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Protective effect of pioglitazone on sepsis-induced intestinal injury in a rodent model Min Gao, MD, PhD,a,b Yu Jiang, MD,c Xuefei Xiao, MD, PhD,a Yue Peng, MD,a,b Xianzhong Xiao, MD, PhD,b,c and Mingshi Yang, MDa,b,* a
Department of Emergency and Critical Care Medicine, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China b Translational Medicine Center of Sepsis, Department of Pathophysiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China c Laboratory of Shock, Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, People’s Republic of China
article info
abstract
Article history:
Background: Pathogenesis and treatment of inflammatory gut barrier failure is an important
Received 16 December 2014
problem in critical care. In this study, we examined the role of pioglitazone, an agonist of
Received in revised form
peroxisome proliferator-activated receptor gamma, in gut barrier failure during
4 February 2015
experimental peritonitis in rats.
Accepted 6 February 2015
Materials and methods: Male rats were randomly divided into three groups as follows: sham,
Available online xxx
sepsis, and sepsis þ pioglitazone. Sepsis was achieved by means of the cecal ligation and puncture (CLP). Pioglitazone was administered intraperitoneally (10 mg/kg/d) for 7 d before
Keywords:
the experiment. Animals were killed at 24 h or followed 72 h for survival. The tissue level of
Pioglitazone
tumor necrosis factor-a, interleukin-6, superoxide dismutase, malondialdehyde, and
Sepsis
myeloperoxidase was measured. Intestinal mucosa injury was assessed histologically. The
Cecal ligation and puncture
plasma fluorescein isothiocyanate-dextran, D-lactic acid, and intestinal diamine oxidase
Intestinal injury
were determined to evaluate the permeability and integrity of intestinal mucosal epithelium. Vena cava blood and tissue samples were used to monitor bacterial translocation. Results: Intestinal inflammation, oxidize stress, neutrophil infiltration, morphology injury, and impaired permeability of the small intestine in the CLP group were found more severe than those in the sham group. Application of pioglitazone not only minimized all the indicators of intestinal injury and barrier failure but also improved the survival of septic rats induced by CLP. Conclusions: Our novel findings suggest that pioglitazone could protect against intestinal injury and maintain intestinal barrier integrity and might be a useful strategy to ameliorate intestinal failure in polymicrobial sepsis. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Sepsis is a complex syndrome resulting from a systemic inflammatory response to infection and is the leading cause of
death in critically ill patients [1]. Despite the importance of early antibiotic therapy, there is a pressing and currently unmet need to validate additional strategies to mitigate the effects of severe sepsis [2]. The gastrointestinal tract is known
* Corresponding author. Department of Emergency and Critical Care Medicine, The Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Yuelu District, Changsha, Hunan, People’s Republic of China. Tel./fax: þ86 731 88618171. E-mail address: [email protected] (M. Yang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.02.007
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to be a large pool of bacteria and endotoxin, and the intestinal mucosa possesses a barrier function to protect against the escape of intraluminal microorganisms and endotoxin through the intestinal wall into the bloodstream and lymph. Emerging information suggests that the small intestine plays a central role in the pathophysiology of sepsis and has been referred to as the “motor” of the systemic inflammatory response [3,4]. Derangement of the gut barrier with its associated bacterial translocation is common and plays an important role in the pathogenesis of sepsis in critically ill patients [5e8]. Furthermore, studies suggest that increased intestinal permeability is independently associated with the development of multiple organ dysfunction syndrome in patients with critical illness [5,9]. Finding ways to prevent or ameliorate the inflammatory gut barrier failure is therefore an important area of ongoing basic and clinical research. Peroxisome proliferator-activated receptors are members of the nuclear receptor supergene family that function through ligand-mediated transcription [10,11]. The peroxisome proliferator-activated receptor gamma (PPARg) subtype is predominantly expressed in adipose tissue and colon and, to a lesser extent, in macrophages, kidney, liver, small intestine, and pancreas [11,12]. Besides its well-known role in lipid and glucose metabolism, there are more and more reports suggesting that PPARg plays a crucial role in the development of immune and/or inflammation responses [11,13e16]. Many recent reports describing the beneficial effects of PPARg activation on inflammation of intestines have been published. It has been shown that synthetic PPARg agonists attenuate colonic damage in several models of colitis [12,17], whereas targeted deletion of PPARg in either intestinal epithelial cells [18] or inflammatory cells [19] lead to further aggravation of dextran sulfate sodium-induced colitis. PPARg/ mice exhibited much more severe injury in an experimental model of intestinal ischemiaereperfusion injury, whereas local and remote tissue injury was markedly attenuated by PPARg agonist treatment [20]. The literature available demonstrated that PPARg agonists could inhibit the inflammatory response by decreasing tumor necrosis factor-a (TNF-a), interleukin (IL)1b, IL-6, myeloperoxidase (MPO), IL-8, monocyte chemotactic protein-1, and inducible nitric oxide synthase expression [20e22]. It is also reported that PPARg ligands, through selective blockage of nuclear factor kappa-B signaling pathway in vitro, lead to a significant decrease in inflammatory IL-8 and monocyte chemotactic protein-1 expression [23,24]. Because of the immune regulatory functions of PPARg and the positive influence of the PPARg agonist on suppression of the intestinal inflammatory process, we speculated that PPARg agonist may have a protective effect on the sepsisinduced intestinal injury. Previous studies have shown that pioglitazone treatment increases serum adiponectin level and improves survival after cecal ligation and puncture (CLP) by ameliorating excessive inflammatory host responses in mice [25]; and pioglitazone effectively prevents lung injury caused by CLP-induced sepsis by maintaining the anti-inflammatory status of visceral adipose tissue as well [26]. Yet there has not been any evidence showing that the PPARg agonist could affect the sepsis-induced intestinal injury. The present study aims to evaluate the effect of the PPARg agonist (pioglitazone) on the course of CLP-induced intestinal injury in rats.
2.
Materials and methods
2.1.
Animal model and subgroups
Male, 8-wk-old, specific, pathogen-free SpragueeDawley rats, each weighing 230e260 g, were purchased from the Laboratory Animals Center of Central South University (Changsha, China). The rats were housed in mesh cages in a humidified room maintained at 25 C, illuminated with 12:12-h lightedark cycles, and provided with standard rodent chow and water ad libitum. The animals were randomly divided into three groups as follows: rats undergoing sham CLP (sham group); rats undergoing CLP (CLP group), and rats undergoing CLP and treated with pioglitazone (CLP þ P group). Pioglitazone hydrochloride, 5-[[4-[2-(5-Ethyl-2pyridinyl)ethoxy]phenyl]methyl]-2,4-thiazolidinedione hydrochloride (Fig. 1), was purchased from SigmaeAldrich, St. Louis, MO. Pioglitazone hydrochloride has the molecular formula C19H20N2O3S $ HCl and its molar mass is 392.90 g/ mol. It was dissolved in 0.5% methylcellulose (Sigmae Aldrich) and injected intraperitoneally (10 mg/kg/d) for 7 d before the operation. Rats in the sham and CLP groups received methylcellulose in a volume equivalent to that used to dissolve pioglitazone. The dose of pioglitazone used in this study was based on previous experiments [25,26]. Sepsis was induced in rats via CLP. Under anesthesia (ketamine 60 mg/kg plus xylazine 10 mg/kg, intramuscular injection), a 2-cm midline abdominal incision was made, and the cecum was ligated immediately distal to the ileocecal valve. The cecum was then punctured twice with an 18-gauge needle, gently squeezed to extrude a small amount of stool, and replaced in the abdomen that was closed in layers. Sham rats were treated identically with the exception that the cecum was neither ligated nor punctured. All animals were injected subcutaneously with 30 mL/kg body weight of normal saline to account for insensible fluid losses that occurred during surgery. Under anesthesia, rats were sacrificed at 24 h after the CLP. Blood and tissue samples were obtained. 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.
2.2.
Biochemical measurement of intestinal injury
2.2.1. Measurement of intestinal cytokines by enzyme-linked immunosorbent assay Intestinal tissue samples were weighed and homogenized (1:10, wt/vol) in 0.1 M phosphate buffer (pH 7.4) in an ice bath. The samples were centrifuged for collecting supernatants and
Fig. 1 e Molecular structure of pioglitazone hydrochloride.
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stored at 20 C until analysis of TNF-a and IL-6 by using enzyme-linked immunosorbent assay kits (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. All samples were measured in triplicate.
2.2.2.
Measurement of malondialdehyde
Intestinal 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 instructions. In brief, the reaction of thiobarbituric acid (TBA) with MDA at 90 Ce100 C produces a pink color. The TBA was added to homogenized samples to precipitate protein. After centrifugation, the supernatant was mixed with an equal volume of 0.67% (wt/vol) TBA and incubated in 90 C for 10 min. The pink color 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.
2.2.3.
Measurement of superoxide dismutase
The superoxide dismutase (SOD) activity was estimated using a commercially available assay kit (Jiancheng Bioengineering Institute) following the manufacturer’s instructions. In brief, epinephrine undergoes autoxidation rapidly at pH 10.0 to produce adrenochrome; a pink color 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.
2.2.4.
