK-MB at 6, 12, 24, and 48 h after CLP (Fig. 2D). The serum level of ALT and AST was significantly increased at 3 h after CLP compared with that after the sham operation. ALT level peaked at (98.37 9.35 U/L) 24 h, and AST level peaked at (225.36 18.85 U/L) 24 h after CLP, and treatment with astaxanthin significantly attenuated this increase (Fig. 2E and F).
3.2. Effect of astaxanthin on multiorgan injury-associated enzymatic indicators
3.3. Effect of astaxanthin on multiorgan injury-associated oxidative stress indicators
The serum level of BUN was significantly increased at 6 h after CLP and reached a peak of (15.25 1.95 mmol/L) at 12 h and sustained at a high level until 48 h after CLP. Astaxanthin markedly suppressed the increase of BUN at 6, 12, 24, and 48 h after CLP (Fig. 2A). The serum level of Cr in the CLP group was significantly increased at 3 h after CLP and was maximal near 12 h (32.16 3.54 mmol/L) and sustained at a high level until 48 h after CLP. Astaxanthin significantly attenuated the increase induced by CLP at 3, 6, 12, and 24 h after CLP (Fig. 2B). The serum level of LDH was significantly higher than that of the sham group at all time points and was maximal near 12 h (1625.48 148.28 U/L) and sustained at a high level until
Twenty-four hours after CLP, rats were sacrificed, and tissue samples of the heart, kidney, liver, and lung were removed for indicators of oxidative stress determination. MDA, an indicator of lipid peroxidation levels, increased in the CLP group compared with the sham group (P < 0.01), whereas the increase was significantly attenuated by astaxanthin treatment (P < 0.01), suggesting that lipid peroxidate level was suppressed by astaxanthin (Fig. 3A). SOD activity was significantly downregulated at 24 h after CLP (P < 0.01), and astaxanthin treatment upregulated SOD activity against the CLP challenge (P < 0.01; Fig. 3B). This suggested that astaxanthin could suppress the oxidative stress induced by CLP via upregulation of SOD activity in rats.
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Fig. 2 e Effect of astaxanthin on serum levels of multiorgan injury-associated enzymatic indicators including BUN (A), Cr (B), LDH (C), CK-MB (D), ALT (E), and AST (F). Astaxanthin was administered by oral gavage for 7 d (100 mg/kg/d) before the operation. The results are presented as mean ± standard deviation. Of 5 to 6 animals per group. ** compared with the sham group P < 0.01; # or ## compared with the CLP D V group P < 0.05 or P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
3.4.
Effect of astaxanthin on multiorgan histology
The histologic features reveal normal cell structure in the heart, kidney, liver, and lung of sham-operated rats (Fig. 4). CLP induced marked histopathologic changes (congestion, inflammatory cell infiltration, necrosis, and degeneration) in tissue sections from septic rats. These pathologic changes were inhibited by treatment with astaxanthin.
3.5.
Effect of astaxanthin on peritoneal bacterial load
The effect of astaxanthin on bacterial clearance was determined by counting CFUs. Twenty-four hours after CLP operation, the intraperitoneal bacterial counts were significantly
increased compared with those in the sham group. Astaxanthin significantly decreased the CFUs in rats’ peritoneal fluid 24 h after CLP (Fig. 5).
3.6.
Effect of astaxanthin on CLP-induced lethality
CLP-induced sepsis showed 50% survival rate on the first 24 h of observation and reached a stable 30% survival rate at 48 h after CLP. Log-rank analysis of the 72-h survival curves demonstrated that astaxanthin at doses of 100 mg/kg pretreatment for 7 d provided a significant level of protection. Astaxanthin treatment group showed 90% survival rate on the first 24 h and reached a plateau at 60 h after CLP with a survival rate of 80% (P ¼ 0.0169; Fig. 6).
Fig. 3 e Effect of astaxanthin on MDA content (A) and SOD (B) activity in heart, kidney, liver, and lung tissues. Astaxanthin was administered by oral gavage for 7 d (100 mg/kg/d) before the operation. The results are presented as mean ± standard deviation. Of 5 to 6 animals per group. * or ** compared with the sham group P < 0.05 or P < 0.01; # or ## compared with the CLP D V group P < 0.05 or P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
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Fig. 4 e Effects of astaxanthin on multiorgan histology. (A) Hematoxylin-eosinestained heart, kidney, liver, and lung sections from septic rats. Representative images were chosen from the different experimental groups (magnification 3200). (B) Semiquantitative analysis of multiorgan injury. Of 5 to 6 animals per group. ** compared with the sham group P < 0.01; ## compared with the CLP D V group P < 0.01. Sham [ sham-operated group; CLP D V [ cecal ligation and puncture with vehicle treatment; CLP D Asta [ cecal ligation and puncture with astaxanthin treatment. (Color version of the figure is available online.)
4.
Discussion
Sepsis is a severe clinical syndrome that results from systemic host response to infection. Despite improvements in the management of septic patients through systemic antibiotics, aggressive surgical intervention, and careful monitoring, septic shock and MODS continue to be the most common cause of death in surgical care units [26]. Because the inflammatory and immune response during the whole stage of sepsis involves a vast array of mediators, thus, natural products that have various components that act on different cascades are likely to be more beneficial for sepsis treatment than drugs targeting a single mediator. Astaxanthin is a natural compound extracted from algae, crustaceans, shellfish, and various plants. It has been widely studied in the past years in different research fields. Studies indicated that astaxanthin has anti-inflammatory, antioxidative, anticancer, and immunomodulatory properties [12e20]. No evidence has been published, however, whether it has protective effects on sepsis.
