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 1 8 7 ( 2 0 1 4 ) 6 1 0 e6 1 5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Protective effect of leflunomide against oxidative intestinal injury in a rodent model of sepsis Erdogan Ozturk, MD,a,* Murat Surucu, MD,b Abdurrahman Karaman, MD,c Emine Samdancı, MD,d and Ersin Fadillioglu, MDe a
Department of Anesthesiology and Reanimation, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey Sehit Kamil Devlet Hastanesi, Gaziatep, Turkey c Department of Pediatric Surgery, Faculty of Medicine, Inonu University, Malatya, Turkey d Department of Pathology, Faculty of Medicine, Inonu University, Malatya, Turkey e Department of Physiology, Hacettepe University, Ankara, Turkey b
article info
abstract
Article history:
Background: Sepsis is defined as an uncontrolled inflammatory response in a host. The
Received 7 August 2013
process may lead to severe sepsis, multisystem organ failure and even death. Leflunomide
Received in revised form
has important immunomodulatory and anti-inflammatory effects, which may mitigate
27 October 2013
host response to bacterial translocation. The goal of our study was to measure the effects
Accepted 6 November 2013
leflunomide administration had on a variety of biochemical markers upregulated in sys-
Available online 26 November 2013
temic inflammatory response syndrome, sepsis, and multiple organ failure syndrome. Materials and methods: Wistar albino type rats were randomly divided into five groups:
Keywords:
control, sham, leflunomide, sepsis, and sepsis þ leflunomide. Sepsis was achieved by
Sepsis
means of the cecal ligation and puncture method. Leflunomide 2 10 mg/kg/d was
Leflunomide
administered before the experiment. At the end of 24 h, the tissue levels of superoxide
Oxidative stress
dismutase, catalase activity, malondialdehyde, nitric oxide, and protein carbonyl were measured. Results: The level of the bowel superoxide dismutase and catalase levels of the sepsis group is significantly lower than those of the control, sham, and leflunomide groups (P < 0.05). Malondialdehyde, nitric oxide, and protein carbonyl levels are significantly higher in sepsis compared with other groups (P < 0.05). Conclusions: Leflunomide’s prevention of protein and lipid peroxidation was observed in septic bowel tissue. Use of leflunomide could have protective effects against both the onset and the progressive stages of sepsis. Crown Copyright ª 2014 Published by Elsevier Inc. All rights reserved.
1.
Introduction
Sepsis is a leading cause of morbidity and mortality in intensive care units. The gastrointestinal tract plays a critical role in sepsis pathogenesis [1]. A main function of the intestine is
absorption of nutrients by epithelial cells lining the lumen of the bowel (gut). Normally in the bowel, homeostasis exists between the intraluminal bacteria, their products, and the intestinal mucosal barrier [2]. Patients with abdominal inflammation have mucosal barrier dysfunction, so that
* Corresponding author. Department of Anesthesiology and Reanimation, Faculty of Medicine, Bezmialem Vakif University, 34093 Fatih, Istanbul, Turkey. Tel.: þ0 212 453 1700; fax: þ0 212 621 7580. E-mail address: [email protected] (E. Ozturk). 0022-4804/$ e see front matter Crown Copyright ª 2014 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.11.005
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 1 8 7 ( 2 0 1 4 ) 6 1 0 e6 1 5
intestinal bacteria and toxins can trigger, maintain, and exacerbate the systemic inflammatory response syndrome and multiple organ failure syndrome [3]. During a sepsis crisis, ileus and intestinal mucosal barrier dysfunction are the most frequently encountered gastrointestinal tract responses. Ileus promotes development of secondary infections and multiple organ failure through bacterial stasis, bacterial overgrowth, and bacterial translocation [4]. Oxidative stress is defined as an imbalance between the production of oxygen radicals and antioxidant defenses system. Oxidants are generated as a result of the inflammatory response by phagocytic cells. Oxidative stress in laboratory animal models of sepsis has been good defined. Oxidative stress is initiated by reactive oxygen species (ROS) including superoxide anion (O 2 ), hydrogen peroxide (H2O2), and hydroxyl radical (OH). The formation of O 2 , H2O2, and OH is the main pathway responsible for the production of ROS. Pathophysiological mechanisms that cause the injury of the mucosal barrier are the adhesion of polymorphonuclear neutrophils and activation of their release of proinflammatory cytokines, as well as the formation of O 2 , OH, H2O2, and reactive nitrogen species [2]. Leflunomide (N-[4-triflurormethylphenyl]-5-methylisoxazol4-carboxamide) is an orally administrated prodrug. The active drug A771726 (teriflunomide) formed nonenzymatically in the intestinal mucosa from leflunomide, has immunosuppressive, immunoregulatory, and anti-inflammatory effects against various disease including rheumatoid arthritis [5]. Several molecular pathways of inflammatory and cellular injury have been made responsible in the pathogenesis of sepsis, septic shock, and multiple organ failure. Some of these mechanisms include associated with excessive production of cytokines, ROS, and eicosanoids [2,3]. We designed this study to investigate the antioxidant properties of leflunomide, as immunoregulatory agent, on induced abdominal sepsis. The intestinal mucosa was examined by histologic and biochemical approaches such as the measurement of superoxide dismutase (SOD), catalase (CAT) activity, nitric oxide (NO), malondialdehyde (MDA), and protein carbonyl (PC) levels.