Measurement of MPO
The MPO activity was estimated according to a standard protocol using a commercially available assay kit (Jiancheng Bioengineering Institute). In brief, the intestine homogenate supernatant fluids containing MPO was incubated in a 50-mM KPO4 buffer containing the substrate H2O2 (1.5 M) and o-Dianisidine dihydrochloride (167 mg/mL; SigmaeAldrich) for 30 min. The enzymatic activity was determined spectrophotometrically by measuring the change in absorbance at 460 nm using a 96-well plate reader. The MPO activity was expressed as units per gram protein.
2.3. Measurement of intestinal permeability and integrity 2.3.1.
2.3.2.
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Measurement of diamine oxidase
The diamine oxidase (DAO) content was estimated using a commercially available assay kit (Jiancheng Bioengineering Institute) following the manufacturer’s instruction. In brief, to 0.1 mL 1:10 homogenate of intestinal mucosa, 3 mL phosphate-buffered saline (0.2 M, pH 7.2), 0.1 mL horseradish peroxidase (4 mg), 0.1 mL DAO (500 mg), 0.5 mL dilated standard solution, and 0.1 mL Dianisidine was added. After being mixed, the mixture was incubated in a water bath at 37 C for 30 min, and then the optical density value at 340 nm was recorded after resting for 5 min in the air. The DAO content was calculated according to the standard curve. Determination of protein content of the tissue homogenate was determined with Bradford method.
2.3.3.
In vivo permeability assay
In vivo intestinal permeability was measured using the fluorescein isothiocyanate-labeled dextran (FD-4) method as described [27] with modification. Rats were administered 500 mL of FD-4 (300 mg/mL; SigmaeAldrich) by oral gavage at 18 h after CLP and sham. Six hours later, blood was harvested and serum was isolated. Serial dilutions of FD-4 were made to generate a standard curve, and serum concentrations of FD-4 were determined using a Fluorescence Microplate Reader (BioTek) with an excitation wavelength of 485 nm and emission wavelength of 525 nm. Serum concentrations of FD-4 after CLP were normalized to FD-4 concentrations after the sham operation.
2.4.
Histologic evaluation
The intestinal tissue was promptly rinsed with cold 0.9% saline solution and immediately fixed in 10% buffered formalin phosphate (pH 7.0) until processing for histologic sections. The formalin-fixed samples were embedded in paraffin and sectioned. After deparaffinization and dehydration, the sections were stained with hematoxylin and eosin for histologic assessment of intestinal mucosa by two pathologists blinded to the experimental groups. Tissue injury was quantified in a masked manner by adoption of a preexisting scoring system as described previously [28]. Histologic scoring was performed based on the depth of inflammation and amount of crypt damage. Briefly, scores were graded as follows: (1) inflammation depth: none, 0; mucosa, 1; mucosa and submucosa, 2; transmural, 3; (2) crypt damage: none, 0; basal one-third damaged, 1; basal two-thirds damaged, 2; only surface epithelium intact, 3; entire crypt and epithelium lost, 4. Sections were scored for each feature separately, and the scores were added to reach the final histologic scoring for individual colon specimen.
Measurement of D-lactic acid
The level of D-lactic acid in plasma was estimated according to a standard protocol using a commercially available assay kit (BioVision Incorporated, Milpitas, CA). In this fast kit, D-lactic acid is specially oxidized by D-lactate dehydrogenase and generates proportional color. The D-lactate concentration was determined spectrophotometrically by measuring the change in absorbance at 460 nm using a 96well plate reader. The D-lactic acid was expressed as nanomoles per milliliter.
2.5.
Bacterial translocation analysis
A sample of vena cava blood (100 mL) was extracted under sterile conditions and inoculated on blood agar medium. Additionally, a piece of liver tissue samples or mesenteric lymph nodes (MLNs) were harvested and weighed and then ground with disposable tissue grinders and suspended in 1 mL of sterile normal saline. A 20-mL aliquot of the suspension was inoculated for blood agar culture and was incubated overnight
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at 37 C. Bacterial growth and bacterial species were checked 24 h after incubation. Colony counts were expressed as colony-forming units per milliliter of blood or colony-forming unit per milligram of tissue.
2.6.
Survival studies
To determine the effect of pioglitazone pretreatment on mortality from CLP-induced sepsis, survival studies were performed. Rats were randomly divided into three experimental groups (n ¼ 8e12 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.
2.7.
Statistical analysis
Data are presented as mean and standard error. Multiple comparisons were analyzed for significant differences by using the one-way analysis of variance with Tukey post hoc test. The ManneWhitney test was applied to data with nonnormal distributions. 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.
3.
Results
3.1. Effect of pioglitazone on sepsis-induced intestinal injury Intestinal TNF-a and IL-6 concentrations were very low in sham-operated rats. In contrast, intestinal TNF-a and IL-6 concentrations in rats undergoing CLP increased greatly at 24 h after CLP. The increase in TNF-a and IL-6 concentrations was attenuated by pioglitazone pretreatment (P < 0.01; Fig. 2). MDA, an indicator of lipid peroxidation level, increased in the CLP group compared with that in the sham group (P < 0.01), whereas the increase was significantly attenuated by pioglitazone pretreatment (P < 0.01), suggesting that lipid peroxidate level was suppressed by pioglitazone (Fig. 3). SOD activity was significantly downregulated at 24 h after CLP (P < 0.01), and pioglitazone pretreatment upregulated SOD activity against CLP challenge (P < 0.01; Fig. 3). This suggested that pioglitazone could suppress the oxidative stress induced by CLP via upregulation of SOD activity in rats. Intestinal levels of MPO, a marker of neutrophils infiltration, were increased significantly in the CLP group compared with those in the sham group (P < 0.01). Pioglitazone pretreatment decreased CLP-induced MPO levels in the intestine (P < 0.01; Fig. 4), indicating that pioglitazone could decrease intestinal infiltration of neutrophils in abdominal sepsis.
Fig. 2 e Effect of pioglitazone on local production of TNF-a and IL-6. Pioglitazone was injected intraperitoneally for 7 d (10 mg/kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01, ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
3.2. Effect of pioglitazone on sepsis-induced impaired intestinal permeability and integrity To determine whether pioglitazone pretreatment exerts protective effect on intestinal epithelial barrier dysfunction, plasma D-lactic acid, intestinal DAO content, and intestinal permeability to FD-4 were examined. CLP caused a significant increase of D-lactate levels in rat plasma, whereas the intestinal DAO content reduced dramatically than in the sham group. Pioglitazone pretreatment greatly inhibited the increase of D-lactate levels in rat plasma and preserved the intestinal DAO content (P < 0.01; Fig. 5A and B). CLP caused more than a 4-fold increase in serum FD-4 concentration, and pioglitazone pretreatment decreased CLP-induced serum FD-4 levels by 50% (P < 0.01; Fig. 5C).
Fig. 3 e Effect of pioglitazone on MDA content and SOD activity in the intestinal tissue. Pioglitazone was injected intraperitoneally for 7 d (10 mg/kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01; ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
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epithelial appearance with crypts and mild inflammatory cell infiltration in the sections of pioglitazone-treated rats (Fig. 6). The histologic evaluation showed there was a significant improvement in the pioglitazone pretreatment group compared with those rats in the vehicle-treated CLP group (P < 0.01; Fig. 6).
3.4.
Fig. 4 e Effect of pioglitazone on MPO activity in the intestinal tissue. Pioglitazone was injected intraperitoneally for 7 d (10 mg/kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01; # compared with the CLP group P < 0.05. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
3.3.
Effect of pioglitazone on intestinal histology
Histopathologic evaluation of stained sections showed normal intestinal mucosa and submucosa in the control group (Fig. 6). On the other hand, the sepsis group exhibited severe inflammatory cell infiltration and locally necrotic areas (Fig. 6) with a significant increase in the mean grading scores in comparison with those of the controls (P < 0.01). There was a regular
Effect of pioglitazone on bacterial translocation
To determine whether pioglitazone pretreatment impacted systemic bacterial load in septic rats, blood cultures were obtained 24 h after CLP. Sepsis induced bacteremia in both CLP groups; pioglitazone pretreatment decreased the blood bacterial burden significantly (P < 0.01, Fig. 7). Bacterial translocation to MLNs and liver was also used as readout for intestinal barrier integrity; CLP caused obvious bacterial translocation to the MLN and liver, and pioglitazone pretreatment provided a significant protection against it (P < 0.01, Fig. 7B and C).
3.5. Effect of pioglitazone on the survival rate of CLPinduced septic rats To determine the effect of pioglitazone on CLP-induced mortality, the survival rate after CLP was calculated. All rats in the vehicle and/or sham-operation group survived for 7 d. CLPinduced sepsis showed 50% survival rate on the first 24 h of observation and reached a stable 25% survival rate at 48 h after CLP. Log-rank analysis of the 72-h survival curves demonstrated that pioglitazone at doses of 10 mg/kg pretreatment for 7 d provided a significant level of protection. Pioglitazone treatment group showed 91.67% survival rate in the first 24 h
Fig. 5 e Effect of pioglitazone on indicators of intestinal epithelial barrier dysfunction, including plasma D-lactic acid (A), intestinal DAO content (B), and intestinal permeability to FD-4 (C). Pioglitazone was injected intraperitoneally for 7 d (10 mg/ kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group * or ** compared with the sham group P < 0.05 or P < 0.01; # or ## compared with the CLP group P < 0.05 or P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
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Fig. 6 e Effects of pioglitazone on intestinal histology. Hematoxylin and eosinestained intestine sections from rats of different experimental groups. Representative images were chosen from the different experimental groups (magnification 340, 3100, or 3200 at the upper, middle, and bottom panels). CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group. (Color version of figure is available online.)