Here, the present study was aimed to investigate the potential effect of astaxanthin on sepsis and sepsis-induced MODS. In the present study, we found that astaxanthin exerted markedly protective effect against CLP, attenuating multiorgan injury, as confirmed by biochemical assays and histopathologic study, and improved the survival of septic rats. This protection is due primarily to the inhibition of inflammation and oxidative stress, which are two of the most important mechanisms of organ injury of polymicrobial sepsis. As revealed clearly by our findings, pretreatment with astaxanthin significantly inhibited the elevation of serum TNF-a, IL-1b, and IL-6 caused by CLP, upregulated SOD activity, and resulted in decrease in MDA content in the heart, kidney, liver, and lung tissue. We also observed a significant decrease in myeloperoxidase in multiorgan tissue in the astaxanthin-treated CLP rats compared with the vehicle-treated rats, demonstrating the protective capacity of astaxanthin in septic rats. Previous studies have demonstrated that TNF-a, IL-1b, and IL-6 are the most strongly associated cytokines with sepsis
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Fig. 5 e Effects of astaxanthin on peritoneal bacterial clearance. Bacterial count (CFUs) in peritoneal fluid 24 h after CLP. Of 5 to 6 animals per group. **, compared with the sham group P < 0.01; ##, compared with the CLP D V group P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
syndrome. The overproduction of these inflammatory mediators induces endothelial and epithelial injury, vascular leakage, edema, and vasodilatation, subsequently causing the development of MODS and even death. Therefore, interfering with the cytokine overproduction during early sepsis may improve sepsis outcome. Astaxanthin is a potent antioxidant to terminate the induction of inflammation in biological systems. Astaxanthin acts against inflammation. It was reported that astaxanthin suppressed serum nitric oxide, TNF-a, and IL-1b in mice injected with LPS, astaxanthin also inhibited the expression or production of these proinflammatory mediators and cytokines in both LPS-stimulated RAW264.7 cells and primary macrophages [18]. Astaxanthin also protected U937 cells from LPS-induced inflammatory and oxidative stress [19]. Astaxanthin inhibited LPS-stimulated IL-6 messenger RNA and protein in BV-2 microglial cells by suppressing ERK-, MSK-, and NF-kBemediated signals [20]. In the present study,
Fig. 6 e Effect of astaxanthin on CLP-induced lethality. Of 10 animals per group. # compared with the CLP D V group P < 0.05. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
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treatment with astaxanthin significantly inhibited the elevation of TNF-a, IL-1b, and IL-6 levels at different time points caused by CLP; it is consistent with other reports that demonstrated the potent inhibitory effect of astaxanthin [18e20]. The role of oxidative stress in the pathogenesis of sepsis has been widely reported [6,27e29]. Previous studies have shown that sepsis is associated with enhanced generation of ROS, which are highly reactive and react with biological macromolecules, producing lipid peroxides, inactivating proteins, and mutating DNA [6,27e29]. During sepsis, overproduction of ROS is highly toxic to host tissues, and their interactions with various cellular macromolecules can result in severe pathophysiologic consequences such as MODS [6,27e29]. Antioxidants might counteract the toxicity of ROS and that free-radical ablation for the treatment of sepsis could be useful in the clinical setting of sepsis-induced MODS. Astaxanthin is a carotenoid containing an additional carboxyl group on each ring structure at the two extremities. It is a more effective antioxidant than vitamin E, by 100e1000 times [30]. Previous studies have revealed that astaxanthin, with its antioxidative property, is beneficial as a therapeutic agent for various diseases both in vivo and in vitro without any side effects or toxicity [16,18,19,30e33]. In the present study, we examined the SOD activity and MDA content in multiorgan to evaluate the oxidative stress of the septic rats. SOD is the only antioxidant enzyme that can scavenge superoxide and MDA is an indicator of lipid peroxidation levels, which were commonly known as markers of oxidative stress and antioxidant status. We found that astaxanthin treatment upregulated SOD activity and resulted in decrease in MDA content in the heart, kidney, liver, and lung tissue expectedly. As a consequence of an inappropriate response to an infection, sepsis could compromise the function of distinct organ systems, leading to MODS [5]. Organ failure often begins with respiratory failure, followed by intestinal, hepatic, renal, hematologic, and cardiac failure; the exact order may vary because of preexisting disease or the precipitating insult. Mortality is strongly correlated with the number of organ systems failing, as well as age and duration of organ failure. A definite explanation for the pathophysiology of MODS has yet to be elucidated. In the present study, we examined the degree of renal, heart, and liver dysfunction by monitoring serum levels of organ injury-associated enzymatic indicators including BUN, Cr, LDH, CK-MB, ALT, and AST for 48 h after CLP. The levels of BUN, Cr, and LDH peaked 12 h after CLP operation, whereas the peak level of CK-MB, ALT, and AST appeared at 24 h after CLP. This time-dependent CLP-induced enzyme profile may indicate differences in the vulnerabilities of various organs affected by sepsis. These increases were significantly attenuated by astaxanthin treatment, which provided significant protection from acute organ dysfunction. This multiorgan function improvement with astaxanthin treatment was also confirmed by assessing histologic analyses. In kidney, liver, and lung samples, histopathologic changes caused by CLP such as congestion, inflammatory cell infiltration, necrosis, and degeneration were ameliorated by astaxanthin treatment. The CLP model used in our present study constitutes arguably the best surrogate for abdominal polymicrobial
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human sepsis to date. CLP is generally recognized as a reliable and clinically relevant animal model of the human septic condition. It reflects the progress of clinical human sepsis that occurs as a consequence of invasion of the body by gramnegative or gram-positive bacteria, fungi, and, probably, viruses and parasites. In our study, astaxanthin effectively reduced CFUs in the peritoneal cavity in CLP-induced septic rats, and this result enabled us to suggest that astaxanthin could be used as a potential agent against sepsis. In addition to its bactericidal effect, astaxanthin improved survival in septic rats. Because we have used a severe CLP model, 72-h survival studies allowed us to clearly observe the protective effect of astaxanthin. In CLP groups, the survival rate decreased dramatically to 50% at the first 24 h, and to the 48 h, the rate plateaued at 30%. Astaxanthin treatment significantly inhibited the lethality in septic animals, it showed 90% survival rate on the first 24 h and reached a plateau at 60 h after CLP with a survival rate of 80%, which indicates that astaxanthin may be a potent and efficacious agent to treat sepsis.
5.