2.
Materials and methods
2.1.
Animals and specimens
Male Wistar albino rats (250e300 g) were used in the present study. The all procedures were done according to local ethical committee (Inonu University School of Medicine’s Animal Care and Use Committee). The individual cages with constant temperature and darkelight cycles were used for maintenance. The animals were fed with standard rat chow and tap water ad libitum. The rats were fasted before 12 h the experiment, except water. A total of 50 rats were equally divided into control, sham, leflunomide, sepsis, and sepsis þ leflunomide groups. Before the experiments, the rats within leflunomide and sepsis þ leflunomide groups were received 10 mg/kg intragastric leflunomide (Arava; Aventis Pharmaceuticals, Compiegne, France) for two doses with an 8 h interval [5]. Equal volume of saline was applied to control group’s rats with the same interval.
2.2.
611
Surgical procedure
Bacterial translocation causes sepsis with a mechanism contains intestinal obstruction, obstructive jaundice, intraabdominal hypertension, intestinal ischemiaereperfusion injury, and ileus. Polymicrobial sepsis was induced by the cecal ligation and puncture (CLP) technique described by Wichterman et al. [6]. The CLP model is an intra-abdominal sepsis model that has been used frequently [7]. After ketamine (75 mg/kg)exylazine (8 mg/kg) anesthesia, povidone-iodine was used for disinfection and the surgical procedures were done under sterile conditions. The midline laparotomy was performed to reach the cecum. The ligation of cecum was done just below the ileocecal valve with 3-0 silk sutures. During this ligation, intestinal continuity was conserved. The perforation was done on two locations of antimesenteric surface of the cecum with an 18-gauge needle. The fecal matter was gently extruded with compression on cecum. Then the bowel was replaced into the abdomen. After the operation, 3 mL/100 g body weight of saline was given subcutaneously. The animals were allowed to drink water, but not food. The laparotomy without perforation was performed in sham group. The laparotomy was performed in sham group and the cecum was manipulated without ligation or perforation. There were no antibiotic applications to rats. At the end of the 24 h of the surgical application, the rats were sacrificed under anesthesia. The bowel of all groups were removed and washed with ice-cold saline. The samples were kept for biochemical and light microscopic examinations. The samples for the measurements of oxidant/antioxidant parameters were kept at 80 C until processing.
2.3.