Fig. 7 e Effects of pioglitazone on bacterial translocation to systemic circulation (A), MLN (B), and liver (C). The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01; ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, shamoperated group.
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Fig. 8 e Effect of pioglitazone on survival rate of CLPinduced septic rats. Of 8e12 animals per group ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
and reached a plateau at 60 h after CLP with a survival rate of 75% (P < 0.01, Fig. 8).
4.
Discussion
Clinical management of septic patients is mainly limited to supportive care, and novel therapeutic options are needed to improve the outcome of patients with abdominal sepsis. The gut forms a dynamic barrier regulating the passage of luminal content, such as microbes and their products. Pathologic inflammation in the gastrointestinal tract and breakdown of the gut barrier function are key hallmarks in sepsis [3e8]. Increased intestinal permeability is known to promote translocation and invasion of bacteria and their toxins into the systemic circulation [5,9]. Pathogenesis and treatment of inflammatory gut barrier failure is an important problem in critical care. The PPARg agonist pioglitazone is the most widely used thiazolidinedione (TZD) antidiabetic drug and currently the only TZD available without regulatory restrictions [29]. It is an oral drug usually provided in the form of a tablet, and no intravenous forms are available in clinical medicine for now. The cost of pioglitazone is relatively low and could be afforded by most of the patients. The precise mechanism of action of TZDs remains unclear, but the current consensus is that TZDs target the transcription factor PPARg to regulate transcription of genes involved in metabolic homeostasis and then improve insulin sensitivity. Besides its well-known role in lipid and glucose metabolism, PPARg represses transcription of genes related to inflammation via transrepression and antagonizes other transcription factors that activate inflammatory pathways, such as nuclear factor kappa-B and adaptor-related protein complex 1 [30]. In recent years, many scientific reports describing the influence of PPARg ligands on inflammation of intestines have been published. It is reported that PPARg ligands could protect animals against intestinal injury caused by different models of experimental colitis [12,17e19,21e23], Crohn disease [24], intestinal ischemiaereperfusion injury [20], necrotizing enterocolitis [31], and postoperative ileus [32]. Yet there has not been any evidence showing that the PPARg agonist can affect the sepsis-induced intestinal injury. We
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hypothesized pioglitazone may be therapeutically effective for attenuating sepsis-induced intestinal injury. We therefore evaluated the effects of pioglitazone in a rat model of CLPinduced intestinal injury. In the present study, we show for the first time that pioglitazone protects against intestinal injury in abdominal sepsis. At first, proinflammatory cytokines including TNF-a and IL-6 concentrations in the intestine were detected to evaluate the local inflammation of the intestine. CLP caused significant increase in TNF-a and IL-6 expressions in rat intestine at 24 h compared with the sham group as expected. Pioglitazone pretreatment decreased local production of TNF-a and IL-6 compared with the vehicle-treated CLP group. This result is consistent with other reports that demonstrated the potent inhibitory effect of pioglitazone on intestinal inflammation by decreasing cytokines expression [20e22]. Then, oxidative stress in the intestinal tissue was determined as another index to evaluate the CLP-induced intestinal injury. The role of oxidative stress in the pathogenesis of sepsis has been widely reported. Overproduction of reactive oxygen species is highly toxic to host tissues, and their interactions with various biological macromolecules can result in severe pathophysiologic consequences leading to multiple organ dysfunction syndrome [33,34]. SOD is the only antioxidant enzyme that can scavenge superoxide, and MDA is an indicator of lipid peroxidation level; these two were commonly known as markers of oxidative stress and antioxidant status. The present results showed that CLP caused significant increase in MDA content in intestinal tissue, whereas the SOD activity decreased greatly; pioglitazone pretreatment upregulated SOD activity and resulted in decrease in the MDA content expectedly. It is well-known that PPARg agonists have anti-inflammatory and antioxidant effects [35e37]. Our findings are in accordance with these reports that demonstrated excellent antioxidant capacity of pioglitazone. MPO, a marker of neutrophil recruitment, was also determined to evaluate the CLP-induced intestinal injury. In spite of its critical role in mounting host defense reactions, neutrophils can also cause significant tissue damage in inflammatory disease. Neutrophils can secrete destructive elastases and lipid mediators and reactive oxygen species with the capacity to damage both endothelial and epithelial cell function [38,39]. Convincing data have established neutrophil recruitment as a fundamental component in the pathophysiology of sepsis-induced intestinal injury [40,41]. The present results showed that CLP caused significant increase in the level of MPO content in intestinal tissue; pioglitazone pretreatment significantly decreased intestinal level of MPO, indicating that pioglitazone can decrease intestinal infiltration of neutrophils in abdominal sepsis. Sepsis is associated with disturbed gut barrier function, which facilitates translocation of pathogens and their toxins into the systemic circulation. In the present study, we examined the plasma D-lactic acid, intestinal DAO content, intestinal permeability to FD-4 and bacterial translocation to systemic circulation (blood), lymphatic organs (MLN), and liver to evaluate whether pioglitazone pretreatment exerts protective effect on intestinal epithelial barrier dysfunction for the first time. Plasma D-lactic acid and intestinal DAO are sensitive markers of intestinal permeability. D-lactic acid is a
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metabolic product of bacteria, and it is solely present in the intestinal lumen. When the integrity of intestinal mucosa is damaged, intestinal permeability increases and D-lactic acid could permeate through the bowel to get access to the bloodstream [42]. DAO is an intracellular enzyme with high activity existing in intestinal villous cells in both human beings and all other mammalians. Its activity is especially high in the jejunum and ileum. It is a relatively stable marker of maturation and integrity of intestinal mucosa cells [43]. The activity of DAO in intestinal mucosa decreases when its cells are injured. FD4 is commonly used to study passage of high molecular weight substances across the epithelium. The present results showed that on CLP challenge, there were multiple signs of intestinal barrier failure including high levels of plasma D-lactic acid, significant reduction of intestinal DAO content, and increased permeability to fluorescein isothiocyanate-dextran and bacterial translocation to blood, MLN, and liver. Pioglitazone pretreatment minimized all the mentioned signs of intestinal barrier failure, suggesting that PPARg plays an important role in septic intestinal damage, and pioglitazone might be a useful tool to maintain gut barrier integrity in abdominal sepsis. Morphology alteration of the intestine was also examined by hematoxylin and eosin staining. There was a great pathologic alteration that occurred at 24 h after development of CLPinduced sepsis, including the infiltration of inflammatory cells, intestinal mucosal villous edema, epithelial cells sloughing, villus tip destruction, villous necrosis, and mucosal erosion. Compared with vehicle-treated CLP group, a mild alteration of mucosa edema, exudation, microvilli structure, intestinal wall erosion, and invasion of inflammatory cells was observed in the pioglitazone pretreatment CLP group. The present data showed that histologic damage of intestinal mucosa is accompanied by the intestinal injury and barrier failure, which is consistent with other previous studies [44,45]. Taken together, the present study shows that pioglitazone decreases sepsis-induced inflammatory response, oxidative stress, and neutrophil infiltration in the intestine. Moreover, pioglitazone markedly reduced intestinal permeability and leakage, suggesting that it might be a useful tool to maintain intestinal barrier integrity and protect against sepsis-induced intestinal injury. Although we confirmed the beneficial effect of pioglitazone on sepsis-induced intestinal injury in a rodent model, the present study has some limitations that need to be addressed. First, we used CLP in this study because it is considered the gold standard model of sepsis and is thought to mimic a perforated viscous, endogenous fecal contamination, and variable progression of disease, which resembles clinical sepsis [46,47]. However, we recognize the difficulty in translating animal studies to humans; and it was conducted in previously healthy animals under highly controlled circumstances, in contrast to the clinical setting in which patients often have underlying illness and co-morbidities. Therefore, clinical trials are necessary to fully realize the potential use of pioglitazone in sepsis-induced intestinal injury. Second, the pioglitazone was administrated intraperitoneally (10 mg/kg/d) for 7 d before the CLP procedure in the present study and it does not represent medical practice. The use of pioglitazone at the time of CLP or for a longer period after CLP might have different results. Therefore, these results cannot be directly directed to
clinical use unless similar interventions and further studies have been taken in this sepsis model. Finally, randomized studies with larger numbers of animals and different dosages are needed to better clarify the role of pioglitazone in sepsis.
Acknowledgment This study was financially supported by grants from National Natural Science Foundation of China (81201486, 81373147). This study was performed at the Translational Medicine Center of Sepsis, Department of Pathophysiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China. Authors’ contributions: M.Y., X.X., and M.G. contributed to the conception and design. M.G. and Y.J. conducted the animal experiment, X.X. and Y.P. conducted the molecular biological detection. M.G. and Y.J. analyzed the data and drafted the article. All authors reviewed the article and approved of it.