Conclusions
In conclusion, the present study shows that astaxanthin exerts impressively protective effects on polymicrobial sepsis by decreasing proinflammatory cytokines, suppressing ROS generation, enhancing antioxidant status and bactericidal ability, and ultimately leading to the alleviation of MODS. It is for the first time we found that astaxanthin exerted impressively protective effects on CLP-induced multiple organ injury and improved the survival of sepsis. In conjunction with previous studies on astaxanthin in different fields, we believe that astaxanthin may be a potential therapeutic candidate for sepsis. Although we confirmed the beneficial effect of astaxanthin on sepsis, the precise molecular mechanisms and signaling pathway still need to be fully studied. And pretreatment with astaxanthin for 7 d before the CLP procedure in the present study does not represent medical practice. The use of astaxanthin after CLP might have different results. Therefore, randomized studies with larger numbers of animals and different astaxanthin dosages administrated after CLP are needed to better clarify the role of astaxanthin in sepsis. Also, there is a significant difference in homology between rodent inflammation and injury response and human inflammation. Clinical trials are necessary to fully realize the potential use of astaxanthin in sepsis.
Acknowledgment This study was financially supported by grants from National Natural Science Foundation of China (81201487) and the Science and Technology Planning Project of Hunan Province (2012F J3142). Authors’ contributions: A.W. and L.Z. contributed to the conception and design. M.G. and Z.X. conducted the animal experiment. J.Z. and X.L. conducted the molecular biological
detection. L.Z. and M.G. analyzed the data and drafted the article. All authors reviewed the article and approved of it. This study was performed at the Central laboratory of Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China.
Disclosure The authors have not disclosed any potential conflicts of interest. And the work described here has not been published previously, and not under consideration for publication elsewhere, in whole or in part.
references
[1] Iwashyna TJ, Netzer G, Langa KM, et al. Spurious inferences about long-term outcomes: the case of severe sepsis and geriatric conditions. Am J Respir Crit Care Med 2012;185:835. [2] Wang HE, Shapiro NI, Angus DC, et al. National estimates of severe sepsis in United States emergency departments. Crit Care Med 2007;35:1928. [3] Hall MJ, Williams SN, DeFrances CJ, et al. Inpatient care for septicemia or sepsis: a challenge for patients and hospitals. NCHS Data Brief; 2011:1. [4] Heyland DK, Hopman W, Coo H, et al. Long-term healthrelated quality of life in survivors of sepsis. Short form 36: a valid and reliable measure of health-related quality of life. Crit Care Med 2000;28:3599. [5] Matsuda N, Hattori Y. Systemic inflammatory response syndrome (SIRS): molecular pathophysiology and gene therapy. J Pharmacol Sci 2006;101:189. [6] Galley HF. Oxidative stress and mitochondrial dysfunction in sepsis. Br J Anaesth 2011;107:57. [7] Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea FM. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006;46:185. [8] Delgado-Vargas F, Paredes-Lo’pez O. Natural colorant for food and nutraceutical uses. Boca Raton: CRC Press; 2003. p. 137. [9] McCoy M. Astaxanthin market a hard one to crack. Chem Eng News 1999;77:15. [10] Roche F. Astaxanthin: human food safety summary. In: Astaxanthin As a Pigmenter in Salmon Feed, Color Additive Petition 7C02 1 1. Basel, Switzerland: United States Food and Drug Administration; Hoffman-La Roche Ltd.; 1987. p. 43. [11] Guerin M, Huntley ME, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol 2003;21:210. [12] Mortensen A, Skibsted LH, Truscott TG. The interaction of dietary carotenoids with radical species. Arch Biochem Biophys 2001;385:13. [13] Ambati RR, Phang SM, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applicationsea review. Mar Drugs 2014;12:128. [14] Sila A, Ghlissi Z, Kamoun Z, et al. Astaxanthin from shrimp by-products ameliorates nephropathy in diabetic rats. Eur J Nutr 2015;54:301. [15] Fassett RG, Coombes JS. Astaxanthin in cardiovascular health and disease. Molecules 2012;17:2030. [16] Liu X, Osawa T. Astaxanthin protects neuronal cells against oxidative damage and is a potent candidate for brain food. Forum Nutr 2009;61:129.
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[17] Chew BP, Mathison BD, Hayek MG, et al. Dietary astaxanthin enhances immune response in dogs. Vet Immunol Immunopathol 2011;140:199. [18] Lee SJ, Bai SK, Lee KS, et al. Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation. Mol Cells 2003;16:97. [19] Franceschelli S, Pesce M, Ferrone A, et al. Astaxanthin treatment confers protection against oxidative stress in U937 cells stimulated with lipopolysaccharide reducing O2production. PLoS One 2014;9:e88359. [20] Kim YH, Koh HK, Kim DS. Down-regulation of IL-6 production by astaxanthin via ERK-, MSK-, and NF-kappaBmediated signals in activated microglia. Int Immunopharmacol 2010;10:1560. [21] Ohno M, Darwish WS, Ikenaka Y, et al. Astaxanthin can alter CYP1A-dependent activities via two different mechanisms: induction of protein expression and inhibition of NADPH P450 reductase dependent electron transfer. Food Chem Toxicol 2011;49:1285. [22] Su X, Wang L, Song Y, et al. Inhibition of inflammatory responses by ambroxol, a mucolytic agent, in a murine model of acute lung injury induced by lipopolysaccharide. Intensive Care Med 2004;30:133. [23] Coimbra R, Porcides RD, Melbostad H, et al. Nonspecific phosphodiesterase inhibition attenuates liver injury in acute endotoxemia. Surg Infect 2005;6:73. [24] Chen YT, Sun CK, Lin YC, et al. Adipose-derived mesenchymal stem cell protects kidneys against ischemiareperfusion injury through suppressing oxidative stress and inflammatory reaction. J Transl Med 2011;9:51.
