Sepsis and multiple organ dysfunction syndrome (MODS) are common problems that cause frequent morbidity and mortality despite advances in critical care therapy and improved antibiotic regimens (1). Antibiotics have a primary role in therapy but are often inadequate when used alone in severely affected septic patients. Although the etiology of MODS is multifactorial, a proposed common denominator of the sepsis syndrome and organ failure is the release of excessive cytokines, predominantly by the macrophage (2). A number of immunomodulatory therapies aimed at decreasing the dysregulated inflammatory response have been examined in patients with severe sepsis (3,4). The failure of these therapies to improve outcome in sepsis has led many researchers to investigate additional models of therapy.
Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the active ingredient accounting for the pungency of hot peppers. Apart from being used as a food additive and flavoring principle, the compound has important pharmacological actions. Capsaicin is uniquely selective for stimulation and then blockade of a subset of mammalian afferent neurons of dorsal root ganglia with C and A δ fibers. In response to stimulation, peptide mediators are released from the central and peripheral nerve endings of these neurons. Most of capsaicin-sensitive afferents contain calcitonin gene-related peptide (CGRP) and/or substance P (5). Capsaicin has also been shown to have complex pharmacological actions. Several studies indicate that macrophage functions were altered by capsaicin. For example, rat peritoneal macrophages preincubated with capsaicin inhibited the incorporation of arachidonic acid into membrane lipids (6). Capsaicin also inhibited the secretion of hydrolytic enzymes (collagenase, elastase and hyaluronidase) by rat peritoneal macrophages (6). These in vitro studies demonstrated that capsaicin can control the release of inflammatory mediators and lysosomal enzyme secretions by macrophages. In the present study, in in vivo conditions, the influence of small- and large-dose capsaicin in regulating the systemic inflammatory responses was investigated during different stages of sepsis in rats.
Experiments were performed in compliance with guidelines prescribed by the Institutional Animal Care and Use Committee at the İnönü University School of Medicine. Female Sprague-Dawley rats, 200–230 g, were purchased from the Experimental Research Laboratory of Dicle University, Turkey, and maintained under standard laboratory conditions. Rats were housed five to a cage and given access to food and drink ad libitum. After 7 days of acclimatization, we followed the experimental protocol.
We randomly divided 120 rats into 6 groups of 20 animals each. The animals of the first group were sham operated (control, group C). In the second group (group S) of rats, sepsis was induced by cecal ligation and puncture (CLP). Rats in the CLC group (control + small-dose capsaicin) received capsaicin (1 mg/kg subcutaneously [s.c.]) 3 h before surgery and underwent only laparotomy. Rats in the SLC group (sepsis + small-dose capsaicin) received capsaicin 3 h before surgery at a dose of 1 mg/kg s.c. and were made septic by CLP. Rats in the CHC group (control + large-dose capsaicin) received capsaicin in 3 consecutive doses of 25, 75, and 50 mg/kg (a total of 150 mg/kg s.c.) 2 weeks before the surgery and underwent only laparotomy. Rats in the SHC group (sepsis + large-dose capsaicin) received capsaicin in the same doses as the CHC rats and were made septic by CLP. To study the inflammatory responses during different stages of sepsis, each group was randomly subdivided into 2 subgroups (early and late sepsis) consisting of 10 rats killed at 9 or 18 h, respectively, after the surgical procedure (7). All rats in the late sepsis group survived 18 h after CLP. Rats were killed by cervical dislocation under ether anesthesia. Blood was immediately collected through heart puncture, and the liver, lung, and heart samples were rapidly collected in all rats.
Sepsis was induced by cecal ligation and two-hole puncture, as described by Wichterman et al. (8), with minor modification. Briefly, under ether anesthesia, the peritoneum was opened in a sterile manner and the cecum was tied with a 3–0 silk suture 1 cm from the distal end. Care was taken not to obstruct the ileocecal valve. Two punctures were made with an 18-gauge needle through the cecum distal to the point of ligation. The needle was removed and a small amount of stool expelled from the puncture to ensure patency. The cecum was replaced into the peritoneal cavity and the anterior peritoneal wall and skin were closed with silk sutures. For sham operations, a laparotomy was performed in a similar manner without CLP. Saline (5 mL/100 g of body weight) was given s.c. to each rat at the time of surgery and also at 6 h postoperatively for fluid resuscitation. Animals were fasted but had free access to water after operative procedures.
