- Email: [email protected]
Effects of Nuclear Factor-κB Inhibitors on Colon Anastomotic Healing in Rats1
Journal of Surgical Research 171, 355–360 (2011) doi:10.1016/j.jss.2010.01.028
Effects of Nuclear Factor-kB Inhibitors on Colon Anastomotic Healing in Rats1 Abdulkadir Bedirli, M.D.,*,2 Bulent Salman, M.D.,* Hatice Pasaoglu, M.D.,† Ebru Ofluoglu, Ph.D.,† and Omer Sakrak, M.D.* *Department of General Surgery; and †Department of Biochemistry, Gazi University Medical School, Ankara, Turkey Submitted for publication November 30, 2009
Background. Nuclear factor (NF)-kB plays an essential role in inflammation. We tested this role by administering NF-kB-inhibitors into rats undergoing a well-established model of colonic anastomotic healing. Methods. Wistar rats underwent laparotomy, descending colonic transection, and handsewn reanastomosis. The animals were randomized to receive either a selective NF-kB inhibitor (parthenolide 0.5 mg/kg or resveratrol 0.5 mg/kg) or an equal volume of water by gavages before operation and then daily after surgery. Animals were sacrificed either immediately after anastomotic construction (d 0) or at the third, fifth, or seventh postoperative day. Results. Both parthenolide and resveratrol treatment led to early significant increases in plasma levels of IL-6. On d 7, hydroxyproline levels were significantly higher in the parthenolide and resveratrol groups. A similar pattern was observed with the bursting pressure. In contrast, gelatinase activity (MMP-2 and MMP-9 expression) was significantly higher in the control group on postoperative d 3. On d 3, expression of NF-kB activity was up-regulated in the anastomotic area. Both parthenolide and resveratrol completely attenuated NF-kB activity. Study groups also developed more marked inflammatory cell infiltration and collagen deposition on histology analysis. Conclusions. Parthenolide and resveratrol significantly improved healing and mechanical stability of colonic anastomoses in rats during the early postoperative period. Both agents may be acting to accelerate the host reparative process as well as to enhance pro-
1 Presented at the 42nd Congress of the European Society for Surgical Research, May 23–26, 2007, Rotterdam, The Netherlands. 2 To whom correspondence and reprint requests should be addressed at A.Taner Kislali Mah, Siyasal Villari, No. 44, 06810, Cayyolu, Ankara, Turkey. E-mail: [email protected].
tection of the anastomotic wound bed.
Ó 2011 Elsevier Inc.
All rights reserved.
Key Words: anastomosis; healing; colon; NF-kB; matrix metalloproteinases.
INTRODUCTıON
Wound healing is a complex insult-initiated biologic process that involves coordination and recruitment of multiple cellular and molecular events, and affects growth factor-mediated extracellular matrix (ECM) homeostasis [1]. An ECM is a dynamic superstructure of self-aggregating macromolecules, including fibronectin, collagen, and proteoglycans, to which cells attach by means of surface receptors called integrins, forming a three-dimensional supporting scaffold that isolates tissue compartments, mediates cell attachment, and determines tissue architecture [2]. Anastomotic wound healing in the gastrointestinal tract differs from the previously described process in that a controlled, full-thickness injury of limited duration is applied. Wound strength is determined by the amount and quality of newly-synthesized and deposited collagen, and by the degradation of preformed collagen. Collagenase activity is important in determining anastomotic integrity and suture-holding capacity during the first few days of healing. Remodeling of ECM by the matrix metalloproteinases (MMPs), a large family of zinc dependent neutral endopeptidases, is central to a wide number of physiologic and pathologic processes [3]. The induction of MMP gene expression, including MMP-1 (interstitial collagenase), MMP-3 (stromelysin-1), and MMP-9 (gelatinase B), can occur due to a wide range of stimuli, including inflammatory cytokines, growth
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FIG. 1. The classical NF-kB pathway is activated by a variety of inflammatory signals, resulting in coordinate expression of multiple inflammatory and innate immune genes. The proinflammatory cytokines IL-1b and TNF-a activate NF-kB, and their expression is induced in response to NF-kB activation, thus forming an amplifying feed forward loop.
factors, phorbol esters, oxidant stress, and mechanical injury [4, 5]. These cytokines and growth factors that are up-regulated in epithelial cells further contribute to tissue inflammation. A majority of inflammatory cytokines use the nuclear factor (NF)-kB pathway for signaling on ligand binding to cell surface receptor (Fig. 1). IL-1b and TNF-a rapidly activate NF-kB, a regulator of genes involved in inflammation and immune responses [6, 7]. Levels of these inflammatory cytokines and active NF-kB are elevated during proliferative inflammatory conditions, including wound healing, rheumatoid arthritis, and atherosclerosis, and correlate with increased levels of MMP-1, MMP-3, and MMP-9 enzymes [4, 8–10]. We therefore hypothesized that blocking the NF-kB pathway might be beneficial in strengthening colonic anastomoses. In addition, measurements were taken to assess whether NF-kB inhibitors increased the presence of MMP-2 and MMP-9 in the anastomotic areas. MATERIALS AND METHODS The experimental protocols were conducted with the approval of the Animal Research Committee at Gazi University, Ankara. All animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Animals and Experimental Design Male Wistar rats (n ¼ 120; weight 230–280 g) were housed two per cage and allowed to become acclimated with laboratory conditions for
7 d before the experiment. The animal rooms were windowless with temperature (22 6 20 C) and lighting controls. The rats were divided into three groups (n ¼ 30 per group) in a randomized manner: control, parthenolide, and resveratrol. The control group received isovolumetric saline solution, the parthenolide group received 0.5 mg/kg parthenolide (Sigma-Aldrich Corp., St. Louis, MO) and the resveratrol group received 0.5 mg/kg resveratrol (Sigma-Aldrich Corp.) orally by gavages for 7 d before the operation and then daily after surgery until the time of sacrifice. The animals were fasted overnight before the experiments but were given free access to water. They were anesthetized with ketamine 100 mg/kg and xylazine 20 mg/kg body weight, intraperitoneally. They were left to breathe spontaneously throughout the procedures. The abdominal skin was disinfected with 70% alcohol. All procedures were performed under sterile conditions by a surgeon who was blinded to the treatment groups the animals. For the determination of plasma interleukin (IL)-6 levels, 0.5 mL of blood was collected from all animals by transecting the tip of their tail. Then a midline incision was made, approximately 3 cm long, sufficient to expose the colon and rectum. For the colonic anastomosis, approximately 3 cm proximal to the peritoneal reflection, a 1 cm segment was resected and colonic continuity was restored by an end-to-end anastomosis, using eight to 10 inverting interrupted sutures (Ethilon 8/0; Ethicon, Norderstedt, Germany) depending on the diameter of the colon. The abdomen was washed out with sterile saline and closed with a running suture using 4-0 vicryl (Ethicon). After the operation, animals were kept in their cages, and they were allowed food and drink ad libitum.