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[25] Sener G, Toklu H, Ercan F, et al. Protective effect of betaglucan against oxidative organ injury in a rat model of sepsis. Int Immunopharmacol 2005;5:1387. [26] Jones AE, Brown MD, Trzeciak S, et al. The effect of a quantitative resuscitation strategy on mortality in patients with sepsis: a meta-analysis. Crit Care Med 2008; 36:2734. [27] Koksal GM, Sayilgan C, Aydin S, et al. Correlation of plasma and tissue oxidative stresses in intra-abdominal sepsis. J Surg Res 2004;122:180. [28] Sakaguchi S, Furusawa S. Oxidative stress and septic shock: metabolic aspects of oxygen-derived free radicals generated in the liver during endotoxemia. FEMS Immunol Med Microbiol 2006;47:167. [29] Sheu SS, Nauduri D, Anders MW. Targeting antioxidants to mitochondria: a new therapeutic direction. Biochim Biophys Acta 2006;1762:256. [30] Ye Q, Huang B, Zhang X, et al. Astaxanthin protects against MPP(þ)-induced oxidative stress in PC12 cells via the HO-1/NOX2 axis. BMC Neurosci 2012;13:156. [31] Dong LY, Jin J, Lu G, et al. Astaxanthin attenuates the apoptosis of retinal ganglion cells in db/db mice by inhibition of oxidative stress. Mar Drugs 2013;11:960. [32] Augusti PR, Conterato GM, Somacal S, et al. Astaxanthin reduces oxidative stress, but not aortic damage in atherosclerotic rabbits. J Cardiovasc Pharmacol Ther 2009; 14:314. [33] Tripathi DN, Jena GB. Astaxanthin intervention ameliorates cyclophosphamide-induced oxidative stress, DNA damage and early hepatocarcinogenesis in rat: role of Nrf2, p53, p38 and phase-II enzymes. Mutat Res 2010;696:69.
Available online at www.sciencedirect.com
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Protective effect of astaxanthin against multiple organ injury in a rat model of sepsis Liping Zhou, MM,a Min Gao, MD, PhD,b Zhiming Xiao, MD, PhD,c Juan Zhang, MD, PhD,a Xiangmin Li, MD, PhD,a and Aimin Wang, MD, PhDa,* a
Department of Emergency Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China b Department of Critical Care Medicine, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China c Department of Gastroenterology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China
article info
abstract
Article history:
Background: Astaxanthin, a xanthophyll carotenoid, holds exceptional promise as an
Received 27 November 2014
antioxidant, anti-inflammatory, and anticancer agent. No evidence has been published
Received in revised form
whether it has protective effects on sepsis. The study aimed to investigate the potential
10 February 2015
effects of astaxanthin on sepsis and multiple organ dysfunctions.
Accepted 12 February 2015
Materials and methods: Sepsis was induced by cecal ligation and puncture (CLP) in Spraguee
Available online xxx
Dawley rats. Animals subjected to CLP and sham-operated control rats were given vehicle or astaxanthin 100 mg/kg/d by oral gavage for 7 d before the operation. The rats were killed
Keywords:
at the indicated time points, and the specimen was collected. Cytokines and multiorgan
Astaxanthin
injury-associated enzymatic and oxidative stress indicators were investigated. Multiorgan
Sepsis
tissues were assessed histologically, the peritoneal bacterial load and the 72-h survival was
Cecal ligation and puncture
observed too.
Multiple organ dysfunction
Results: Sepsis resulted in a significant increase in serum tumor necrosis factor-a, inter-
syndrome
leukin-1b, and interleukin-6 levels showing systemic inflammatory response; it also caused a remarkable decrease in the superoxide dismutase activity and a significant increase in the malondialdehyde content showing oxidative damage; sepsis caused a great increase in organ injury-associated indicators, including blood urea nitrogen, creatinine, lactate dehydrogenase, creatine kinase isoenzyme-MB isotype, alanine aminotransferase, and aspartate aminotransferase, which was confirmed by histologic examination. And there was a dramatical increase of colony-forming units in the peritoneal cavity in septic rats. Astaxanthin reversed these inflammatory and oxidant response, alleviated the organ injury, reduced the peritoneal bacterial load, and improved the survival of septic rats induced by CLP. Conclusions: Astaxanthin exerts impressively protective effects on CLP-induced multiple organ injury. It might be used as a potential treatment for clinical sepsis. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Emergency Medicine, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, People’s Republic of China. Tel./fax: þ86 731 84327089. E-mail address: [email protected] (A. Wang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.02.026
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1.
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Introduction
Sepsis is a devastating and complex syndrome with a high mortality rate and limited therapeutic options. Despite recent advances in surgical techniques and critical care medicine, overall case mortality from septic events is still high, ranging between 30% and 50% [1]. Septic causes are responsible for 200,000 deaths per year in the United States [2], making it a leading cause of death in hospitals of the 21st century. In words, the burden of morbidity, mortality, reduced quality of life, and excessive cost of sepsis on the healthcare system ($14e16 billion/year) [3] are obvious indicators of how much of an unmet medical challenge this condition truly represents [4]. Because of increase in the aging population, immunosuppressive therapies and invasive procedures, the incidence and mortality of sepsis are likely to increase despite the recent medical advances. Thus, new therapeutic perspectives are highly warranted. Sepsis is a complex clinical syndrome that is caused by a harmful host response to infection. Over the past decades, our collective knowledge regarding the pathophysiology of sepsis has grown exponentially. During the development of sepsis, bacterial components, such as lipopolysaccharide (LPS), may activate an inflammatory cascade, which results in the release of inflammatory mediators, including tumor necrosis factor-a (TNF-a), interleukin-1b (IL)-1b, IL-6, and so on [5]. The overproduction of inflammatory mediators induces endothelial and epithelial injury, vascular leakage, edema, and vasodilatation, subsequently causing the development of multiple organ dysfunction syndrome (MODS). Marked oxidative stress as a result of the inflammatory responses inherent with sepsis also initiates changes in mitochondrial function, which may result in organ damage and MODS [6]. Thus, the development of new drugs with an effective anti-inflammatory and antioxidant profile to reduce the incidence and mortality associated with this devastating condition would be valuable. Astaxanthin is a xanthophyll carotenoid, which is found in various microorganisms and marine animals [7]. It plays biological roles and possesses a number of desired features for food applications, such as natural origin, nil toxicity, high versatility, and both hydro and liposolubility [8,9]. The United States Food and Drug Administration has approved the use of astaxanthin as a food colorant in animal and fish feed [10]. The European Commission considers natural astaxanthin as a food dye [11]. In addition to its pigmentation function, astaxanthin has shown a variety of physiologic and pharmacologic important properties such as antioxidant, anti-inflammatory, and anticancer activity [12,13]. It has been reported that astaxanthin is effective for the prevention or treatment of diabetes [14], cardiovascular diseases [15], and neurodegenerative disorders [16], and also stimulates immunization [17]. Moreover, research has so far reported no significant side effects of astaxanthin consumption in animals and humans. Previous studies have shown that astaxanthin can decrease inflammation by inhibiting reactive oxygen species (ROS)-induced nuclear factor kappa-B (NF-kB) activation [18], it also has therapeutic properties protecting U937 cells from LPS-induced inflammatory and oxidative stress [19]. Astaxanthin inhibited LPS-stimulated IL-6 messenger RNA and protein in BV-2
microglial cells by suppressing extracellular signal-regulated kinase (ERK-), mitogen- and stress-activated protein kinase (MSK-), and NF-kBemediated signals [20]. No evidence has been published, however, whether it has protective effects on sepsis mortality and associated organ dysfunction. Therefore, the present study was designed to investigate the potential effect of astaxanthin on sepsis and sepsis-induced MODS.