Capsaicin was obtained from Sigma (St. Louis, MO). Stem solution of capsaicin (1%) was dissolved in 10% ethanol, 10% Tween 80, 80% isotonic NaCl solution, and further diluted in isotonic saline. Deactivation of primary afferent nerves by s.c. administration of a neurotoxic dose of capsaicin was performed as described previously (9). Briefly, this neural deactivation was achieved with capsaicin in the total dose of 150 mg/kg injected over 3 consecutive days starting with the doses of 10 and 15 mg/kg given on the first day, 25 and 50 mg/kg on the second day, and 50 mg/kg on the third day. Capsaicin was injected s.c. in the dorsal thoracic region under light ether anesthesia. Control rats received the equivalent volume of the solvent solution. To check the effectiveness of the capsaicin denervation, a drop of 0.1 mg/mL solution of capsaicin was instilled into one eye of each rat and protective wiping movements were counted as previously reported (9). All animals pretreated with a large dose of capsaicin reacted negatively to the wiping movement test, confirming functional ablation of the capsaicin-sensitive afferent sensory nerves. Capsaicin was injected s.c. 3 h before the surgery to activate the sensory nerves at a dose of 1 mg/kg. Control rats were injected with solvent solution alone. The preliminary experiment showed that pretreatment with 1 mg/kg capsaicin significantly increased the plasma concentration of CGRP after 9 and 18 h (0.38 ± 0.02 versus 0.73 ± 0.03 and 0.94 ± 0.03, n = 3).
Rat kits of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were obtained from Biosource International, Inc. (Camarillo, CA). Rat kits of CGRP were obtained from Phoenix Peptide (Belmont, CA). The other reagents were of the best quality commercially available. Rat TNF-α, IL-6 and IL-10 were measured with the micro ELISA method according to the manufacturer’s instructions. Cytokines are expressed in pg/mL. CGRP was measured with the micro ELISA method (competitive enzyme immunoassay kit) and expressed in ng/mL.
Blood was centrifuged at 1000g for 15 min, and plasma was stored at −80°C until assayed. The method for total nitrite/nitrate (NOx) level was based on the Griess reaction (10). Nitrite and nitrate were measured after enzymatic conversion of nitrate to nitrite by nitrate reductase (from Aspergillus) in the presence of NADPH as described by Bories and Bories (11). The oxidation of the coenzyme was monitored by the decrease in absorbance at 340 nm. Results are expressed as micromole/l.
At the end of each experimental period, the lung, liver, and heart were removed and rapidly washed with ice-cold saline, dissected out, and folded with aluminum foil. They were labeled and stored at −80°C until processing. Tissues were homogenized in Tris-HCL buffered saline (1:5 w/v; pH 7.4; Potter Elvehjem glass homogenizer) and centrifuged at 3500g for 45 min at 4°C. One volume of supernatant was mixed with one volume of chloroform and ethanol mixture (3/5 v/v) and centrifuged at 3500g for 45 min at 4°C. The uppermost ethanol layer of supernatant was assayed for superoxide dismutase (SOD) activity and protein level. The activity of SOD was measured spectrophotometrically as described by Sun et al. (12). Briefly, xanthine-xanthine oxidase was used to generate a superoxide flux. Reduction of nitrobluetetrazolium (NBT) by superoxide anion to blue formazan was determined at 560 nm. One unit of enzyme activity was defined as the amount of protein causing 50 percent inhibition in NBT reduction by superoxide. All protein determinations in samples were performed according to Lowry et al. (13) using bovine serum albumin as standard. For malondialdehyde (MDA) determination, all tissues were first weighed, homogenized with cold 1.15% KCl and diluted (1/9 w/v) to make 10% homogenate. Samples were then centrifuged at 3500 g for 45 min at 4°C. Supernatants were taken and analyzed for MDA. MDA concentration was measured as the total thiobarbituric acid reactive substances (TBARS) or as MDA equivalents, as described by Uchiyama and Mihara (14). A Shimadzu 1601 UV-VIS spectrophotometer (Shimadzu, Kyoto, Japan) was used for protein, MDA and SOD assays. SOD and MDA results were expressed as U/mg protein and nmol/g tissue, respectively.