Macroscopic Intra-abdominal Analysis Ten rats from each group were sacrificed by intracardiac puncture after anesthesia with ketamine 100 mg/kg and xylazine 20 mg/kg either immediately after anastomotic construction (d 0) or at the third, fifth, or seventh postoperative day. The abdomen was reopened and inspected for anastomotic dehiscence, peritonitis, ileus, and the formation of adhesions. Peritonitis was defined as macroscopic signs of inflammation, fibrin deposition, and hypervascularity. Mechanical obstruction was defined as obstruction of the intestine caused by the suture, with distention proximal to the anastomosis but not distal to the anastomosis. In addition, all surrounding structures adhesive to the anastomosis (colon, intestine, omentum, and pelvic fatty tissue) were considered separate adhesions.
Plasma Interleukin-6 Levels Serum samples for IL-6 were frozen at –70 C until they were assayed in triplicate. The IL-6 concentrations of serum were determined by enzym e-linked immunosorbent assay (ELISA; R&D Systems, Munich, Germany).
Mechanical Analysis Anastomotic segments, approximately 4 cm in length, with the suture line in the middle, were carefully resected, including surrounding tissues and adhesions, and washed in saline. To measure bursting pressure, the segments were infused (2 mL/min) with 0.9% NaCl containing methylene blue. The maximum pressure (mm Hg) recorded immediately before sudden loss of pressure was taken as the bursting pressure. The site of rupture (within or outside the anastomotic line) was noted. Once the bursting pressure was recorded, the anastomosis site was resected along with a 5 mm distal and proximal portion of the colon, and anastomosis was opened longitodinally and divided into two equal longitudinal segments. One segment was placed in 4% formalin solution for histologic assessment. The second segment of the anastomosis site was frozen in liquid nitrogen and stored at –80 C until further processing.
BEDIRLI ET AL.: EFFECTS OF NUCLEAR FACTOR-kB INHIBITORS
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Determination of Hydroxyproline, MMP-2, and MMP-9 Levels Anastomotic hydroxyproline content was measured according to the method of Jamall et al. [11]. Briefly, 25 mg of homogenate taken from hydrolyzation was lyophilisated and dissolved in the 1 mL 50% (vol/vol) isopropyl alcohol. Ten minutes later, chloramine T was added to these samples. Next, the samples were incubated for 90 min at 50 C after adding 1 mL Erlich’s reagent. Absorbance was measured at 560 nm, and the amount of hydroxyproline was calculated from a standard curve constructed using hydroxy-L-proline at concentrations of 0.25 to 40 mg/mL. Results were expressed as mg/mg tissue. MMP-2 and MMP-9 concentrations in tissue supernatants were determined with enzyme immunoassays performed according to the instructions of the manufacturers. Rat MMP-2 and MMP-9 activity assay systems were purchased from Amersham Biosciences Corp., Piscataway, NJ. Duplicate evaluations were performed for each sample. Quantification of the reaction product was achieved by measuring the absorbance at 405 nm using a spectrophotometer.
Tissue NF-kB Activity Tissues were homogenized in 10 volumes of cold 0.01 M Tris-HCI Buffer (pH 7.4) using an automatic homogenizer. NF-kB activity in tissue supernatants were determined with enzyme immunoassays by the use of a commercial enzyme-linked immunosorbent assay (ELISA) kit (Panomics Inc., Redwood City, CA) according to the manufacturer’s guidelines. All samples were tested in duplicate.
Histology The sections were stained with hematoxylin and eosin and the area of anastomosis was graded histologically in a blind fashion using a numerical scale. Inflammatory cell infiltration, fibroblast activity, and collogen deposition were graded from 0 to 4 as follows: 0 ¼ no evidence, 1 ¼ occasional evidence, 2 ¼ light scattering, 3 ¼ abundant evidence, and 4 ¼ confluent cells or fibers.
Statistical Analysis All values were expressed as the mean 6 SD. Data were compared by analysis of variance with post hoc analysis using the NewmanKeuls test. When a difference was found, specific differences were identified by using the Kruskal-Wallis test. Statistical evaluation was carried out using SPSS 10.0 software (SPSS, Chicago, IL). Values of P < 0.05 were accepted as significant.