2.
Materials and methods
2.1.
Animal preparation
All experimental protocols used for animals were approved by the Animal Care and Use Committee of Xiangya School of Medicine, Central South University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male 8-wk-old specific pathogen-free SpragueeDawley rats, each weighing 220e250 g, were purchased from the Laboratory Animals Center of Central South University (Changsha, China). They were acclimated in a humidified room and maintained on a standard pellet diet before the experiment. The temperature in both the feeding room and the operation room was maintained at about 25 C.
2.2.
Experimental design
2.2.1.
Animal groups
Rats were randomly divided into three groups as follows: rats undergoing sham cecal ligation and puncture (CLP) operation (sham group); rats undergoing CLP and treated with vehicle (CLP þ V group), and rats undergoing CLP and treated with astaxanthin (CLP þ Asta group). Astaxanthin was purchased from SigmaeAldrich, St. Louis, MO. It was diluted in olive oil (100 mg/mL) immediately before use. Either astaxanthin (100 mg/kg) or an equal volume of olive oil was administered by oral gavage for 7 d before the operation. Rats in the sham group received olive oil in a volume equivalent to that used to dissolve astaxanthin. The dose of astaxanthin used in this study was based on previous experiments [21].
2.2.2.
Sepsis model: CLP
Animals were anesthetized with ketamine (60 mg/kg, intramuscular) plus xylazine (10 mg/kg, intramuscular). And then a 2-cm midline abdominal incision was performed. The cecum was then exposed, ligated just distal to the ileocecal valve to avoid intestinal obstruction, punctured twice with an 18-gauge needle, and returned to the abdominal cavity. The incision was then closed in layers. In sham group rats, the cecum was exposed and the bowel was massaged as described previously, but it was not ligated or punctured. The rats were resuscitated with 30 mL/kg body weight normal saline subcutaneously immediately after surgery. The animals were then returned to their cages.
2.2.3.
Specimen collection
Under anesthesia, blood samples were collected from the external carotid vein at 1, 3, 6, 12, 24, and 48 h after CLP. The
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serum was separated by centrifugation at 3000g for 15 min at 4 C and then stored at 80 C before cytokines and multiorgan injury-associated enzymatic indicators analysis. Twenty-four hours after CLP, rats were anesthetized and peritoneal fluid was collected. And tissue samples of the heart, kidney, liver, and lung were removed for indicators of oxidative stress determination and histologic analysis.
2.3. Measurement of systemic cytokines by enzyme-linked immunosorbent assay The concentrations of TNF-a, IL-1b, and IL-6 were determined using enzyme-linked immunosorbent assay kits (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. All samples were measured in triplicate.
2.4. Measurement of multiorgan injury-associated enzymatic indicators The serum levels of multiorgan injury-associated enzymatic indicators, including blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase isoenzyme-MB isotype (CK-MB), were evaluated by the Olympus AU5400 Automatic Biochemical Analyzer (Olympus, Tokyo, Japan) using commercially available clinical assay kits.
2.5.
Measurement of oxidative stress in multiple organs
2.5.1.
Measurement of malondialdehyde
The multiorgan tissue samples were weighed and homogenized (1:10, wt/vol) in 0.1 M phosphate buffer (pH 7.4) in an ice bath. The homogenate was centrifuged at 3000g for 20 min at 4 C. Subsequently, malondialdehyde (MDA) content in the supernatants was measured using a commercially available MDA assay kit (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instruction. Absorbance was measured at 532 nm with a 96-well plate reader (Bio-Tek Instruments Inc, Winooski, VT), and MDA concentration was determined and expressed as nanomoles per gram tissue.
2.5.2.
Measurement of superoxide dismutase
The superoxide dismutase (SOD) activity was estimated using a commercially available assay kit (Jiancheng Bioengineering Institute) following the manufacturer’s instruction. In brief, epinephrine undergoes autoxidation rapidly at pH 10.0 to produce adrenochrome, a pink-colored product that was detected at 480 nm in kinetic mode using ultraviolet-visible spectrophotometer. The amount of enzyme required to produce 50% inhibition was defined as one unit of enzyme activity. The SOD activity was expressed as units per milligram protein.
2.6.