Disclosure The authors have not disclosed any potential conflicts of interest. The work described here has not been published previously and is not under consideration for publication elsewhere, in whole or in part.
references
[1] Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003;348:138. [2] Wenzel RP, Edmond MB. Septic shockdevaluating another failed treatment. N Engl J Med 2012;366:2122. [3] Clark JA, Coopersmith CM. Intestinal crosstalk: a new paradigm for understanding the gut as the “motor” of critical illness. Shock 2007;28:384. [4] Hassoun HT, Kone BC, Mercer DW, et al. Post-injury multiple organ failure: the role of the gut. Shock 2001;15:1. [5] Fink MP, Delude RL. Epithelial barrier dysfunction: a unifying theme to explain the pathogenesis of multiple organ dysfunction at the cellular level. Crit Care Clin 2005;21:177. [6] Gatt M, Reddy BS, MacFie J. Review article: bacterial translocation in the critically illdevidence and methods of prevention. Aliment Pharmacol Ther 2007;25:741. [7] Alverdy JC, Chang EB. The re-emerging role of the intestinal microflora in critical illness and inflammation: why the gut hypothesis of sepsis syndrome will not go away. J Leukoc Biol 2008;83:461. [8] Deitch EA. Gut-origin sepsis: evolution of a concept. Surgeon 2012;10:350. [9] Deitch EA. Role of the gut lymphatic system in multiple organ failure. Curr Opin Crit Care 2001;7:92. [10] Schoonjans K, Martin G, Staels B, Auwerx J. Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol 1997;8:159. [11] Clark RB. The role of PPARs in inflammation and immunity. J Leukoc Biol 2002;71:388. [12] Dubuquoy L, Rousseaux C, Thuru X, et al. PPARgamma as a new therapeutic target in inflammatory bowel diseases. Gut 2006;55:1341. [13] Kiec-Wilk B, Dembinska-Kiec A, Olszanecka A, Bodzioch M, Kawecka-Jaszcz K. The selected pathophysiological aspects of PPARs activation. J Physiol Pharmacol 2005;56:149.
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
[14] Ptak-Belowska A, Pawlik MW, Krzysiek-Maczka G, Brzozowski T, Pawlik WW. Transcriptional upregulation of gastrin in response to peroxisome proliferator-activated receptor gamma agonist triggers cell survival pathways. J Physiol Pharmacol 2007;58:793. [15] Konturek PC, Dembinski A, Warzecha Z, et al. Pioglitazone, a specific ligand of peroxisome proliferator-activated receptorgamma, protects pancreas against acute cerulein-induced pancreatitis. World J Gastroenterol 2005;11:6322. [16] Konturek PC, Brzozowski T, Engel M, et al. Ghrelin ameliorates colonic inflammation. Role of nitric oxide and sensory nerves. J Physiol Pharmacol 2009;60:41. [17] Linard C, Gremy O, Benderitter M. Reduction of peroxisome proliferation-activated receptor gamma expression by gammairradiation as a mechanism contributing to inflammatory response in rat colon: modulation by the 5-aminosalicylic acid agonist. J Pharmacol Exp Ther 2008;324:911. [18] Adachi M, Kurotani R, Morimura K, et al. Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease. Gut 2006;55:1104. [19] Shah YM, Morimura K, Gonzalez FJ. Expression of peroxisome proliferator-activated receptor-gamma in macrophage suppresses experimentally induced colitis. Am J Physiol Gastrointest Liver Physiol 2007;292:G657. [20] Nakajima A, Wada K, Miki H, et al. Endogenous PPAR gamma mediates anti-inflammatory activity in murine ischemiareperfusion injury. Gastroenterology 2001;120:460. [21] Arita M, Yoshida M, Hong S, et al. Resolvin E1, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Proc Natl Acad Sci U S A 2005; 102:7671. [22] Ten HT, Corbaz A, Amitai H, et al. Blockade of endogenous IL-18 ameliorates TNBS-induced colitis by decreasing local TNF-alpha production in mice. Gastroenterology 2001;121: 1372. [23] Nakai M, Sudo K, Yamada Y, et al. The role of the tumor necrosis factor receptor in 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis in mice. Dig Dis Sci 2005;50:1669. [24] Hollenbach E, Vieth M, Roessner A, Neumann M, Malfertheiner P, Naumann M. Inhibition of RICK/nuclear factor-kappaB and p38 signaling attenuates the inflammatory response in a murine model of Crohn disease. J Biol Chem 2005;280:14981. [25] Tsujimura Y, Matsutani T, Matsuda A, et al. Effects of pioglitazone on survival and omental adipocyte function in mice with sepsis induced by cecal ligation and puncture. J Surg Res 2011;171:e215. [26] Kutsukake M, Matsutani T, Tamura K, et al. Pioglitazone attenuates lung injury by modulating adipose inflammation. J Surg Res 2014;189:295. [27] Clark JA, Gan H, Samocha AJ, Fox AC, Buchman TG, Coopersmith CM. Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis. Am J Physiol Gastrointest Liver Physiol 2009;297:G471. [28] Kajiya M, Silva MJ, Sato K, Ouhara K, Kawai T. Hydrogen mediates suppression of colon inflammation induced by dextran sodium sulfate. Biochem Biophys Res Commun 2009; 386:11. [29] Kawaguchi-Suzuki M, Frye RF. Current clinical evidence on pioglitazone pharmacogenomics. Front Pharmacol 2013;4:147.
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[30] Cariou B, Charbonnel B, Staels B. Thiazolidinediones and PPARg agonists: time for a reassessment. Trends Endocrinol Metab 2012;23:205. [31] Baregamian N, Mourot JM, Ballard AR, Evers BM, Chung DH. PPAR-gamma agonist protects against intestinal injury during necrotizing enterocolitis. Biochem Biophys Res Commun 2009;379:423. [32] De Backer O, Elinck E, Priem E, Leybaert L, Lefebvre RA. Peroxisome proliferator-activated receptor gamma activation alleviates postoperative ileus in mice by inhibition of Egr-1 expression and its downstream target genes. J Pharmacol Exp Ther 2009;331:496. [33] Koksal GM, Sayilgan C, Aydin S, Oz H, Uzun H. Correlation of plasma and tissue oxidative stresses in intra-abdominal sepsis. J Surg Res 2004;122:180. [34] 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. [35] Zou C, Hu H, Xi X, Shi Z, Wang G, Huang X. Pioglitazone protects against renal ischemia-reperfusion injury by enhancing antioxidant capacity. J Surg Res 2013;184:1092. [36] El-Sheikh AA, Rifaai RA. Peroxisome proliferator activator receptor (PPAR)-gamma ligand, but not PPAR-alpha, ameliorates cyclophosphamide-induced oxidative stress and inflammation in rat liver. PPAR Res 2014;2014:626319. [37] Al RF, El EE. PPAR-gamma activator induces neuroprotection in hypercholesterolemic rats subjected to global cerebral ischemia/reperfusion injury: in vivo and in vitro inhibition of oxidative stress. Exp Gerontol 2014;51:1. [38] Swirski FK, Wildgruber M, Ueno T, et al. Myeloperoxidaserich Ly-6Cþ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice. J Clin Invest 2010;120:2627. [39] Grommes J, Vijayan S, Drechsler M, et al. Simvastatin reduces endotoxin-induced acute lung injury by decreasing neutrophil recruitment and radical formation. PLoS One 2012;7:e38917. [40] Asaduzzaman M, Rahman M, Jeppsson B, Thorlacius H. Pselectin glycoprotein-ligand-1 regulates pulmonary recruitment of neutrophils in a platelet-independent manner in abdominal sepsis. Br J Pharmacol 2009;156:307. [41] Zhang S, Zhang S, Rahman M, Herwald H, Thorlacius H. Streptococcal M1 protein-induced lung injury is independent of platelets in mice. Shock 2011;35:86. [42] Smith SM, Eng RH, Buccini F. Use of D-lactic acid measurements in the diagnosis of bacterial infections. J Infect Dis 1986;154:658. [43] Thompson JS, Vaughan WP, Forst CF, Jacobs DL, Weekly JS, Rikkers LF. The effect of the route of nutrient delivery on gut structure and diamine oxidase levels. JPEN J Parenter Enteral Nutr 1987;11:28. [44] Shebrain S, Ramjit A. Radicicol, a Hsp90 inhibitor, inhibits intestinal inflammation and leakage in abdominal sepsis. J Surg Res 2013;185:e53. [45] Short SS, Wang J, Castle SL, et al. Low doses of celecoxib attenuate gut barrier failure during experimental peritonitis. Lab Invest 2013;93:1265. [46] Wichterman KA, Baue AE, Chaudry IH. Sepsis and septic shockda review of laboratory models and a proposal. J Surg Res 1980;29:189. [47] Hubbard WJ, Choudhry M, Schwacha MG, et al. Cecal ligation and puncture. Shock 2005;24(Suppl 1):52.