FIG. 2. Anastomotic bursting pressure in rats after anastomosis. Each bar represents the mean 6 SD. *P < 0.01, versus control group.
third postoperative day. At this time, anastomotic rupture occurred within the suture line in all animals. The anastomotic bursting pressure showed an increase from d 3 onwards in all groups (Fig. 2). There was no difference between the control and parthenolide-or resveratrol-treated animals at postoperative d 3 and 5. In contrast, the average bursting pressure was significantly higher in the parthenolide and resveratrol groups than in the control group 7 d postoperatively (P < 0.01). During the bursting pressure measurement, all ruptures occurred outside the suture line on d 7. Plasma Interleukin-6 Levels
Plasma IL-6 levels are shown in Fig. 3. On postoperative d 3, concentration of IL-6 were significantly elevated in groups parthenolide and resveratrol, compared with control group (P < 0.01). However, on postoperative d 5–7, no significant difference was found among the groups. Hydroxyproline Concentration
RESULTS General Observations
There were no significant differences between the groups in preoperative weight, dietary intake, or pattern of weight change after surgery. Four animals in the control group developed colonic anastomotic complications compared with none in the groups receiving parthenolide or resveratrol (P ¼ 0.042). Two developed colonic perianastomotic abscesses, one developed mechanical obstruction by d 3, and one had colonic anastomotic dehiscence by d 7. Anastomotic Strength
The strength of the anastomoses as determined by measuring colonic bursting pressure was low on the
The hydroxyproline concentration in the 5-mm tissue containing the actual anastomosis was assayed as a measure for the presence of collagen. Figure 4 shows that administration of selective NF-kB inhibitors led to an increased anastomotic hydroxyproline concentration on d 7 (P < 0.05). Average hydroxyproline concentrations were similar between the parthenolide and resveratrol groups at all time points. MMP-2 and MMP-9 Expression
MMP activity measured in the colonic anastomoses was similar in all groups on d 3. After treatment with NF-kB inhibitors, there was a significant decrease compared with the control in mean MMP-2 and MMP-9 expression on d 5 (P < 0.05) (Figs. 5 and 6).
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FIG. 3. Interleukin (IL)-6 expression in plasma. Each bar represents the mean 6 SD. *P < 0.05, versus control group.
Expression of NF-kB
NF-kB concentration in the parthenolide- or resveratrol-treated rats decreased by approximately 65% on postoperative d 3 and 5 compared with values in the control group. No differences between resveratrol-treated animals and parthenolide-treated animals were noted for either day (Fig. 7). Histology
The wound healing process as assessed from inflammatory cell infiltration, collagen deposition, and fibroblast activity is presented in Table 1. Specifically, the parthenolide and resveratrol groups developed more marked fibroblast activity and collagen deposition than the control group (P < 0.05). Similarly, parthenolide or resveratrol treatment developed significantly more marked inflammatory cell infiltration than the control group (P < 0.05). DISCUSSION
In these experiments, we investigated the effects of parthenolide and resveratrol on cellular and molecular
FIG. 4. Data represent hydroxyproline concentration (mg/mg tissue) after anastomosis and are expressed as mean 6 SD. *P < 0.05, versus control group.
FIG. 5. MMP-2 expression in anastomotic tissue. Each bar represents the mean 6 SD. *P < 0.05, versus control group.
events in the early inflammatory stage of anastomotic wound healing in rats. Our data demonstrate that parthenolide and resveratrol treatment increased anastomotic hydroxyproline concentrations and reduced specific markers of the inflammatory response, including MMP-2 and MMP-9 expression, during the early inflammatory stage of wound healing. Moreover, these experiments demonstrated that parthenolide and resveratrol treatment had beneficial effects on the colonic anastomotic complications and the strength of the colonic anastomoses in rats. Wound healing consists of three phases: inflammation, proliferation, and maturation and remodeling. These phases proceed, overlapping each other, and are regulated by a complicated array of cytokines and growth factors secreted by inflammatory and resident cells [12, 13]. The first phase of the healing, described as the inflammatory phase, is seen on the first 3 d of the healing process. IL-6 is a pleiotropic cytokine produced by inflammatory and resident cells and has a crucial role in the pathogenesis of various inflammations [14, 15]. Sato previously observed the up-regulation of IL-6 at wound sites [16]. Gallucci and his colleagues [17] have recently reported that IL-6 KO mice exhibited
FIG. 6. MMP-9 expression in anastomotic tissue. Each bar represents the mean 6 SD. *P < 0.05, versus control group.
BEDIRLI ET AL.: EFFECTS OF NUCLEAR FACTOR-kB INHIBITORS
FIG. 7. Tissue NF-kB activity. Each bar represents the mean 6 SD. *P < 0.01, versus control group.
impairment in wound healing with reduced activation of a transcription factor AP-1 at the wound sites. In this study, we examined the effects of NF-kB inhibitors on the effector molecules in the wound-healing process, including cytokines. The plasma level of IL-6 significantly increased in animals treated with NF-kB inhibitors after 3 d. Our results showed that the the bankruptcy of the NF-kB pathway that induced early production of IL-6 might participate in improvement. A component of optimal healing is regulation of collagen formation. The process of collagen cleavage, another fundamental branch that determines connective tissue quality, is mainly regulated by proteases, specifically MMPs, which are involved in various processes during wound healing, such as debridement, angiogenesis, and matrix remodeling [18]. Immunohistochemical and in situ hybridization studies have shown that collagenase and other MMPs are in close vicinity to the suture line in uncomplicated anastomotic healing [19, 20]. Furthermore, experimental evidence suggests that inhibition of MMPs enhances the breaking strength of colonic anastomoses [21]. After surgery, TABLE 1 The Wound Healing Process Control Inflammatory cell infiltration POD 3 POD 5 POD 7 Collagen deposition POD 3 POD 5 POD 7 Fibroblast activity POD 3 POD 5 POD 7
Resveratrol Parthenolide
1.1 (0.2) 1.7 (0.5) 1.8 (0.6)
1.4 (0.3) 2.6 (0.6)* 2.7 (0.4)*
1.3 (0.2) 2.5 (0.4)* 2.8 (0.7)*
1.3 (0.4) 2.1 (0.6) 2.3 (0.7)
1.8 (0.5) 2.5 (0.7) 3.1 (1.0)*
1.6 (0.3) 2.4 (0.4) 2.8 (0.7)*
1.2 (0.3) 1.5 (0.6) 1.7 (0.4)
1.8 (0.6) 2.3 (0.7) 3.2 (1.1)*
1.7 (0.4) 2.4 (0.5) 3.5 (0.9)*
POD ¼ postoperative day. Values are means (SD). * P < 0.05, versus control group.