Statistical analyses of the study were performed using SPSS for Windows, version 10.0 (SPSS Inc., Chicago, IL). The data among the groups were analyzed by analysis of variance with Tukey’s post hoc testing. The within-group data were analyzed using repeated-measures analysis of variance followed by Tukey’s post hoc testing. A P value of <0.05 was considered to be statistically significant. Results were expressed as the mean ± sem.
As shown in Figures 1 and 2, the septic rats pretreated with small-dose capsaicin demonstrated a significant (approximately 30%) decrease in the plasma proinflammatory cytokine concentrations when compared with the group S and SHC rats. In contrast to proinflammatory cytokines, there was an apparent increase in plasma concentration of IL-10 (an antiinflammatory cytokine) in septic rats pretreated with small-dose capsaicin (P < 0.05). Although an additional increase in circulating cytokine levels was also observed in large-dose capsaicin-pretreated septic rats, these differences did not achieve statistical significance when compared with group S.
Septic rats had increased levels of NOx compared with controls (Figs. 1 and 2). Serum levels of NOx were significantly affected by small-dose capsaicin pretreatment. NOx levels were markedly reduced in the septic rats pretreated with small-dose capsaicin when compared with groups S and SHC during the late sepsis period (P < 0.05).
To determine whether capsaicin stimulation or deactivation had any affect on antioxidant status to sepsis, SOD levels of tissue were also monitored during both early and late sepsis periods in all groups of rats (Tables 1 and 2). The highest level of SOD was observed in the septic rats pretreated with small-dose capsaicin (P < 0.05). MDA levels in the liver and lung increased in group S when compared with group C during the late sepsis period (P < 0.05). Small-dose capsaicin pretreatment significantly prevented the increase of MDA levels in the liver and lung (P < 0.05).
Figure 3 shows the effects of capsaicin stimulation and deactivation on plasma CGRP concentrations in all groups of rats (both early and late sepsis groups). In group CLC, the plasma CGRP concentrations significantly increased compared with groups C, CHC, and SHC (P < 0.05). The CGRP concentration tended to increase after CLP in group SLC, both in the early and late sepsis periods, but it was not statistically significant. Deactivation of afferent sensory nerves with large-dose capsaicin treatment resulted in a significant decrease in CGRP concentration in group CHC (P < 0.05). In the group SHC rats, exposure to CLP-induced sepsis evoked no increase in CGRP concentrations in both the early and late sepsis periods.
These data indicate that capsaicin alters the host response to CLP-induced sepsis in a manner that significantly changes cytokine and reactive nitrogen species (RNS) production, tissue antioxidant level, and lipid peroxidation. In this study, the CLP method was used as the sepsis animal model because its biphasic clinical manifestations and early (hyperdynamic) and late (hypodynamic) phases mimic those in humans. It has been demonstrated that the hyperdynamic state persists from 2 to 10 h and that the hypodynamic state occurs at 16 to 20 h after CLP (7,8). Therefore, we chose 9 h for early sepsis groups and 18 h for late sepsis groups.
As stated before, in vitro studies show that capsaicin exhibits antiinflammatory properties. The prevention of release of proinflammatory mediators, eicosanoids, and hydrolytic enzymes is associated with the antiinflammatory properties of capsaicin. Rat peritoneal macrophages preincubated with 10 μM capsaicin for 1 h inhibited the incorporation of arachidonic acid into membrane lipids, prostaglandin E2, leukotriene B4, and leukotriene C4 by 76%, 48%, 46%, and 48%, respectively (6). In our in vivo study, we showed that small-dose capsaicin pretreatment caused a significant decrease in the production of proinflammatory cytokines (TNF, IL-6) and was also associated with a marked increase in the production of the antiinflammatory cytokine IL-10 in the rat model of sepsis. Our results suggest that in vivo pretreatment with small-dose capsaicin exerts a wide range of antiinflammatory properties of capsaicin. Such a decrease in proinflammatory cytokine levels could also explain, at least in part, why small-dose capsaicin treatment resulted in the ability of these animals to produce more of the IL-10 in response to CLP-induced sepsis.