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Protective effect of pioglitazone on sepsis-induced intestinal injury in a rodent model Min Gao, MD, PhD,a,b Yu Jiang, MD,c Xuefei Xiao, MD, PhD,a Yue Peng, MD,a,b Xianzhong Xiao, MD, PhD,b,c and Mingshi Yang, MDa,b,* a
Department of Emergency and Critical Care Medicine, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China b Translational Medicine Center of Sepsis, Department of Pathophysiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China c Laboratory of Shock, Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, People’s Republic of China
article info
abstract
Article history:
Background: Pathogenesis and treatment of inflammatory gut barrier failure is an important
Received 16 December 2014
problem in critical care. In this study, we examined the role of pioglitazone, an agonist of
Received in revised form
peroxisome proliferator-activated receptor gamma, in gut barrier failure during
4 February 2015
experimental peritonitis in rats.
Accepted 6 February 2015
Materials and methods: Male rats were randomly divided into three groups as follows: sham,
Available online xxx
sepsis, and sepsis þ pioglitazone. Sepsis was achieved by means of the cecal ligation and puncture (CLP). Pioglitazone was administered intraperitoneally (10 mg/kg/d) for 7 d before
Keywords:
the experiment. Animals were killed at 24 h or followed 72 h for survival. The tissue level of
Pioglitazone
tumor necrosis factor-a, interleukin-6, superoxide dismutase, malondialdehyde, and
Sepsis
myeloperoxidase was measured. Intestinal mucosa injury was assessed histologically. The
Cecal ligation and puncture
plasma fluorescein isothiocyanate-dextran, D-lactic acid, and intestinal diamine oxidase
Intestinal injury
were determined to evaluate the permeability and integrity of intestinal mucosal epithelium. Vena cava blood and tissue samples were used to monitor bacterial translocation. Results: Intestinal inflammation, oxidize stress, neutrophil infiltration, morphology injury, and impaired permeability of the small intestine in the CLP group were found more severe than those in the sham group. Application of pioglitazone not only minimized all the indicators of intestinal injury and barrier failure but also improved the survival of septic rats induced by CLP. Conclusions: Our novel findings suggest that pioglitazone could protect against intestinal injury and maintain intestinal barrier integrity and might be a useful strategy to ameliorate intestinal failure in polymicrobial sepsis. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Sepsis is a complex syndrome resulting from a systemic inflammatory response to infection and is the leading cause of
death in critically ill patients [1]. Despite the importance of early antibiotic therapy, there is a pressing and currently unmet need to validate additional strategies to mitigate the effects of severe sepsis [2]. The gastrointestinal tract is known
* Corresponding author. Department of Emergency and Critical Care Medicine, The Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Yuelu District, Changsha, Hunan, People’s Republic of China. Tel./fax: þ86 731 88618171. E-mail address: [email protected] (M. Yang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.02.007
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to be a large pool of bacteria and endotoxin, and the intestinal mucosa possesses a barrier function to protect against the escape of intraluminal microorganisms and endotoxin through the intestinal wall into the bloodstream and lymph. Emerging information suggests that the small intestine plays a central role in the pathophysiology of sepsis and has been referred to as the “motor” of the systemic inflammatory response [3,4]. Derangement of the gut barrier with its associated bacterial translocation is common and plays an important role in the pathogenesis of sepsis in critically ill patients [5e8]. Furthermore, studies suggest that increased intestinal permeability is independently associated with the development of multiple organ dysfunction syndrome in patients with critical illness [5,9]. Finding ways to prevent or ameliorate the inflammatory gut barrier failure is therefore an important area of ongoing basic and clinical research. Peroxisome proliferator-activated receptors are members of the nuclear receptor supergene family that function through ligand-mediated transcription [10,11]. The peroxisome proliferator-activated receptor gamma (PPARg) subtype is predominantly expressed in adipose tissue and colon and, to a lesser extent, in macrophages, kidney, liver, small intestine, and pancreas [11,12]. Besides its well-known role in lipid and glucose metabolism, there are more and more reports suggesting that PPARg plays a crucial role in the development of immune and/or inflammation responses [11,13e16]. Many recent reports describing the beneficial effects of PPARg activation on inflammation of intestines have been published. It has been shown that synthetic PPARg agonists attenuate colonic damage in several models of colitis [12,17], whereas targeted deletion of PPARg in either intestinal epithelial cells [18] or inflammatory cells [19] lead to further aggravation of dextran sulfate sodium-induced colitis. PPARg/ mice exhibited much more severe injury in an experimental model of intestinal ischemiaereperfusion injury, whereas local and remote tissue injury was markedly attenuated by PPARg agonist treatment [20]. The literature available demonstrated that PPARg agonists could inhibit the inflammatory response by decreasing tumor necrosis factor-a (TNF-a), interleukin (IL)1b, IL-6, myeloperoxidase (MPO), IL-8, monocyte chemotactic protein-1, and inducible nitric oxide synthase expression [20e22]. It is also reported that PPARg ligands, through selective blockage of nuclear factor kappa-B signaling pathway in vitro, lead to a significant decrease in inflammatory IL-8 and monocyte chemotactic protein-1 expression [23,24]. Because of the immune regulatory functions of PPARg and the positive influence of the PPARg agonist on suppression of the intestinal inflammatory process, we speculated that PPARg agonist may have a protective effect on the sepsisinduced intestinal injury. Previous studies have shown that pioglitazone treatment increases serum adiponectin level and improves survival after cecal ligation and puncture (CLP) by ameliorating excessive inflammatory host responses in mice [25]; and pioglitazone effectively prevents lung injury caused by CLP-induced sepsis by maintaining the anti-inflammatory status of visceral adipose tissue as well [26]. Yet there has not been any evidence showing that the PPARg agonist could affect the sepsis-induced intestinal injury. The present study aims to evaluate the effect of the PPARg agonist (pioglitazone) on the course of CLP-induced intestinal injury in rats.
2.
Materials and methods
2.1.
Animal model and subgroups
Male, 8-wk-old, specific, pathogen-free SpragueeDawley rats, each weighing 230e260 g, were purchased from the Laboratory Animals Center of Central South University (Changsha, China). The rats were housed in mesh cages in a humidified room maintained at 25 C, illuminated with 12:12-h lightedark cycles, and provided with standard rodent chow and water ad libitum. The animals were randomly divided into three groups as follows: rats undergoing sham CLP (sham group); rats undergoing CLP (CLP group), and rats undergoing CLP and treated with pioglitazone (CLP þ P group). Pioglitazone hydrochloride, 5-[[4-[2-(5-Ethyl-2pyridinyl)ethoxy]phenyl]methyl]-2,4-thiazolidinedione hydrochloride (Fig. 1), was purchased from SigmaeAldrich, St. Louis, MO. Pioglitazone hydrochloride has the molecular formula C19H20N2O3S $ HCl and its molar mass is 392.90 g/ mol. It was dissolved in 0.5% methylcellulose (Sigmae Aldrich) and injected intraperitoneally (10 mg/kg/d) for 7 d before the operation. Rats in the sham and CLP groups received methylcellulose in a volume equivalent to that used to dissolve pioglitazone. The dose of pioglitazone used in this study was based on previous experiments [25,26]. Sepsis was induced in rats via CLP. Under anesthesia (ketamine 60 mg/kg plus xylazine 10 mg/kg, intramuscular injection), a 2-cm midline abdominal incision was made, and the cecum was ligated immediately distal to the ileocecal valve. The cecum was then punctured twice with an 18-gauge needle, gently squeezed to extrude a small amount of stool, and replaced in the abdomen that was closed in layers. Sham rats were treated identically with the exception that the cecum was neither ligated nor punctured. All animals were injected subcutaneously with 30 mL/kg body weight of normal saline to account for insensible fluid losses that occurred during surgery. Under anesthesia, rats were sacrificed at 24 h after the CLP. Blood and tissue samples were obtained. 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.
2.2.
Biochemical measurement of intestinal injury
2.2.1. Measurement of intestinal cytokines by enzyme-linked immunosorbent assay Intestinal tissue samples were weighed and homogenized (1:10, wt/vol) in 0.1 M phosphate buffer (pH 7.4) in an ice bath. The samples were centrifuged for collecting supernatants and
Fig. 1 e Molecular structure of pioglitazone hydrochloride.
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stored at 20 C until analysis of TNF-a and IL-6 by using enzyme-linked immunosorbent assay kits (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. All samples were measured in triplicate.
2.2.2.
Measurement of malondialdehyde
Intestinal 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 instructions. In brief, the reaction of thiobarbituric acid (TBA) with MDA at 90 Ce100 C produces a pink color. The TBA was added to homogenized samples to precipitate protein. After centrifugation, the supernatant was mixed with an equal volume of 0.67% (wt/vol) TBA and incubated in 90 C for 10 min. The pink color 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.
2.2.3.
Measurement of superoxide dismutase
The superoxide dismutase (SOD) activity was estimated using a commercially available assay kit (Jiancheng Bioengineering Institute) following the manufacturer’s instructions. In brief, epinephrine undergoes autoxidation rapidly at pH 10.0 to produce adrenochrome; a pink color 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.
2.2.4.