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degradation of mature collagen begins in the first 24 h and predominates for the first 4 d. This is caused by the up-regulation of MMPs, which are an important class of enzymes involved in collagen metabolism. By postoperative d 7, collagen synthesis becomes the dominant force, particularly proximal to the anastomosis. Many collagen formation-associated genes, such as IL6 and iNOS, have promoter or enhancer elements for NF-kB [22]. The present paper studied the link between the NF-kB pathway and altered collagen formation, and its association with MMP production. Our hypothesis in the present study was that parthenolide and resveratrol treatment can alter collagen formation during colonic anastomotic healing by decreasing NF-kB expression, which has cross-interaction with the NF-kB pathway and MMPs. The gelatinases MMP-2 and MMP-9, in particular, have been studied in the present study because of their specificity for breaking down the collagen of basement membranes. Various techniques were used to confirm the detection of MMPs, namely zymography, ELISA, and novel activity assay systems. Zymography is an electrophoretic technique that relies on the gelatinolytic activity of MMPs. Although sensitive, this method is time-consuming, lacks specificity, and is only semiquantitative. Our previous studies revealed that by using the activity assay systems, we were able to measure MMP-2 and MMP-9 in samples from rat tissue supernatants [23, 24]. Whereas on postoperative d 3 there were no differences between the groups, we found a significant increase in MMP-2 and MMP-9 expression in control animals on d 5 after surgery. To further investigate the signaling mechanism in wound healing, we examined the activation of NF-kB in parthenolide- or resveratrol-treated rats. NF-kB plays a pivotal role during the inflammatory stage, and NF-kB activation is thus proposed to be a critical event of inflammatory cell migration and synthesis of matrix repair related proteins. Preliminary experiments have shown that coordinated up-regulation of MMPs and enhanced matrix remodeling occur at sites, such as healing wounds, where growth factors and inflammatory cytokines coexist [25, 26]. Studies by Bond et al. in rabbit dermal fibroblasts have suggested that NF-kB activity is essential for MMP-1 and MMP-3 up-regulation [4]. Our study showed a suppression of MMP-2 and MMP-9 expression in colonic anastomoses treated with parthenolide or resveratrol. There is a possibility that expression of other MMPs might also be down-regulated by parthenolide or resveratrol, although we did not examine expression of all members of the MMP family. In conclusion, the activation of the NF-kB signaling pathway contributes to the induction of MMP-2 and MMP-9 expression in colonic anastomoses. Our data confirmed that parthenolide and resveratrol block
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NF-kB activation. The findings of the present study may provide a theoretical basis for the use of NF-kB inhibitors in colonic anastomoses. Further study is needed to establish the clinical utility of blocking NF-kB signaling in anastomotic wound healing. REFERENCES 1. Singer AJ. Cutaneous wound healing. N Engl J Med 1999; 341:738. 2. Dubay DA, Franz MG. Acute wound healing: The biology of acute wound failure. Surg Clin North Am 2003;83:463. 3. Birkedal-Hansen H, Moore WG, Bodden MK, et al. Matrix metalloproteinases: A review. Crit Rev Oral Biol Med 1993;4:19. 4. Bond M, Baker AH, Newby AC. Nuclear factor kB activity is essential for matrix metalloproteinase-1 and -3 upregulation in rabbit dermal fibroblasts. Biochem Biophys Res Commun 1999; 264:561. 5. Chakraborti S, Mandal M, Das S, et al. Regulation of matrix metalloproteinases: An overview. Mol Cell Biochem 2003;253:269. 6. Chen LF, Greene WC. Shaping the nuclear action of NF-kB. Nat Rev Mol Cell Biol 2004;5:392. 7. Schmidt C, Peng B, Li Z, et al. Mechanisms of proinflammatory cytokine-induced biaphasic NF-kB activation. Mol Cell 2003; 12:1287. 8. Tsutsumi R, Ito H, Hiramitsu T, et al. Celecoxib inhibits production of MMp and NO via down-regulation of NF-kB and JNK in a PGE2 independent manner in human articular chondrocytes. Rheumatol Int 2008;28:727. 9. Schmidt R, Bultmann A, Fischel S, et al. Extracellular matrix metalloproteinase inducer (CD147) is a novel receptor on platelets, activates plateles, and augments nuclear facor kB-dependent inflammation in monocytes. Circ Res 2008;102:302. 10. Saika S, Miyamoto T, Yamanaka O, et al. Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-kB, in treatment of corneal alkali burns in mice. Am J Pathol 2005; 166:1393. 11. Jamall IS, Finelli VN, Que Hee SS. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues. Anal Biochem 1981;112:70. 12. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341:738.