Biochemical measures
The tissue samples were homogenized in Tris-HCL buffer (1:5 wt/vol, pH 7.4; Potter Elvehjem glass homogenizer) for measurements of oxidant/antioxidant parameters. The homogenized samples were centrifuged at 3500 g for 45 min at 4 C to separate supernatant. The supernatant was mixed with same volume of chloroform and ethanol mixture (3/5 vol/vol) and centrifuged at 3500 g for 45 min at 4 C to separate the extracted part. The determinations of protein were performed according to Lowry et al. [8] using bovine serum albumin as standard. Total (CueZn and Mn) SOD (EC 1.15.1.1) activity was determined within ethanol phase of the extracted samples [9]. The method is mainly based on the inhibition of nitrobluetetrazolium reduction. Fifty percent inhibition of nitrobluetetrazolium reduction rate was used as one unit enzyme activity. The CAT (EC 1.11.1.6) activity of bowel tissue was measured with a method based on decomposition rate of the H2O2 [10] and it was evaluated in whole bowel homogenates. The measurement of NO level was based on the Griess’ reaction [11]. Somogyi reagent was used for deproteinization of the homogenized samples. The total nitrite (nitrite þ nitrate) was measured at 545 nm with the conversion of nitrate to nitrite by copperized cadmium granules. The bowel PC was determined in the homogenized samples after the reaction of the carbonyl groups of proteins with
612
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 1 8 7 ( 2 0 1 4 ) 6 1 0 e6 1 5
Table 1 e Histologic grading system. Grade
Histologic appearance
0 1 2 3 4 5
Normal Subepithelial edema, partial separation of apical cells Epithelial cells slough from tips of villi Progression of slough to base of villi Partial mucosal necrosis of lamina propria Total mucosal necrosis
2,4-dinitrophenylhydrazine [12]. The end of this reaction, 2,4dinitrophenylhydrazone, was measured by spectrophotometer. The tissue MDA level represents lipid peroxidation. The reaction of thiobarbituric acid (TBA) with MDA at 90e100 C produces a pink color [13]. The trichloroacetic acid 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.
2.4.
Histopathology
Before the dehydration of the histologic samples, 2-cm long ileum specimens were fixed with 10% formaldehyde, and then embedded in paraffin wax. The samples were sectioned (5 mm) and stained with hematoxylin and eosin (H&E). Two separate investigators using light microscopy performed the blind histologic evaluation [14] (Table 1).
2.5.
Statistical analysis
Data were analyzed with a commercially available statistics software package (SPSS for Windows 10.0, Chicago). KolmogoroveSmirnov test was used to analyze the distributions of the groups. The comparisons of the groups were done with one-way analysis of variance (ANOVA) test with Least Significant Difference Test (LSD) as post hoc multiple comparisons. The results are presented as mean standard deviation and p values
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Protective effect of leflunomide against oxidative intestinal injury in a rodent model of sepsis Erdogan Ozturk, MD,a,* Murat Surucu, MD,b Abdurrahman Karaman, MD,c Emine Samdancı, MD,d and Ersin Fadillioglu, MDe a
Department of Anesthesiology and Reanimation, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey Sehit Kamil Devlet Hastanesi, Gaziatep, Turkey c Department of Pediatric Surgery, Faculty of Medicine, Inonu University, Malatya, Turkey d Department of Pathology, Faculty of Medicine, Inonu University, Malatya, Turkey e Department of Physiology, Hacettepe University, Ankara, Turkey b
article info
abstract
Article history:
Background: Sepsis is defined as an uncontrolled inflammatory response in a host. The
Received 7 August 2013
process may lead to severe sepsis, multisystem organ failure and even death. Leflunomide
Received in revised form
has important immunomodulatory and anti-inflammatory effects, which may mitigate
27 October 2013
host response to bacterial translocation. The goal of our study was to measure the effects
Accepted 6 November 2013
leflunomide administration had on a variety of biochemical markers upregulated in sys-
Available online 26 November 2013
temic inflammatory response syndrome, sepsis, and multiple organ failure syndrome. Materials and methods: Wistar albino type rats were randomly divided into five groups:
Keywords:
control, sham, leflunomide, sepsis, and sepsis þ leflunomide. Sepsis was achieved by
Sepsis
means of the cecal ligation and puncture method. Leflunomide 2 10 mg/kg/d was
Leflunomide
administered before the experiment. At the end of 24 h, the tissue levels of superoxide
Oxidative stress
dismutase, catalase activity, malondialdehyde, nitric oxide, and protein carbonyl were measured. Results: The level of the bowel superoxide dismutase and catalase levels of the sepsis group is significantly lower than those of the control, sham, and leflunomide groups (P < 0.05). Malondialdehyde, nitric oxide, and protein carbonyl levels are significantly higher in sepsis compared with other groups (P < 0.05). Conclusions: Leflunomide’s prevention of protein and lipid peroxidation was observed in septic bowel tissue. Use of leflunomide could have protective effects against both the onset and the progressive stages of sepsis. Crown Copyright ª 2014 Published by Elsevier Inc. All rights reserved.