Measurement of MPO
The MPO activity was estimated according to a standard protocol using a commercially available assay kit (Jiancheng Bioengineering Institute). In brief, the intestine homogenate supernatant fluids containing MPO was incubated in a 50-mM KPO4 buffer containing the substrate H2O2 (1.5 M) and o-Dianisidine dihydrochloride (167 mg/mL; SigmaeAldrich) for 30 min. The enzymatic activity was determined spectrophotometrically by measuring the change in absorbance at 460 nm using a 96-well plate reader. The MPO activity was expressed as units per gram protein.
2.3. Measurement of intestinal permeability and integrity 2.3.1.
2.3.2.
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Measurement of diamine oxidase
The diamine oxidase (DAO) content was estimated using a commercially available assay kit (Jiancheng Bioengineering Institute) following the manufacturer’s instruction. In brief, to 0.1 mL 1:10 homogenate of intestinal mucosa, 3 mL phosphate-buffered saline (0.2 M, pH 7.2), 0.1 mL horseradish peroxidase (4 mg), 0.1 mL DAO (500 mg), 0.5 mL dilated standard solution, and 0.1 mL Dianisidine was added. After being mixed, the mixture was incubated in a water bath at 37 C for 30 min, and then the optical density value at 340 nm was recorded after resting for 5 min in the air. The DAO content was calculated according to the standard curve. Determination of protein content of the tissue homogenate was determined with Bradford method.
2.3.3.
In vivo permeability assay
In vivo intestinal permeability was measured using the fluorescein isothiocyanate-labeled dextran (FD-4) method as described [27] with modification. Rats were administered 500 mL of FD-4 (300 mg/mL; SigmaeAldrich) by oral gavage at 18 h after CLP and sham. Six hours later, blood was harvested and serum was isolated. Serial dilutions of FD-4 were made to generate a standard curve, and serum concentrations of FD-4 were determined using a Fluorescence Microplate Reader (BioTek) with an excitation wavelength of 485 nm and emission wavelength of 525 nm. Serum concentrations of FD-4 after CLP were normalized to FD-4 concentrations after the sham operation.
2.4.
Histologic evaluation
The intestinal tissue was promptly rinsed with cold 0.9% saline solution and immediately fixed in 10% buffered formalin phosphate (pH 7.0) until processing for histologic sections. The formalin-fixed samples were embedded in paraffin and sectioned. After deparaffinization and dehydration, the sections were stained with hematoxylin and eosin for histologic assessment of intestinal mucosa by two pathologists blinded to the experimental groups. Tissue injury was quantified in a masked manner by adoption of a preexisting scoring system as described previously [28]. Histologic scoring was performed based on the depth of inflammation and amount of crypt damage. Briefly, scores were graded as follows: (1) inflammation depth: none, 0; mucosa, 1; mucosa and submucosa, 2; transmural, 3; (2) crypt damage: none, 0; basal one-third damaged, 1; basal two-thirds damaged, 2; only surface epithelium intact, 3; entire crypt and epithelium lost, 4. Sections were scored for each feature separately, and the scores were added to reach the final histologic scoring for individual colon specimen.
Measurement of D-lactic acid
The level of D-lactic acid in plasma was estimated according to a standard protocol using a commercially available assay kit (BioVision Incorporated, Milpitas, CA). In this fast kit, D-lactic acid is specially oxidized by D-lactate dehydrogenase and generates proportional color. The D-lactate concentration was determined spectrophotometrically by measuring the change in absorbance at 460 nm using a 96well plate reader. The D-lactic acid was expressed as nanomoles per milliliter.
2.5.
Bacterial translocation analysis
A sample of vena cava blood (100 mL) was extracted under sterile conditions and inoculated on blood agar medium. Additionally, a piece of liver tissue samples or mesenteric lymph nodes (MLNs) were harvested and weighed and then ground with disposable tissue grinders and suspended in 1 mL of sterile normal saline. A 20-mL aliquot of the suspension was inoculated for blood agar culture and was incubated overnight
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at 37 C. Bacterial growth and bacterial species were checked 24 h after incubation. Colony counts were expressed as colony-forming units per milliliter of blood or colony-forming unit per milligram of tissue.
2.6.
Survival studies
To determine the effect of pioglitazone pretreatment on mortality from CLP-induced sepsis, survival studies were performed. Rats were randomly divided into three experimental groups (n ¼ 8e12 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.
2.7.
Statistical analysis
Data are presented as mean and standard error. Multiple comparisons were analyzed for significant differences by using the one-way analysis of variance with Tukey post hoc test. The ManneWhitney test was applied to data with nonnormal distributions. 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.
3.
Results
3.1. Effect of pioglitazone on sepsis-induced intestinal injury Intestinal TNF-a and IL-6 concentrations were very low in sham-operated rats. In contrast, intestinal TNF-a and IL-6 concentrations in rats undergoing CLP increased greatly at 24 h after CLP. The increase in TNF-a and IL-6 concentrations was attenuated by pioglitazone pretreatment (P < 0.01; Fig. 2). MDA, an indicator of lipid peroxidation level, increased in the CLP group compared with that in the sham group (P < 0.01), whereas the increase was significantly attenuated by pioglitazone pretreatment (P < 0.01), suggesting that lipid peroxidate level was suppressed by pioglitazone (Fig. 3). SOD activity was significantly downregulated at 24 h after CLP (P < 0.01), and pioglitazone pretreatment upregulated SOD activity against CLP challenge (P < 0.01; Fig. 3). This suggested that pioglitazone could suppress the oxidative stress induced by CLP via upregulation of SOD activity in rats. Intestinal levels of MPO, a marker of neutrophils infiltration, were increased significantly in the CLP group compared with those in the sham group (P < 0.01). Pioglitazone pretreatment decreased CLP-induced MPO levels in the intestine (P < 0.01; Fig. 4), indicating that pioglitazone could decrease intestinal infiltration of neutrophils in abdominal sepsis.
Fig. 2 e Effect of pioglitazone on local production of TNF-a and IL-6. Pioglitazone was injected intraperitoneally for 7 d (10 mg/kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01, ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
3.2. Effect of pioglitazone on sepsis-induced impaired intestinal permeability and integrity To determine whether pioglitazone pretreatment exerts protective effect on intestinal epithelial barrier dysfunction, plasma D-lactic acid, intestinal DAO content, and intestinal permeability to FD-4 were examined. CLP caused a significant increase of D-lactate levels in rat plasma, whereas the intestinal DAO content reduced dramatically than in the sham group. Pioglitazone pretreatment greatly inhibited the increase of D-lactate levels in rat plasma and preserved the intestinal DAO content (P < 0.01; Fig. 5A and B). CLP caused more than a 4-fold increase in serum FD-4 concentration, and pioglitazone pretreatment decreased CLP-induced serum FD-4 levels by 50% (P < 0.01; Fig. 5C).
Fig. 3 e Effect of pioglitazone on MDA content and SOD activity in the intestinal tissue. Pioglitazone was injected intraperitoneally for 7 d (10 mg/kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01; ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
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epithelial appearance with crypts and mild inflammatory cell infiltration in the sections of pioglitazone-treated rats (Fig. 6). The histologic evaluation showed there was a significant improvement in the pioglitazone pretreatment group compared with those rats in the vehicle-treated CLP group (P < 0.01; Fig. 6).
3.4.
Fig. 4 e Effect of pioglitazone on MPO activity in the intestinal tissue. Pioglitazone was injected intraperitoneally for 7 d (10 mg/kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01; # compared with the CLP group P < 0.05. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
3.3.
Effect of pioglitazone on intestinal histology
Histopathologic evaluation of stained sections showed normal intestinal mucosa and submucosa in the control group (Fig. 6). On the other hand, the sepsis group exhibited severe inflammatory cell infiltration and locally necrotic areas (Fig. 6) with a significant increase in the mean grading scores in comparison with those of the controls (P < 0.01). There was a regular
Effect of pioglitazone on bacterial translocation
To determine whether pioglitazone pretreatment impacted systemic bacterial load in septic rats, blood cultures were obtained 24 h after CLP. Sepsis induced bacteremia in both CLP groups; pioglitazone pretreatment decreased the blood bacterial burden significantly (P < 0.01, Fig. 7). Bacterial translocation to MLNs and liver was also used as readout for intestinal barrier integrity; CLP caused obvious bacterial translocation to the MLN and liver, and pioglitazone pretreatment provided a significant protection against it (P < 0.01, Fig. 7B and C).
3.5. Effect of pioglitazone on the survival rate of CLPinduced septic rats To determine the effect of pioglitazone on CLP-induced mortality, the survival rate after CLP was calculated. All rats in the vehicle and/or sham-operation group survived for 7 d. CLPinduced sepsis showed 50% survival rate on the first 24 h of observation and reached a stable 25% survival rate at 48 h after CLP. Log-rank analysis of the 72-h survival curves demonstrated that pioglitazone at doses of 10 mg/kg pretreatment for 7 d provided a significant level of protection. Pioglitazone treatment group showed 91.67% survival rate in the first 24 h
Fig. 5 e Effect of pioglitazone on indicators of intestinal epithelial barrier dysfunction, including plasma D-lactic acid (A), intestinal DAO content (B), and intestinal permeability to FD-4 (C). Pioglitazone was injected intraperitoneally for 7 d (10 mg/ kg/d) before the operation. The results are presented as mean ± standard error. Of five to eight animals per group * or ** compared with the sham group P < 0.05 or P < 0.01; # or ## compared with the CLP group P < 0.05 or P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
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Fig. 6 e Effects of pioglitazone on intestinal histology. Hematoxylin and eosinestained intestine sections from rats of different experimental groups. Representative images were chosen from the different experimental groups (magnification 340, 3100, or 3200 at the upper, middle, and bottom panels). CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group. (Color version of figure is available online.)