13. Martin P. Wound healing-aiming for perfect skin regeneration. Science 1997;276:75. 14. Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: A paradigm for cytokines. Science 1992;258:593. 15. Akira S, Kishimoto T. IL-6 and NF-IL6 in acute-phase response and viral infection. Immunol Rev 1992;127:25. 16. Sato Y, Ohshima T. The expression of mRNA of proinflammatory cytokines during skin wound healing in mice: A preliminary study for forensic wound age estimation (II). Int J Legal Med 2000;113:140. 17. Gallucci RM, Simeonova PP, Matheson JM, et al. Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice. FASEB J 2000;14:2525. 18. Parks WC. Matrix metalloproteinases in repair. Wound Repair Regen 1999;7:423. 19. Savage FJ, Lacombe DL, Boulos PB, et al. Role of matrix metalloproteinases in healing of colonic anastomosis. Dis Colon Rectum 1997;40:962. 20. Vaalamo M, Karjalainen-Lindsberg ML, Puolakkainen P, et al. Distinct expression profiles of stromelysin-2 (MMP-10), collagenase-3 (MMP-13), macrophage metalloelastase (MMP-12), and tissue inhibitor of metalloproteinases-3 (TIMP-3) in intestinal ulceration. Am J Pathol 1998;152:1005. 21. De Hingh IH, de Man BM, Lomme RM, et al. Colonic anastomotic strength and matrix metalloproteinase activity in an experimental model of bacterial peritonitis. Br J Surg 2003;90:981. 22. Morris KR, Lutz RD, Choi HS, et al. Role of the NF-kB signaling pathway and kB cis-regulatory elements on the IRF-1 and iNOS promoter regions in mycobacterial lipoarabinomannan induction of nitric oxide. Infect Immun 2003;71:1442. 23. Kerem M, Bedirli A, Karahacioglu E, et al. Effects of soluble fiber on matrix metalloproteinase-2 activity and healing of colon anastomosis in rats given radiotherapy. Clin Nutr 2006;25:661. 24. Bedirli A, Kerem M, Karahacioglu E, et al. Effects of two conventional preoperative radiation schedules on anastomotic healing in the rat colon. Eur Surg Res 2007;39:141. 25. Bond M, Chase AJ, Baker AH, et al. Inhibition of transcription factor NF-kB reduces matrix metalloproteinase-1, -3 and -9 production by vascular smooth muscle cells. Cardiovasc Res 2001; 50:556. 26. Takada Y, Aggarwal BB. Betulinic acid suppresses carcinogeninduced NF-kB activation through inhibition of I kB alpha kinase and p65 phosphorylation: Abrogation of cyclooxygenase-2 and matrix metalloprotease-9. J Immunol 2003;171:3278.
Effects of Nuclear Factor-kB Inhibitors on Colon Anastomotic Healing in Rats1 Abdulkadir Bedirli, M.D.,*,2 Bulent Salman, M.D.,* Hatice Pasaoglu, M.D.,† Ebru Ofluoglu, Ph.D.,† and Omer Sakrak, M.D.* *Department of General Surgery; and †Department of Biochemistry, Gazi University Medical School, Ankara, Turkey Submitted for publication November 30, 2009
Background. Nuclear factor (NF)-kB plays an essential role in inflammation. We tested this role by administering NF-kB-inhibitors into rats undergoing a well-established model of colonic anastomotic healing. Methods. Wistar rats underwent laparotomy, descending colonic transection, and handsewn reanastomosis. The animals were randomized to receive either a selective NF-kB inhibitor (parthenolide 0.5 mg/kg or resveratrol 0.5 mg/kg) or an equal volume of water by gavages before operation and then daily after surgery. Animals were sacrificed either immediately after anastomotic construction (d 0) or at the third, fifth, or seventh postoperative day. Results. Both parthenolide and resveratrol treatment led to early significant increases in plasma levels of IL-6. On d 7, hydroxyproline levels were significantly higher in the parthenolide and resveratrol groups. A similar pattern was observed with the bursting pressure. In contrast, gelatinase activity (MMP-2 and MMP-9 expression) was significantly higher in the control group on postoperative d 3. On d 3, expression of NF-kB activity was up-regulated in the anastomotic area. Both parthenolide and resveratrol completely attenuated NF-kB activity. Study groups also developed more marked inflammatory cell infiltration and collagen deposition on histology analysis. Conclusions. Parthenolide and resveratrol significantly improved healing and mechanical stability of colonic anastomoses in rats during the early postoperative period. Both agents may be acting to accelerate the host reparative process as well as to enhance pro-
1 Presented at the 42nd Congress of the European Society for Surgical Research, May 23–26, 2007, Rotterdam, The Netherlands. 2 To whom correspondence and reprint requests should be addressed at A.Taner Kislali Mah, Siyasal Villari, No. 44, 06810, Cayyolu, Ankara, Turkey. E-mail: [email protected].
tection of the anastomotic wound bed.
Ó 2011 Elsevier Inc.
All rights reserved.
Key Words: anastomosis; healing; colon; NF-kB; matrix metalloproteinases.