Histologic analysis
Twenty-four hours after CLP, tissue samples of the heart, kidney, liver, and lung were removed for histologic analysis. Each sample was immersed in 10% neutral-buffered formalin and processed routinely by embedding in paraffin. Sections of tissues were cut at 4e5 mm, mounted on slides, and stained with hematoxylin-eosin. Slides were examined by an
3
experienced pathologist blinded to the treatment, and photographs were representatives of five to six rats per group. Semiquantitative analysis about the histopathologic injury of lungs was evaluated as previously described [22]. Briefly, lung injury was graded from 0 (normal) to 4 (severe) in the following categories: neutrophil infiltration, interstitial edema, congestion, hemorrhage, hyaline membrane formation, and necrosis. Semiquantitative analysis about the histopathologic injury of livers was evaluated as previously described [23]. Briefly, liver injury was graded from 0 (normal) to 4 (severe) in four categories as follows: hepatocellular necrosis, hemorrhage, hepatic parenchymal inflammatory infiltrate, and sinusoidal inflammatory infiltrate. Semiquantitative analysis about the histopathologic injury of kidneys was evaluated as previously described [24]. The score reflected the grading of tubular necrosis, loss of brush border, cast formation, and tubular dilatation, which was judged as follows: 0 (none), 1 (10%), 2 (11%e25%), 3 (26%e45%), 4 (46%e75%), and 5 (76%). Semiquantitative analysis about the histopathologic injury of the heart was evaluated as previously described [25]. Briefly, heart injury was graded from 0 (normal) to 3 (severe) in two criterions as follows: inflammatory cell infiltration and degeneration of muscle fibers. The histologic score of the organ was calculated as the sum of the scores given for each criterion. The histologic changes were evaluated in random, nonconsecutive 200 histologic fields (Olympus BX51/Olympus DP71; Olympus).
2.7.
Measurement of blood and peritoneal bacterial load
Twenty-four hours after CLP, rats were anesthetized and peritoneal fluid was collected. After serial dilutions with phosphate-buffered saline, the peritoneal fluid was cultured overnight on blood-agar base plates (Trypticase Soy Agar Deeps; Becton Dickinson, NJ) at 37 C, and colony-forming units (CFUs) were counted.
2.8.
Survival studies
To determine the effect of astaxanthin pretreatment on mortality from CLP-induced sepsis, survival studies were performed. Rats were randomly divided into three experimental groups (n ¼ 10 per group) as mentioned previously. All rats had free access to water and food and were frequently monitored by dedicated research personnel to determine the 72-h survival statistics.
2.9.
Statistical analysis
Data are presented as mean and standard deviation. Multiple comparisons were analyzed for significant differences using the one-way analysis of variance with Tukey post hoc test for multiple comparisons. KaplaneMeier plots were used to illustrate survival between treatment groups, and statistical assessment was performed by the log-rank test. Animals still alive at 72 h after CLP were censored at 72 h. All tests were two-sided, and significance was accepted at P < 0.05. GraphPad Prism version 5.02 (GraphPad Prism Software Inc, San Diego, CA) was used for data analysis and figure preparation.
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Fig. 1 e Effect of astaxanthin on serum levels of TNF-a (A), IL-1b (B), and IL-6 (C). Astaxanthin was administered by oral gavage for 7 d (100 mg/kg/d) before the operation. The results are presented as mean ± standard deviation. Of 5 to 6 animals per group. * or ** compared with the sham group P < 0.05 or P < 0.01; # or ## compared with the CLP D V group P < 0.05 or P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
3.
Results
3.1.
Effect of astaxanthin on systemic cytokine levels
TNF-a, IL-1b, and IL-6 concentrations were very low in shamoperated rats. In contrast, serum TNF-a and IL-1b concentrations in rats undergoing CLP were increased at 1 h and were maximal at 3 h. The increase in TNF-a and IL-1b concentrations at 1, 3, 6, and 12 h after CLP was attenuated by astaxanthin treatment (P < 0.01; Fig. 1A and B). Serum IL-6 concentrations in rats undergoing CLP were increased at 1 h and were maximal at 6 h, the increased serum level of IL-6 at 1, 3, 6, 12, and 24 h after CLP was significantly suppressed by astaxanthin treatment (P < 0.01 or P < 0.05; Fig. 1C).
48 h after CLP. Treatment with astaxanthin markedly attenuated the increase at 3, 6, 12, and 24 h after CLP (Fig. 2C). In the CLP group, the serum level of CK-MB was not significantly higher than that of the sham group at 1 and 3 h after CLP. However, the CK-MB level began to increase at 6 h after CLP and reached a peak of (765.39 77.38 U/L) at 24 h after CLP. Astaxanthin markedly suppressed the increase of CK-MB at 6, 12, 24, and 48 h after CLP (Fig. 2D). The serum level of ALT and AST was significantly increased at 3 h after CLP compared with that after the sham operation. ALT level peaked at (98.37 9.35 U/L) 24 h, and AST level peaked at (225.36 18.85 U/L) 24 h after CLP, and treatment with astaxanthin significantly attenuated this increase (Fig. 2E and F).
3.2. Effect of astaxanthin on multiorgan injury-associated enzymatic indicators
3.3. Effect of astaxanthin on multiorgan injury-associated oxidative stress indicators
The serum level of BUN was significantly increased at 6 h after CLP and reached a peak of (15.25 1.95 mmol/L) at 12 h and sustained at a high level until 48 h after CLP. Astaxanthin markedly suppressed the increase of BUN at 6, 12, 24, and 48 h after CLP (Fig. 2A). The serum level of Cr in the CLP group was significantly increased at 3 h after CLP and was maximal near 12 h (32.16 3.54 mmol/L) and sustained at a high level until 48 h after CLP. Astaxanthin significantly attenuated the increase induced by CLP at 3, 6, 12, and 24 h after CLP (Fig. 2B). The serum level of LDH was significantly higher than that of the sham group at all time points and was maximal near 12 h (1625.48 148.28 U/L) and sustained at a high level until
Twenty-four hours after CLP, rats were sacrificed, and tissue samples of the heart, kidney, liver, and lung were removed for indicators of oxidative stress determination. MDA, an indicator of lipid peroxidation levels, increased in the CLP group compared with the sham group (P < 0.01), whereas the increase was significantly attenuated by astaxanthin treatment (P < 0.01), suggesting that lipid peroxidate level was suppressed by astaxanthin (Fig. 3A). SOD activity was significantly downregulated at 24 h after CLP (P < 0.01), and astaxanthin treatment upregulated SOD activity against the CLP challenge (P < 0.01; Fig. 3B). This suggested that astaxanthin could suppress the oxidative stress induced by CLP via upregulation of SOD activity in rats.