Fig. 7 e Effects of pioglitazone on bacterial translocation to systemic circulation (A), MLN (B), and liver (C). The results are presented as mean ± standard error. Of five to eight animals per group ** compared with the sham group P < 0.01; ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, shamoperated group.
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Fig. 8 e Effect of pioglitazone on survival rate of CLPinduced septic rats. Of 8e12 animals per group ## compared with the CLP group P < 0.01. CLP D P, cecal ligation and puncture with pioglitazone pretreatment; sham, sham-operated group.
and reached a plateau at 60 h after CLP with a survival rate of 75% (P < 0.01, Fig. 8).
4.
Discussion
Clinical management of septic patients is mainly limited to supportive care, and novel therapeutic options are needed to improve the outcome of patients with abdominal sepsis. The gut forms a dynamic barrier regulating the passage of luminal content, such as microbes and their products. Pathologic inflammation in the gastrointestinal tract and breakdown of the gut barrier function are key hallmarks in sepsis [3e8]. Increased intestinal permeability is known to promote translocation and invasion of bacteria and their toxins into the systemic circulation [5,9]. Pathogenesis and treatment of inflammatory gut barrier failure is an important problem in critical care. The PPARg agonist pioglitazone is the most widely used thiazolidinedione (TZD) antidiabetic drug and currently the only TZD available without regulatory restrictions [29]. It is an oral drug usually provided in the form of a tablet, and no intravenous forms are available in clinical medicine for now. The cost of pioglitazone is relatively low and could be afforded by most of the patients. The precise mechanism of action of TZDs remains unclear, but the current consensus is that TZDs target the transcription factor PPARg to regulate transcription of genes involved in metabolic homeostasis and then improve insulin sensitivity. Besides its well-known role in lipid and glucose metabolism, PPARg represses transcription of genes related to inflammation via transrepression and antagonizes other transcription factors that activate inflammatory pathways, such as nuclear factor kappa-B and adaptor-related protein complex 1 [30]. In recent years, many scientific reports describing the influence of PPARg ligands on inflammation of intestines have been published. It is reported that PPARg ligands could protect animals against intestinal injury caused by different models of experimental colitis [12,17e19,21e23], Crohn disease [24], intestinal ischemiaereperfusion injury [20], necrotizing enterocolitis [31], and postoperative ileus [32]. Yet there has not been any evidence showing that the PPARg agonist can affect the sepsis-induced intestinal injury. We
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hypothesized pioglitazone may be therapeutically effective for attenuating sepsis-induced intestinal injury. We therefore evaluated the effects of pioglitazone in a rat model of CLPinduced intestinal injury. In the present study, we show for the first time that pioglitazone protects against intestinal injury in abdominal sepsis. At first, proinflammatory cytokines including TNF-a and IL-6 concentrations in the intestine were detected to evaluate the local inflammation of the intestine. CLP caused significant increase in TNF-a and IL-6 expressions in rat intestine at 24 h compared with the sham group as expected. Pioglitazone pretreatment decreased local production of TNF-a and IL-6 compared with the vehicle-treated CLP group. This result is consistent with other reports that demonstrated the potent inhibitory effect of pioglitazone on intestinal inflammation by decreasing cytokines expression [20e22]. Then, oxidative stress in the intestinal tissue was determined as another index to evaluate the CLP-induced intestinal injury. The role of oxidative stress in the pathogenesis of sepsis has been widely reported. Overproduction of reactive oxygen species is highly toxic to host tissues, and their interactions with various biological macromolecules can result in severe pathophysiologic consequences leading to multiple organ dysfunction syndrome [33,34]. SOD is the only antioxidant enzyme that can scavenge superoxide, and MDA is an indicator of lipid peroxidation level; these two were commonly known as markers of oxidative stress and antioxidant status. The present results showed that CLP caused significant increase in MDA content in intestinal tissue, whereas the SOD activity decreased greatly; pioglitazone pretreatment upregulated SOD activity and resulted in decrease in the MDA content expectedly. It is well-known that PPARg agonists have anti-inflammatory and antioxidant effects [35e37]. Our findings are in accordance with these reports that demonstrated excellent antioxidant capacity of pioglitazone. MPO, a marker of neutrophil recruitment, was also determined to evaluate the CLP-induced intestinal injury. In spite of its critical role in mounting host defense reactions, neutrophils can also cause significant tissue damage in inflammatory disease. Neutrophils can secrete destructive elastases and lipid mediators and reactive oxygen species with the capacity to damage both endothelial and epithelial cell function [38,39]. Convincing data have established neutrophil recruitment as a fundamental component in the pathophysiology of sepsis-induced intestinal injury [40,41]. The present results showed that CLP caused significant increase in the level of MPO content in intestinal tissue; pioglitazone pretreatment significantly decreased intestinal level of MPO, indicating that pioglitazone can decrease intestinal infiltration of neutrophils in abdominal sepsis. Sepsis is associated with disturbed gut barrier function, which facilitates translocation of pathogens and their toxins into the systemic circulation. In the present study, we examined the plasma D-lactic acid, intestinal DAO content, intestinal permeability to FD-4 and bacterial translocation to systemic circulation (blood), lymphatic organs (MLN), and liver to evaluate whether pioglitazone pretreatment exerts protective effect on intestinal epithelial barrier dysfunction for the first time. Plasma D-lactic acid and intestinal DAO are sensitive markers of intestinal permeability. D-lactic acid is a
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metabolic product of bacteria, and it is solely present in the intestinal lumen. When the integrity of intestinal mucosa is damaged, intestinal permeability increases and D-lactic acid could permeate through the bowel to get access to the bloodstream [42]. DAO is an intracellular enzyme with high activity existing in intestinal villous cells in both human beings and all other mammalians. Its activity is especially high in the jejunum and ileum. It is a relatively stable marker of maturation and integrity of intestinal mucosa cells [43]. The activity of DAO in intestinal mucosa decreases when its cells are injured. FD4 is commonly used to study passage of high molecular weight substances across the epithelium. The present results showed that on CLP challenge, there were multiple signs of intestinal barrier failure including high levels of plasma D-lactic acid, significant reduction of intestinal DAO content, and increased permeability to fluorescein isothiocyanate-dextran and bacterial translocation to blood, MLN, and liver. Pioglitazone pretreatment minimized all the mentioned signs of intestinal barrier failure, suggesting that PPARg plays an important role in septic intestinal damage, and pioglitazone might be a useful tool to maintain gut barrier integrity in abdominal sepsis. Morphology alteration of the intestine was also examined by hematoxylin and eosin staining. There was a great pathologic alteration that occurred at 24 h after development of CLPinduced sepsis, including the infiltration of inflammatory cells, intestinal mucosal villous edema, epithelial cells sloughing, villus tip destruction, villous necrosis, and mucosal erosion. Compared with vehicle-treated CLP group, a mild alteration of mucosa edema, exudation, microvilli structure, intestinal wall erosion, and invasion of inflammatory cells was observed in the pioglitazone pretreatment CLP group. The present data showed that histologic damage of intestinal mucosa is accompanied by the intestinal injury and barrier failure, which is consistent with other previous studies [44,45]. Taken together, the present study shows that pioglitazone decreases sepsis-induced inflammatory response, oxidative stress, and neutrophil infiltration in the intestine. Moreover, pioglitazone markedly reduced intestinal permeability and leakage, suggesting that it might be a useful tool to maintain intestinal barrier integrity and protect against sepsis-induced intestinal injury. Although we confirmed the beneficial effect of pioglitazone on sepsis-induced intestinal injury in a rodent model, the present study has some limitations that need to be addressed. First, we used CLP in this study because it is considered the gold standard model of sepsis and is thought to mimic a perforated viscous, endogenous fecal contamination, and variable progression of disease, which resembles clinical sepsis [46,47]. However, we recognize the difficulty in translating animal studies to humans; and it was conducted in previously healthy animals under highly controlled circumstances, in contrast to the clinical setting in which patients often have underlying illness and co-morbidities. Therefore, clinical trials are necessary to fully realize the potential use of pioglitazone in sepsis-induced intestinal injury. Second, the pioglitazone was administrated intraperitoneally (10 mg/kg/d) for 7 d before the CLP procedure in the present study and it does not represent medical practice. The use of pioglitazone at the time of CLP or for a longer period after CLP might have different results. Therefore, these results cannot be directly directed to
clinical use unless similar interventions and further studies have been taken in this sepsis model. Finally, randomized studies with larger numbers of animals and different dosages are needed to better clarify the role of pioglitazone in sepsis.