INTRODUCTıON
Wound healing is a complex insult-initiated biologic process that involves coordination and recruitment of multiple cellular and molecular events, and affects growth factor-mediated extracellular matrix (ECM) homeostasis [1]. An ECM is a dynamic superstructure of self-aggregating macromolecules, including fibronectin, collagen, and proteoglycans, to which cells attach by means of surface receptors called integrins, forming a three-dimensional supporting scaffold that isolates tissue compartments, mediates cell attachment, and determines tissue architecture [2]. Anastomotic wound healing in the gastrointestinal tract differs from the previously described process in that a controlled, full-thickness injury of limited duration is applied. Wound strength is determined by the amount and quality of newly-synthesized and deposited collagen, and by the degradation of preformed collagen. Collagenase activity is important in determining anastomotic integrity and suture-holding capacity during the first few days of healing. Remodeling of ECM by the matrix metalloproteinases (MMPs), a large family of zinc dependent neutral endopeptidases, is central to a wide number of physiologic and pathologic processes [3]. The induction of MMP gene expression, including MMP-1 (interstitial collagenase), MMP-3 (stromelysin-1), and MMP-9 (gelatinase B), can occur due to a wide range of stimuli, including inflammatory cytokines, growth
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0022-4804/$36.00 Ó 2011 Elsevier Inc. All rights reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 171, NO. 1, NOVEMBER 2011
FIG. 1. The classical NF-kB pathway is activated by a variety of inflammatory signals, resulting in coordinate expression of multiple inflammatory and innate immune genes. The proinflammatory cytokines IL-1b and TNF-a activate NF-kB, and their expression is induced in response to NF-kB activation, thus forming an amplifying feed forward loop.
factors, phorbol esters, oxidant stress, and mechanical injury [4, 5]. These cytokines and growth factors that are up-regulated in epithelial cells further contribute to tissue inflammation. A majority of inflammatory cytokines use the nuclear factor (NF)-kB pathway for signaling on ligand binding to cell surface receptor (Fig. 1). IL-1b and TNF-a rapidly activate NF-kB, a regulator of genes involved in inflammation and immune responses [6, 7]. Levels of these inflammatory cytokines and active NF-kB are elevated during proliferative inflammatory conditions, including wound healing, rheumatoid arthritis, and atherosclerosis, and correlate with increased levels of MMP-1, MMP-3, and MMP-9 enzymes [4, 8–10]. We therefore hypothesized that blocking the NF-kB pathway might be beneficial in strengthening colonic anastomoses. In addition, measurements were taken to assess whether NF-kB inhibitors increased the presence of MMP-2 and MMP-9 in the anastomotic areas. MATERIALS AND METHODS The experimental protocols were conducted with the approval of the Animal Research Committee at Gazi University, Ankara. All animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Animals and Experimental Design Male Wistar rats (n ¼ 120; weight 230–280 g) were housed two per cage and allowed to become acclimated with laboratory conditions for
7 d before the experiment. The animal rooms were windowless with temperature (22 6 20 C) and lighting controls. The rats were divided into three groups (n ¼ 30 per group) in a randomized manner: control, parthenolide, and resveratrol. The control group received isovolumetric saline solution, the parthenolide group received 0.5 mg/kg parthenolide (Sigma-Aldrich Corp., St. Louis, MO) and the resveratrol group received 0.5 mg/kg resveratrol (Sigma-Aldrich Corp.) orally by gavages for 7 d before the operation and then daily after surgery until the time of sacrifice. The animals were fasted overnight before the experiments but were given free access to water. They were anesthetized with ketamine 100 mg/kg and xylazine 20 mg/kg body weight, intraperitoneally. They were left to breathe spontaneously throughout the procedures. The abdominal skin was disinfected with 70% alcohol. All procedures were performed under sterile conditions by a surgeon who was blinded to the treatment groups the animals. For the determination of plasma interleukin (IL)-6 levels, 0.5 mL of blood was collected from all animals by transecting the tip of their tail. Then a midline incision was made, approximately 3 cm long, sufficient to expose the colon and rectum. For the colonic anastomosis, approximately 3 cm proximal to the peritoneal reflection, a 1 cm segment was resected and colonic continuity was restored by an end-to-end anastomosis, using eight to 10 inverting interrupted sutures (Ethilon 8/0; Ethicon, Norderstedt, Germany) depending on the diameter of the colon. The abdomen was washed out with sterile saline and closed with a running suture using 4-0 vicryl (Ethicon). After the operation, animals were kept in their cages, and they were allowed food and drink ad libitum.
Macroscopic Intra-abdominal Analysis Ten rats from each group were sacrificed by intracardiac puncture after anesthesia with ketamine 100 mg/kg and xylazine 20 mg/kg either immediately after anastomotic construction (d 0) or at the third, fifth, or seventh postoperative day. The abdomen was reopened and inspected for anastomotic dehiscence, peritonitis, ileus, and the formation of adhesions. Peritonitis was defined as macroscopic signs of inflammation, fibrin deposition, and hypervascularity. Mechanical obstruction was defined as obstruction of the intestine caused by the suture, with distention proximal to the anastomosis but not distal to the anastomosis. In addition, all surrounding structures adhesive to the anastomosis (colon, intestine, omentum, and pelvic fatty tissue) were considered separate adhesions.
Plasma Interleukin-6 Levels Serum samples for IL-6 were frozen at –70 C until they were assayed in triplicate. The IL-6 concentrations of serum were determined by enzym e-linked immunosorbent assay (ELISA; R&D Systems, Munich, Germany).
Mechanical Analysis Anastomotic segments, approximately 4 cm in length, with the suture line in the middle, were carefully resected, including surrounding tissues and adhesions, and washed in saline. To measure bursting pressure, the segments were infused (2 mL/min) with 0.9% NaCl containing methylene blue. The maximum pressure (mm Hg) recorded immediately before sudden loss of pressure was taken as the bursting pressure. The site of rupture (within or outside the anastomotic line) was noted. Once the bursting pressure was recorded, the anastomosis site was resected along with a 5 mm distal and proximal portion of the colon, and anastomosis was opened longitodinally and divided into two equal longitudinal segments. One segment was placed in 4% formalin solution for histologic assessment. The second segment of the anastomosis site was frozen in liquid nitrogen and stored at –80 C until further processing.