1.
Introduction
Sepsis is a leading cause of morbidity and mortality in intensive care units. The gastrointestinal tract plays a critical role in sepsis pathogenesis [1]. A main function of the intestine is
absorption of nutrients by epithelial cells lining the lumen of the bowel (gut). Normally in the bowel, homeostasis exists between the intraluminal bacteria, their products, and the intestinal mucosal barrier [2]. Patients with abdominal inflammation have mucosal barrier dysfunction, so that
* Corresponding author. Department of Anesthesiology and Reanimation, Faculty of Medicine, Bezmialem Vakif University, 34093 Fatih, Istanbul, Turkey. Tel.: þ0 212 453 1700; fax: þ0 212 621 7580. E-mail address: [email protected] (E. Ozturk). 0022-4804/$ e see front matter Crown Copyright ª 2014 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.11.005
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 1 8 7 ( 2 0 1 4 ) 6 1 0 e6 1 5
intestinal bacteria and toxins can trigger, maintain, and exacerbate the systemic inflammatory response syndrome and multiple organ failure syndrome [3]. During a sepsis crisis, ileus and intestinal mucosal barrier dysfunction are the most frequently encountered gastrointestinal tract responses. Ileus promotes development of secondary infections and multiple organ failure through bacterial stasis, bacterial overgrowth, and bacterial translocation [4]. Oxidative stress is defined as an imbalance between the production of oxygen radicals and antioxidant defenses system. Oxidants are generated as a result of the inflammatory response by phagocytic cells. Oxidative stress in laboratory animal models of sepsis has been good defined. Oxidative stress is initiated by reactive oxygen species (ROS) including superoxide anion (O 2 ), hydrogen peroxide (H2O2), and hydroxyl radical (OH). The formation of O 2 , H2O2, and OH is the main pathway responsible for the production of ROS. Pathophysiological mechanisms that cause the injury of the mucosal barrier are the adhesion of polymorphonuclear neutrophils and activation of their release of proinflammatory cytokines, as well as the formation of O 2 , OH, H2O2, and reactive nitrogen species [2]. Leflunomide (N-[4-triflurormethylphenyl]-5-methylisoxazol4-carboxamide) is an orally administrated prodrug. The active drug A771726 (teriflunomide) formed nonenzymatically in the intestinal mucosa from leflunomide, has immunosuppressive, immunoregulatory, and anti-inflammatory effects against various disease including rheumatoid arthritis [5]. Several molecular pathways of inflammatory and cellular injury have been made responsible in the pathogenesis of sepsis, septic shock, and multiple organ failure. Some of these mechanisms include associated with excessive production of cytokines, ROS, and eicosanoids [2,3]. We designed this study to investigate the antioxidant properties of leflunomide, as immunoregulatory agent, on induced abdominal sepsis. The intestinal mucosa was examined by histologic and biochemical approaches such as the measurement of superoxide dismutase (SOD), catalase (CAT) activity, nitric oxide (NO), malondialdehyde (MDA), and protein carbonyl (PC) levels.
2.
Materials and methods
2.1.
Animals and specimens
Male Wistar albino rats (250e300 g) were used in the present study. The all procedures were done according to local ethical committee (Inonu University School of Medicine’s Animal Care and Use Committee). The individual cages with constant temperature and darkelight cycles were used for maintenance. The animals were fed with standard rat chow and tap water ad libitum. The rats were fasted before 12 h the experiment, except water. A total of 50 rats were equally divided into control, sham, leflunomide, sepsis, and sepsis þ leflunomide groups. Before the experiments, the rats within leflunomide and sepsis þ leflunomide groups were received 10 mg/kg intragastric leflunomide (Arava; Aventis Pharmaceuticals, Compiegne, France) for two doses with an 8 h interval [5]. Equal volume of saline was applied to control group’s rats with the same interval.
2.2.