Oxidative damage is probably one of several factors that lead to cell damage, organ dysfunction, and death. There is convincing evidence of severe oxidative stress in patients with sepsis (15). Reactive oxygen species (ROS) and RNS, such as superoxide anions, peroxides, hydroxyl radicals, and nitric oxide (NO) generated by activated macrophages for defense mechanisms of the host, can also act as mediators of inflammation if produced in an uncontrolled manner. These radicals can react with cellular components like lipids, proteins, and nucleic acids, resulting in increased levels of lipid peroxides and alterations in the functions of proteins, and may also cause DNA damage (15). In vitro studies show that capsaicin has a potent antioxidant effect. Capsaicin inhibits generation of ROS (16). Preincubation of macrophages with 10 μM capsaicin completely inhibited the superoxide anions, hydrogen peroxide, and nitrite radical production in vitro by macrophages. In addition, it was reported that capsaicin potentially inhibits various lipid peroxidations. Capsaicin was found to scavenge radicals at and near the membrane surface and in the interior of the membrane (17). In the present study, systemic administration of capsaicin at 1 mg/kg before CLP decreased the lipid peroxidation in various tissues, including lung and liver, during the late sepsis period. SOD levels were different between the septic rats pretreated with small-dose capsaicin and those that did not receive pretreatment. We hypothesized that SOD consumption would closely correlate with oxidative stress. Oxidative stress was reduced in the septic rats pretreated with small-dose capsaicin, and hence high plasma levels of this antioxidant are observed in this group of rats.
Sepsis is associated with increased plasma levels of the NO bioreaction products nitrite and nitrate (18). It has been shown that mediators associated with sepsis, such as endotoxin and the proinflammatory cytokines IL-1β, IL-2, IL-6, TNF, and interferon-γ, induce inducible NO synthase (iNOS) and thereby increase RNS production in vitro. INOS is expressed and activated in a variety of cells, including vascular endothelial cells, smooth muscle cells, macrophages, and different parenchymal cells (18). In the SLC group, small-dose capsaicin pretreatment significantly decreased plasma NOx concentrations in comparison with the S group during the late sepsis period. It has been shown that increased plasma NOx concentrations are associated with the development of multiple organ failure in sepsis. In this study, one of the most apparent benefits of the small-dose capsaicin pretreatment in the rats with severe sepsis was an apparent reduction in the plasma levels of NOx. A possible explanation for this observation was that pretreatment with small-dose capsaicin resulted in the ability of these animals to produce smaller quantities of the proinflammatory cytokines in response to CLP-induced sepsis, and hence NOx production was reduced.
CGRP, a potent vasoactive and cardiotonic peptide, interacts with specific G-protein- coupled receptors. CGRP is synthesized and released from small, capsaicin-sensitive sensory neurons (19). CGRP, a principal neurotransmitter in sensory nerves, is widely expressed throughout the body. It was shown that circulating levels of CGRP increase in patients with sepsis (20). Capsaicin has a dual effect on sensory nerves. Repeated administration of large-dose capsaicin causes depletion of neuropeptides from sensory nerves (5). In our study, CGRP levels were high in both early and late sepsis periods in the group S and SLC rats. However, repeated administration of capsaicin and ablation of sensory neurons resulted with decreased CGRP levels in the septic rats. Because ablation of the sensory fibers can result in a marked increase in the severity of inflammation, the sensory neurons have been shown to play a role in maintaining tissue integrity by regulating the local inflammatory response (21). In such a systemic inflammatory context little is known about the effects of this peptide and sensory neurons. There is a growing body of evidence supporting the fact that the CGRP is a potent antiinflammatory mediator. Briefly, the CGRP is thought to inhibit type 1 cytokines (e.g., IL-12 and interferon γ) and to enhance production of IL-10, one of the most immunosuppressive cytokines (22,23). The CGRP also decreased lipopolysaccharide-induced TNF production in rats as well as in humans (24).
In the current study, we also aimed to investigate the effects of afferent sensory ablation and/or activation on systemic inflammatory responses to sepsis. In contrast to small-dose capsaicin, large-dose capsaicin pretreatment resulted in increase of the sepsis-induced proinflammatory cytokine and NOx levels, and lipid peroxidation when compared with the group SLC rats. These results also suggest that, in addition to direct effects of capsaicin, capsaicin-sensitive sensory nerves may also have a protective role in systemic inflammatory syndrome.
In summary, these in vivo observations are consistent with previous in vitro findings showing that capsaicin inhibits the production of proinflammatory mediators and lipid peroxidation and thus exhibited antiinflammatory and immunomodulatory actions in the rat model of sepsis. Capsaicin may be a promising drug candidate for ameliorating excessive inflammatory responses that may be seen in severe septic patients. Further studies are necessary to define the mechanisms by which these alterations occur as well as their resulting congruencies.
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