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Determination of Hydroxyproline, MMP-2, and MMP-9 Levels Anastomotic hydroxyproline content was measured according to the method of Jamall et al. [11]. Briefly, 25 mg of homogenate taken from hydrolyzation was lyophilisated and dissolved in the 1 mL 50% (vol/vol) isopropyl alcohol. Ten minutes later, chloramine T was added to these samples. Next, the samples were incubated for 90 min at 50 C after adding 1 mL Erlich’s reagent. Absorbance was measured at 560 nm, and the amount of hydroxyproline was calculated from a standard curve constructed using hydroxy-L-proline at concentrations of 0.25 to 40 mg/mL. Results were expressed as mg/mg tissue. MMP-2 and MMP-9 concentrations in tissue supernatants were determined with enzyme immunoassays performed according to the instructions of the manufacturers. Rat MMP-2 and MMP-9 activity assay systems were purchased from Amersham Biosciences Corp., Piscataway, NJ. Duplicate evaluations were performed for each sample. Quantification of the reaction product was achieved by measuring the absorbance at 405 nm using a spectrophotometer.
Tissue NF-kB Activity Tissues were homogenized in 10 volumes of cold 0.01 M Tris-HCI Buffer (pH 7.4) using an automatic homogenizer. NF-kB activity in tissue supernatants were determined with enzyme immunoassays by the use of a commercial enzyme-linked immunosorbent assay (ELISA) kit (Panomics Inc., Redwood City, CA) according to the manufacturer’s guidelines. All samples were tested in duplicate.
Histology The sections were stained with hematoxylin and eosin and the area of anastomosis was graded histologically in a blind fashion using a numerical scale. Inflammatory cell infiltration, fibroblast activity, and collogen deposition were graded from 0 to 4 as follows: 0 ¼ no evidence, 1 ¼ occasional evidence, 2 ¼ light scattering, 3 ¼ abundant evidence, and 4 ¼ confluent cells or fibers.
Statistical Analysis All values were expressed as the mean 6 SD. Data were compared by analysis of variance with post hoc analysis using the NewmanKeuls test. When a difference was found, specific differences were identified by using the Kruskal-Wallis test. Statistical evaluation was carried out using SPSS 10.0 software (SPSS, Chicago, IL). Values of P < 0.05 were accepted as significant.
FIG. 2. Anastomotic bursting pressure in rats after anastomosis. Each bar represents the mean 6 SD. *P < 0.01, versus control group.
third postoperative day. At this time, anastomotic rupture occurred within the suture line in all animals. The anastomotic bursting pressure showed an increase from d 3 onwards in all groups (Fig. 2). There was no difference between the control and parthenolide-or resveratrol-treated animals at postoperative d 3 and 5. In contrast, the average bursting pressure was significantly higher in the parthenolide and resveratrol groups than in the control group 7 d postoperatively (P < 0.01). During the bursting pressure measurement, all ruptures occurred outside the suture line on d 7. Plasma Interleukin-6 Levels
Plasma IL-6 levels are shown in Fig. 3. On postoperative d 3, concentration of IL-6 were significantly elevated in groups parthenolide and resveratrol, compared with control group (P < 0.01). However, on postoperative d 5–7, no significant difference was found among the groups. Hydroxyproline Concentration
RESULTS General Observations
There were no significant differences between the groups in preoperative weight, dietary intake, or pattern of weight change after surgery. Four animals in the control group developed colonic anastomotic complications compared with none in the groups receiving parthenolide or resveratrol (P ¼ 0.042). Two developed colonic perianastomotic abscesses, one developed mechanical obstruction by d 3, and one had colonic anastomotic dehiscence by d 7. Anastomotic Strength
The strength of the anastomoses as determined by measuring colonic bursting pressure was low on the
The hydroxyproline concentration in the 5-mm tissue containing the actual anastomosis was assayed as a measure for the presence of collagen. Figure 4 shows that administration of selective NF-kB inhibitors led to an increased anastomotic hydroxyproline concentration on d 7 (P < 0.05). Average hydroxyproline concentrations were similar between the parthenolide and resveratrol groups at all time points. MMP-2 and MMP-9 Expression
MMP activity measured in the colonic anastomoses was similar in all groups on d 3. After treatment with NF-kB inhibitors, there was a significant decrease compared with the control in mean MMP-2 and MMP-9 expression on d 5 (P < 0.05) (Figs. 5 and 6).
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FIG. 3. Interleukin (IL)-6 expression in plasma. Each bar represents the mean 6 SD. *P < 0.05, versus control group.
Expression of NF-kB
NF-kB concentration in the parthenolide- or resveratrol-treated rats decreased by approximately 65% on postoperative d 3 and 5 compared with values in the control group. No differences between resveratrol-treated animals and parthenolide-treated animals were noted for either day (Fig. 7). Histology
The wound healing process as assessed from inflammatory cell infiltration, collagen deposition, and fibroblast activity is presented in Table 1. Specifically, the parthenolide and resveratrol groups developed more marked fibroblast activity and collagen deposition than the control group (P < 0.05). Similarly, parthenolide or resveratrol treatment developed significantly more marked inflammatory cell infiltration than the control group (P < 0.05). DISCUSSION
In these experiments, we investigated the effects of parthenolide and resveratrol on cellular and molecular
FIG. 4. Data represent hydroxyproline concentration (mg/mg tissue) after anastomosis and are expressed as mean 6 SD. *P < 0.05, versus control group.