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Fig. 2 e Effect of astaxanthin on serum levels of multiorgan injury-associated enzymatic indicators including BUN (A), Cr (B), LDH (C), CK-MB (D), ALT (E), and AST (F). Astaxanthin was administered by oral gavage for 7 d (100 mg/kg/d) before the operation. The results are presented as mean ± standard deviation. Of 5 to 6 animals per group. ** compared with the sham group P < 0.01; # or ## compared with the CLP D V group P < 0.05 or P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
3.4.
Effect of astaxanthin on multiorgan histology
The histologic features reveal normal cell structure in the heart, kidney, liver, and lung of sham-operated rats (Fig. 4). CLP induced marked histopathologic changes (congestion, inflammatory cell infiltration, necrosis, and degeneration) in tissue sections from septic rats. These pathologic changes were inhibited by treatment with astaxanthin.
3.5.
Effect of astaxanthin on peritoneal bacterial load
The effect of astaxanthin on bacterial clearance was determined by counting CFUs. Twenty-four hours after CLP operation, the intraperitoneal bacterial counts were significantly
increased compared with those in the sham group. Astaxanthin significantly decreased the CFUs in rats’ peritoneal fluid 24 h after CLP (Fig. 5).
3.6.
Effect of astaxanthin on CLP-induced lethality
CLP-induced sepsis showed 50% survival rate on the first 24 h of observation and reached a stable 30% survival rate at 48 h after CLP. Log-rank analysis of the 72-h survival curves demonstrated that astaxanthin at doses of 100 mg/kg pretreatment for 7 d provided a significant level of protection. Astaxanthin treatment group showed 90% survival rate on the first 24 h and reached a plateau at 60 h after CLP with a survival rate of 80% (P ¼ 0.0169; Fig. 6).
Fig. 3 e Effect of astaxanthin on MDA content (A) and SOD (B) activity in heart, kidney, liver, and lung tissues. Astaxanthin was administered by oral gavage for 7 d (100 mg/kg/d) before the operation. The results are presented as mean ± standard deviation. Of 5 to 6 animals per group. * or ** compared with the sham group P < 0.05 or P < 0.01; # or ## compared with the CLP D V group P < 0.05 or P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
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Fig. 4 e Effects of astaxanthin on multiorgan histology. (A) Hematoxylin-eosinestained heart, kidney, liver, and lung sections from septic rats. Representative images were chosen from the different experimental groups (magnification 3200). (B) Semiquantitative analysis of multiorgan injury. Of 5 to 6 animals per group. ** compared with the sham group P < 0.01; ## compared with the CLP D V group P < 0.01. Sham [ sham-operated group; CLP D V [ cecal ligation and puncture with vehicle treatment; CLP D Asta [ cecal ligation and puncture with astaxanthin treatment. (Color version of the figure is available online.)
4.
Discussion
Sepsis is a severe clinical syndrome that results from systemic host response to infection. Despite improvements in the management of septic patients through systemic antibiotics, aggressive surgical intervention, and careful monitoring, septic shock and MODS continue to be the most common cause of death in surgical care units [26]. Because the inflammatory and immune response during the whole stage of sepsis involves a vast array of mediators, thus, natural products that have various components that act on different cascades are likely to be more beneficial for sepsis treatment than drugs targeting a single mediator. Astaxanthin is a natural compound extracted from algae, crustaceans, shellfish, and various plants. It has been widely studied in the past years in different research fields. Studies indicated that astaxanthin has anti-inflammatory, antioxidative, anticancer, and immunomodulatory properties [12e20]. No evidence has been published, however, whether it has protective effects on sepsis.
Here, the present study was aimed to investigate the potential effect of astaxanthin on sepsis and sepsis-induced MODS. In the present study, we found that astaxanthin exerted markedly protective effect against CLP, attenuating multiorgan injury, as confirmed by biochemical assays and histopathologic study, and improved the survival of septic rats. This protection is due primarily to the inhibition of inflammation and oxidative stress, which are two of the most important mechanisms of organ injury of polymicrobial sepsis. As revealed clearly by our findings, pretreatment with astaxanthin significantly inhibited the elevation of serum TNF-a, IL-1b, and IL-6 caused by CLP, upregulated SOD activity, and resulted in decrease in MDA content in the heart, kidney, liver, and lung tissue. We also observed a significant decrease in myeloperoxidase in multiorgan tissue in the astaxanthin-treated CLP rats compared with the vehicle-treated rats, demonstrating the protective capacity of astaxanthin in septic rats. Previous studies have demonstrated that TNF-a, IL-1b, and IL-6 are the most strongly associated cytokines with sepsis
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Fig. 5 e Effects of astaxanthin on peritoneal bacterial clearance. Bacterial count (CFUs) in peritoneal fluid 24 h after CLP. Of 5 to 6 animals per group. **, compared with the sham group P < 0.01; ##, compared with the CLP D V group P < 0.01. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
syndrome. The overproduction of these inflammatory mediators induces endothelial and epithelial injury, vascular leakage, edema, and vasodilatation, subsequently causing the development of MODS and even death. Therefore, interfering with the cytokine overproduction during early sepsis may improve sepsis outcome. Astaxanthin is a potent antioxidant to terminate the induction of inflammation in biological systems. Astaxanthin acts against inflammation. It was reported that astaxanthin suppressed serum nitric oxide, TNF-a, and IL-1b in mice injected with LPS, astaxanthin also inhibited the expression or production of these proinflammatory mediators and cytokines in both LPS-stimulated RAW264.7 cells and primary macrophages [18]. Astaxanthin also protected U937 cells from LPS-induced inflammatory and oxidative stress [19]. Astaxanthin inhibited LPS-stimulated IL-6 messenger RNA and protein in BV-2 microglial cells by suppressing ERK-, MSK-, and NF-kBemediated signals [20]. In the present study,
Fig. 6 e Effect of astaxanthin on CLP-induced lethality. Of 10 animals per group. # compared with the CLP D V group P < 0.05. Sham [ sham-operated group; CLP [ cecal ligation and puncture; CLP D V [ CLP with vehicle treatment.