Acknowledgment This study was financially supported by grants from National Natural Science Foundation of China (81201486, 81373147). This study was performed at the Translational Medicine Center of Sepsis, Department of Pathophysiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China. Authors’ contributions: M.Y., X.X., and M.G. contributed to the conception and design. M.G. and Y.J. conducted the animal experiment, X.X. and Y.P. conducted the molecular biological detection. M.G. and Y.J. analyzed the data and drafted the article. All authors reviewed the article and approved of it.
Disclosure The authors have not disclosed any potential conflicts of interest. The work described here has not been published previously and is not under consideration for publication elsewhere, in whole or in part.
references
[1] Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003;348:138. [2] Wenzel RP, Edmond MB. Septic shockdevaluating another failed treatment. N Engl J Med 2012;366:2122. [3] Clark JA, Coopersmith CM. Intestinal crosstalk: a new paradigm for understanding the gut as the “motor” of critical illness. Shock 2007;28:384. [4] Hassoun HT, Kone BC, Mercer DW, et al. Post-injury multiple organ failure: the role of the gut. Shock 2001;15:1. [5] Fink MP, Delude RL. Epithelial barrier dysfunction: a unifying theme to explain the pathogenesis of multiple organ dysfunction at the cellular level. Crit Care Clin 2005;21:177. [6] Gatt M, Reddy BS, MacFie J. Review article: bacterial translocation in the critically illdevidence and methods of prevention. Aliment Pharmacol Ther 2007;25:741. [7] Alverdy JC, Chang EB. The re-emerging role of the intestinal microflora in critical illness and inflammation: why the gut hypothesis of sepsis syndrome will not go away. J Leukoc Biol 2008;83:461. [8] Deitch EA. Gut-origin sepsis: evolution of a concept. Surgeon 2012;10:350. [9] Deitch EA. Role of the gut lymphatic system in multiple organ failure. Curr Opin Crit Care 2001;7:92. [10] Schoonjans K, Martin G, Staels B, Auwerx J. Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol 1997;8:159. [11] Clark RB. The role of PPARs in inflammation and immunity. J Leukoc Biol 2002;71:388. [12] Dubuquoy L, Rousseaux C, Thuru X, et al. PPARgamma as a new therapeutic target in inflammatory bowel diseases. Gut 2006;55:1341. [13] Kiec-Wilk B, Dembinska-Kiec A, Olszanecka A, Bodzioch M, Kawecka-Jaszcz K. The selected pathophysiological aspects of PPARs activation. J Physiol Pharmacol 2005;56:149.
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
[14] Ptak-Belowska A, Pawlik MW, Krzysiek-Maczka G, Brzozowski T, Pawlik WW. Transcriptional upregulation of gastrin in response to peroxisome proliferator-activated receptor gamma agonist triggers cell survival pathways. J Physiol Pharmacol 2007;58:793. [15] Konturek PC, Dembinski A, Warzecha Z, et al. Pioglitazone, a specific ligand of peroxisome proliferator-activated receptorgamma, protects pancreas against acute cerulein-induced pancreatitis. World J Gastroenterol 2005;11:6322. [16] Konturek PC, Brzozowski T, Engel M, et al. Ghrelin ameliorates colonic inflammation. Role of nitric oxide and sensory nerves. J Physiol Pharmacol 2009;60:41. [17] Linard C, Gremy O, Benderitter M. Reduction of peroxisome proliferation-activated receptor gamma expression by gammairradiation as a mechanism contributing to inflammatory response in rat colon: modulation by the 5-aminosalicylic acid agonist. J Pharmacol Exp Ther 2008;324:911. [18] Adachi M, Kurotani R, Morimura K, et al. Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease. Gut 2006;55:1104. [19] Shah YM, Morimura K, Gonzalez FJ. Expression of peroxisome proliferator-activated receptor-gamma in macrophage suppresses experimentally induced colitis. Am J Physiol Gastrointest Liver Physiol 2007;292:G657. [20] Nakajima A, Wada K, Miki H, et al. Endogenous PPAR gamma mediates anti-inflammatory activity in murine ischemiareperfusion injury. Gastroenterology 2001;120:460. [21] Arita M, Yoshida M, Hong S, et al. Resolvin E1, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Proc Natl Acad Sci U S A 2005; 102:7671. [22] Ten HT, Corbaz A, Amitai H, et al. Blockade of endogenous IL-18 ameliorates TNBS-induced colitis by decreasing local TNF-alpha production in mice. Gastroenterology 2001;121: 1372. [23] Nakai M, Sudo K, Yamada Y, et al. The role of the tumor necrosis factor receptor in 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis in mice. Dig Dis Sci 2005;50:1669. [24] Hollenbach E, Vieth M, Roessner A, Neumann M, Malfertheiner P, Naumann M. Inhibition of RICK/nuclear factor-kappaB and p38 signaling attenuates the inflammatory response in a murine model of Crohn disease. J Biol Chem 2005;280:14981. [25] Tsujimura Y, Matsutani T, Matsuda A, et al. Effects of pioglitazone on survival and omental adipocyte function in mice with sepsis induced by cecal ligation and puncture. J Surg Res 2011;171:e215. [26] Kutsukake M, Matsutani T, Tamura K, et al. Pioglitazone attenuates lung injury by modulating adipose inflammation. J Surg Res 2014;189:295. [27] Clark JA, Gan H, Samocha AJ, Fox AC, Buchman TG, Coopersmith CM. Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis. Am J Physiol Gastrointest Liver Physiol 2009;297:G471. [28] Kajiya M, Silva MJ, Sato K, Ouhara K, Kawai T. Hydrogen mediates suppression of colon inflammation induced by dextran sodium sulfate. Biochem Biophys Res Commun 2009; 386:11. [29] Kawaguchi-Suzuki M, Frye RF. Current clinical evidence on pioglitazone pharmacogenomics. Front Pharmacol 2013;4:147.
9
[30] Cariou B, Charbonnel B, Staels B. Thiazolidinediones and PPARg agonists: time for a reassessment. Trends Endocrinol Metab 2012;23:205. [31] Baregamian N, Mourot JM, Ballard AR, Evers BM, Chung DH. PPAR-gamma agonist protects against intestinal injury during necrotizing enterocolitis. Biochem Biophys Res Commun 2009;379:423. [32] De Backer O, Elinck E, Priem E, Leybaert L, Lefebvre RA. Peroxisome proliferator-activated receptor gamma activation alleviates postoperative ileus in mice by inhibition of Egr-1 expression and its downstream target genes. J Pharmacol Exp Ther 2009;331:496. [33] Koksal GM, Sayilgan C, Aydin S, Oz H, Uzun H. Correlation of plasma and tissue oxidative stresses in intra-abdominal sepsis. J Surg Res 2004;122:180. [34] 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. [35] Zou C, Hu H, Xi X, Shi Z, Wang G, Huang X. Pioglitazone protects against renal ischemia-reperfusion injury by enhancing antioxidant capacity. J Surg Res 2013;184:1092. [36] El-Sheikh AA, Rifaai RA. Peroxisome proliferator activator receptor (PPAR)-gamma ligand, but not PPAR-alpha, ameliorates cyclophosphamide-induced oxidative stress and inflammation in rat liver. PPAR Res 2014;2014:626319. [37] Al RF, El EE. PPAR-gamma activator induces neuroprotection in hypercholesterolemic rats subjected to global cerebral ischemia/reperfusion injury: in vivo and in vitro inhibition of oxidative stress. Exp Gerontol 2014;51:1. [38] Swirski FK, Wildgruber M, Ueno T, et al. Myeloperoxidaserich Ly-6Cþ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice. J Clin Invest 2010;120:2627. [39] Grommes J, Vijayan S, Drechsler M, et al. Simvastatin reduces endotoxin-induced acute lung injury by decreasing neutrophil recruitment and radical formation. PLoS One 2012;7:e38917. [40] Asaduzzaman M, Rahman M, Jeppsson B, Thorlacius H. Pselectin glycoprotein-ligand-1 regulates pulmonary recruitment of neutrophils in a platelet-independent manner in abdominal sepsis. Br J Pharmacol 2009;156:307. [41] Zhang S, Zhang S, Rahman M, Herwald H, Thorlacius H. Streptococcal M1 protein-induced lung injury is independent of platelets in mice. Shock 2011;35:86. [42] Smith SM, Eng RH, Buccini F. Use of D-lactic acid measurements in the diagnosis of bacterial infections. J Infect Dis 1986;154:658. [43] Thompson JS, Vaughan WP, Forst CF, Jacobs DL, Weekly JS, Rikkers LF. The effect of the route of nutrient delivery on gut structure and diamine oxidase levels. JPEN J Parenter Enteral Nutr 1987;11:28. [44] Shebrain S, Ramjit A. Radicicol, a Hsp90 inhibitor, inhibits intestinal inflammation and leakage in abdominal sepsis. J Surg Res 2013;185:e53. [45] Short SS, Wang J, Castle SL, et al. Low doses of celecoxib attenuate gut barrier failure during experimental peritonitis. Lab Invest 2013;93:1265. [46] Wichterman KA, Baue AE, Chaudry IH. Sepsis and septic shockda review of laboratory models and a proposal. J Surg Res 1980;29:189. [47] Hubbard WJ, Choudhry M, Schwacha MG, et al. Cecal ligation and puncture. Shock 2005;24(Suppl 1):52.