FIG. 5. MMP-2 expression in anastomotic tissue. Each bar represents the mean 6 SD. *P < 0.05, versus control group.
events in the early inflammatory stage of anastomotic wound healing in rats. Our data demonstrate that parthenolide and resveratrol treatment increased anastomotic hydroxyproline concentrations and reduced specific markers of the inflammatory response, including MMP-2 and MMP-9 expression, during the early inflammatory stage of wound healing. Moreover, these experiments demonstrated that parthenolide and resveratrol treatment had beneficial effects on the colonic anastomotic complications and the strength of the colonic anastomoses in rats. Wound healing consists of three phases: inflammation, proliferation, and maturation and remodeling. These phases proceed, overlapping each other, and are regulated by a complicated array of cytokines and growth factors secreted by inflammatory and resident cells [12, 13]. The first phase of the healing, described as the inflammatory phase, is seen on the first 3 d of the healing process. IL-6 is a pleiotropic cytokine produced by inflammatory and resident cells and has a crucial role in the pathogenesis of various inflammations [14, 15]. Sato previously observed the up-regulation of IL-6 at wound sites [16]. Gallucci and his colleagues [17] have recently reported that IL-6 KO mice exhibited
FIG. 6. MMP-9 expression in anastomotic tissue. Each bar represents the mean 6 SD. *P < 0.05, versus control group.
BEDIRLI ET AL.: EFFECTS OF NUCLEAR FACTOR-kB INHIBITORS
FIG. 7. Tissue NF-kB activity. Each bar represents the mean 6 SD. *P < 0.01, versus control group.
impairment in wound healing with reduced activation of a transcription factor AP-1 at the wound sites. In this study, we examined the effects of NF-kB inhibitors on the effector molecules in the wound-healing process, including cytokines. The plasma level of IL-6 significantly increased in animals treated with NF-kB inhibitors after 3 d. Our results showed that the the bankruptcy of the NF-kB pathway that induced early production of IL-6 might participate in improvement. A component of optimal healing is regulation of collagen formation. The process of collagen cleavage, another fundamental branch that determines connective tissue quality, is mainly regulated by proteases, specifically MMPs, which are involved in various processes during wound healing, such as debridement, angiogenesis, and matrix remodeling [18]. Immunohistochemical and in situ hybridization studies have shown that collagenase and other MMPs are in close vicinity to the suture line in uncomplicated anastomotic healing [19, 20]. Furthermore, experimental evidence suggests that inhibition of MMPs enhances the breaking strength of colonic anastomoses [21]. After surgery, TABLE 1 The Wound Healing Process Control Inflammatory cell infiltration POD 3 POD 5 POD 7 Collagen deposition POD 3 POD 5 POD 7 Fibroblast activity POD 3 POD 5 POD 7
Resveratrol Parthenolide
1.1 (0.2) 1.7 (0.5) 1.8 (0.6)
1.4 (0.3) 2.6 (0.6)* 2.7 (0.4)*
1.3 (0.2) 2.5 (0.4)* 2.8 (0.7)*
1.3 (0.4) 2.1 (0.6) 2.3 (0.7)
1.8 (0.5) 2.5 (0.7) 3.1 (1.0)*
1.6 (0.3) 2.4 (0.4) 2.8 (0.7)*
1.2 (0.3) 1.5 (0.6) 1.7 (0.4)
1.8 (0.6) 2.3 (0.7) 3.2 (1.1)*
1.7 (0.4) 2.4 (0.5) 3.5 (0.9)*
POD ¼ postoperative day. Values are means (SD). * P < 0.05, versus control group.
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degradation of mature collagen begins in the first 24 h and predominates for the first 4 d. This is caused by the up-regulation of MMPs, which are an important class of enzymes involved in collagen metabolism. By postoperative d 7, collagen synthesis becomes the dominant force, particularly proximal to the anastomosis. Many collagen formation-associated genes, such as IL6 and iNOS, have promoter or enhancer elements for NF-kB [22]. The present paper studied the link between the NF-kB pathway and altered collagen formation, and its association with MMP production. Our hypothesis in the present study was that parthenolide and resveratrol treatment can alter collagen formation during colonic anastomotic healing by decreasing NF-kB expression, which has cross-interaction with the NF-kB pathway and MMPs. The gelatinases MMP-2 and MMP-9, in particular, have been studied in the present study because of their specificity for breaking down the collagen of basement membranes. Various techniques were used to confirm the detection of MMPs, namely zymography, ELISA, and novel activity assay systems. Zymography is an electrophoretic technique that relies on the gelatinolytic activity of MMPs. Although sensitive, this method is time-consuming, lacks specificity, and is only semiquantitative. Our previous studies revealed that by using the activity assay systems, we were able to measure MMP-2 and MMP-9 in samples from rat tissue supernatants [23, 24]. Whereas on postoperative d 3 there were no differences between the groups, we found a significant increase in MMP-2 and MMP-9 expression in control animals on d 5 after surgery. To further investigate the signaling mechanism in wound healing, we examined the activation of NF-kB in parthenolide- or resveratrol-treated rats. NF-kB plays a pivotal role during the inflammatory stage, and NF-kB activation is thus proposed to be a critical event of inflammatory cell migration and synthesis of matrix repair related proteins. Preliminary experiments have shown that coordinated up-regulation of MMPs and enhanced matrix remodeling occur at sites, such as healing wounds, where growth factors and inflammatory cytokines coexist [25, 26]. Studies by Bond et al. in rabbit dermal fibroblasts have suggested that NF-kB activity is essential for MMP-1 and MMP-3 up-regulation [4]. Our study showed a suppression of MMP-2 and MMP-9 expression in colonic anastomoses treated with parthenolide or resveratrol. There is a possibility that expression of other MMPs might also be down-regulated by parthenolide or resveratrol, although we did not examine expression of all members of the MMP family. In conclusion, the activation of the NF-kB signaling pathway contributes to the induction of MMP-2 and MMP-9 expression in colonic anastomoses. Our data confirmed that parthenolide and resveratrol block
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