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treatment with astaxanthin significantly inhibited the elevation of TNF-a, IL-1b, and IL-6 levels at different time points caused by CLP; it is consistent with other reports that demonstrated the potent inhibitory effect of astaxanthin [18e20]. The role of oxidative stress in the pathogenesis of sepsis has been widely reported [6,27e29]. Previous studies have shown that sepsis is associated with enhanced generation of ROS, which are highly reactive and react with biological macromolecules, producing lipid peroxides, inactivating proteins, and mutating DNA [6,27e29]. During sepsis, overproduction of ROS is highly toxic to host tissues, and their interactions with various cellular macromolecules can result in severe pathophysiologic consequences such as MODS [6,27e29]. Antioxidants might counteract the toxicity of ROS and that free-radical ablation for the treatment of sepsis could be useful in the clinical setting of sepsis-induced MODS. Astaxanthin is a carotenoid containing an additional carboxyl group on each ring structure at the two extremities. It is a more effective antioxidant than vitamin E, by 100e1000 times [30]. Previous studies have revealed that astaxanthin, with its antioxidative property, is beneficial as a therapeutic agent for various diseases both in vivo and in vitro without any side effects or toxicity [16,18,19,30e33]. In the present study, we examined the SOD activity and MDA content in multiorgan to evaluate the oxidative stress of the septic rats. SOD is the only antioxidant enzyme that can scavenge superoxide and MDA is an indicator of lipid peroxidation levels, which were commonly known as markers of oxidative stress and antioxidant status. We found that astaxanthin treatment upregulated SOD activity and resulted in decrease in MDA content in the heart, kidney, liver, and lung tissue expectedly. As a consequence of an inappropriate response to an infection, sepsis could compromise the function of distinct organ systems, leading to MODS [5]. Organ failure often begins with respiratory failure, followed by intestinal, hepatic, renal, hematologic, and cardiac failure; the exact order may vary because of preexisting disease or the precipitating insult. Mortality is strongly correlated with the number of organ systems failing, as well as age and duration of organ failure. A definite explanation for the pathophysiology of MODS has yet to be elucidated. In the present study, we examined the degree of renal, heart, and liver dysfunction by monitoring serum levels of organ injury-associated enzymatic indicators including BUN, Cr, LDH, CK-MB, ALT, and AST for 48 h after CLP. The levels of BUN, Cr, and LDH peaked 12 h after CLP operation, whereas the peak level of CK-MB, ALT, and AST appeared at 24 h after CLP. This time-dependent CLP-induced enzyme profile may indicate differences in the vulnerabilities of various organs affected by sepsis. These increases were significantly attenuated by astaxanthin treatment, which provided significant protection from acute organ dysfunction. This multiorgan function improvement with astaxanthin treatment was also confirmed by assessing histologic analyses. In kidney, liver, and lung samples, histopathologic changes caused by CLP such as congestion, inflammatory cell infiltration, necrosis, and degeneration were ameliorated by astaxanthin treatment. The CLP model used in our present study constitutes arguably the best surrogate for abdominal polymicrobial
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human sepsis to date. CLP is generally recognized as a reliable and clinically relevant animal model of the human septic condition. It reflects the progress of clinical human sepsis that occurs as a consequence of invasion of the body by gramnegative or gram-positive bacteria, fungi, and, probably, viruses and parasites. In our study, astaxanthin effectively reduced CFUs in the peritoneal cavity in CLP-induced septic rats, and this result enabled us to suggest that astaxanthin could be used as a potential agent against sepsis. In addition to its bactericidal effect, astaxanthin improved survival in septic rats. Because we have used a severe CLP model, 72-h survival studies allowed us to clearly observe the protective effect of astaxanthin. In CLP groups, the survival rate decreased dramatically to 50% at the first 24 h, and to the 48 h, the rate plateaued at 30%. Astaxanthin treatment significantly inhibited the lethality in septic animals, it showed 90% survival rate on the first 24 h and reached a plateau at 60 h after CLP with a survival rate of 80%, which indicates that astaxanthin may be a potent and efficacious agent to treat sepsis.
5.
Conclusions
In conclusion, the present study shows that astaxanthin exerts impressively protective effects on polymicrobial sepsis by decreasing proinflammatory cytokines, suppressing ROS generation, enhancing antioxidant status and bactericidal ability, and ultimately leading to the alleviation of MODS. It is for the first time we found that astaxanthin exerted impressively protective effects on CLP-induced multiple organ injury and improved the survival of sepsis. In conjunction with previous studies on astaxanthin in different fields, we believe that astaxanthin may be a potential therapeutic candidate for sepsis. Although we confirmed the beneficial effect of astaxanthin on sepsis, the precise molecular mechanisms and signaling pathway still need to be fully studied. And pretreatment with astaxanthin for 7 d before the CLP procedure in the present study does not represent medical practice. The use of astaxanthin after CLP might have different results. Therefore, randomized studies with larger numbers of animals and different astaxanthin dosages administrated after CLP are needed to better clarify the role of astaxanthin in sepsis. Also, there is a significant difference in homology between rodent inflammation and injury response and human inflammation. Clinical trials are necessary to fully realize the potential use of astaxanthin in sepsis.
Acknowledgment This study was financially supported by grants from National Natural Science Foundation of China (81201487) and the Science and Technology Planning Project of Hunan Province (2012F J3142). Authors’ contributions: A.W. and L.Z. contributed to the conception and design. M.G. and Z.X. conducted the animal experiment. J.Z. and X.L. conducted the molecular biological
detection. L.Z. and M.G. analyzed the data and drafted the article. All authors reviewed the article and approved of it. This study was performed at the Central laboratory of Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China.
Disclosure The authors have not disclosed any potential conflicts of interest. And the work described here has not been published previously, and not under consideration for publication elsewhere, in whole or in part.
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