611
Surgical procedure
Bacterial translocation causes sepsis with a mechanism contains intestinal obstruction, obstructive jaundice, intraabdominal hypertension, intestinal ischemiaereperfusion injury, and ileus. Polymicrobial sepsis was induced by the cecal ligation and puncture (CLP) technique described by Wichterman et al. [6]. The CLP model is an intra-abdominal sepsis model that has been used frequently [7]. After ketamine (75 mg/kg)exylazine (8 mg/kg) anesthesia, povidone-iodine was used for disinfection and the surgical procedures were done under sterile conditions. The midline laparotomy was performed to reach the cecum. The ligation of cecum was done just below the ileocecal valve with 3-0 silk sutures. During this ligation, intestinal continuity was conserved. The perforation was done on two locations of antimesenteric surface of the cecum with an 18-gauge needle. The fecal matter was gently extruded with compression on cecum. Then the bowel was replaced into the abdomen. After the operation, 3 mL/100 g body weight of saline was given subcutaneously. The animals were allowed to drink water, but not food. The laparotomy without perforation was performed in sham group. The laparotomy was performed in sham group and the cecum was manipulated without ligation or perforation. There were no antibiotic applications to rats. At the end of the 24 h of the surgical application, the rats were sacrificed under anesthesia. The bowel of all groups were removed and washed with ice-cold saline. The samples were kept for biochemical and light microscopic examinations. The samples for the measurements of oxidant/antioxidant parameters were kept at 80 C until processing.
2.3.
Biochemical measures
The tissue samples were homogenized in Tris-HCL buffer (1:5 wt/vol, pH 7.4; Potter Elvehjem glass homogenizer) for measurements of oxidant/antioxidant parameters. The homogenized samples were centrifuged at 3500 g for 45 min at 4 C to separate supernatant. The supernatant was mixed with same volume of chloroform and ethanol mixture (3/5 vol/vol) and centrifuged at 3500 g for 45 min at 4 C to separate the extracted part. The determinations of protein were performed according to Lowry et al. [8] using bovine serum albumin as standard. Total (CueZn and Mn) SOD (EC 1.15.1.1) activity was determined within ethanol phase of the extracted samples [9]. The method is mainly based on the inhibition of nitrobluetetrazolium reduction. Fifty percent inhibition of nitrobluetetrazolium reduction rate was used as one unit enzyme activity. The CAT (EC 1.11.1.6) activity of bowel tissue was measured with a method based on decomposition rate of the H2O2 [10] and it was evaluated in whole bowel homogenates. The measurement of NO level was based on the Griess’ reaction [11]. Somogyi reagent was used for deproteinization of the homogenized samples. The total nitrite (nitrite þ nitrate) was measured at 545 nm with the conversion of nitrate to nitrite by copperized cadmium granules. The bowel PC was determined in the homogenized samples after the reaction of the carbonyl groups of proteins with
612
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 1 8 7 ( 2 0 1 4 ) 6 1 0 e6 1 5
Table 1 e Histologic grading system. Grade
Histologic appearance
0 1 2 3 4 5
Normal Subepithelial edema, partial separation of apical cells Epithelial cells slough from tips of villi Progression of slough to base of villi Partial mucosal necrosis of lamina propria Total mucosal necrosis
2,4-dinitrophenylhydrazine [12]. The end of this reaction, 2,4dinitrophenylhydrazone, was measured by spectrophotometer. The tissue MDA level represents lipid peroxidation. The reaction of thiobarbituric acid (TBA) with MDA at 90e100 C produces a pink color [13]. The trichloroacetic acid 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.
2.4.
Histopathology
Before the dehydration of the histologic samples, 2-cm long ileum specimens were fixed with 10% formaldehyde, and then embedded in paraffin wax. The samples were sectioned (5 mm) and stained with hematoxylin and eosin (H&E). Two separate investigators using light microscopy performed the blind histologic evaluation [14] (Table 1).
2.5.
Statistical analysis
Data were analyzed with a commercially available statistics software package (SPSS for Windows 10.0, Chicago). KolmogoroveSmirnov test was used to analyze the distributions of the groups. The comparisons of the groups were done with one-way analysis of variance (ANOVA) test with Least Significant Difference Test (LSD) as post hoc multiple comparisons. The results are presented as mean standard deviation and p values
