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INFLAMMATION/IMMUNITY/MEDIATORS
Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Alabama
Submitted 23 August 2005 ; accepted in final form 28 February 2006
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ABSTRACT |
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,17-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.
liver enzymes; proinflammatory cytokines; neutrophils activation; myeloperoxidase
The depressed organ perfusion and excessive production of proinflammatory mediators may play an important role in the development of multiple organ dysfunctions following hemorrhagic shock and sepsis (15, 21, 31). It has also been reported that systemic IL-6 levels increased after trauma-hemorrhage (T-H), and a sustained elevation in plasma IL-6 levels correlated with the evolving organ dysfunction (2, 9, 17, 38). Clinical data also confirmed that the increased IL-6 production is tied to a poor outcome (24).
Studies have demonstrated that the IL-6-mediated elevation in nitric oxide production is likely one of the detrimental effects induced by this cytokine. It has been reported that nitric oxide is a determinant of IL-6-mediated negative inotropic effects on the heart (10). Yu et al. (39) also demonstrated that IL-6 decreases cardiac contractility and enhances the synthesis of the inducible nitric oxide synthase (iNOS), resulting in an excess of nitric oxide production. In support of these findings, our studies have demonstrated that there was a close correlation between organ dysfunction and the elevation in plasma IL-6 levels (1–4, 13, 14, 17, 19, 38). Our recent study also indicated that the depressed cardiac function following T-H is associated with an increased in situ IL-6 production by the cardiomyocytes (38). The causal role of IL-6 in organ dysfunction has been further established by studies that showed that administration of anti-IL-6 antibodies improved organ function following T-H (34).
Androstenediol (adiol or 5-androstene-3,17-diol), a metabolite of dehydroepiandrosterone (DHEA), was shown to have greater protective capacity than DHEA on lethal bacterial infections and endotoxin shock (4). Additionally, it is reported that the conversion of DHEA leads to an increase in downstream effector hormones in macrophages, which may play an important role in local immunomodulation (25). Administration of adiol following T-H has also been shown to be effective in reducing plasma IL-6 production and improving cardiovascular and hepatic function (29, 30).
Although adiol has been reported to produce the above-mentioned salutary effects following T-H or endotoxin shock, it remains unknown whether its salutary effects are also evident in a two-hit model of T-H and sepsis. Therefore, the present study was undertaken to determine whether administration of adiol has any salutary effects on inflammatory mediators and on the survival of animals following T-H and sepsis. We hypothesized that adiol would have salutary effects on organ function and cytokine production even after a compound insult such as T-H and sepsis.
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MATERIALS AND METHODS |
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Experimental groups. Following cannulation of the femoral arteries and a femoral vein, the animals were divided randomly into two groups, one group undergoing a T-H protocol and the second group in which only a sham operation was performed. These animals were then further divided into two subgroups as follows: at the end of resuscitation (or at the matching time point after sham operation), one subgroup was treated with androstenediol (adiol, 1 mg/kg body wt; Steraloids, Newport, RI), and the other group received the same volume of vehicle (vehicle, Intralipid, 1 ml/kg body wt; Sigma, St. Louis, MO). Twenty hours after the T-H or sham operation, the animals were anesthetized again, and cecal ligation and puncture was performed in both T-H and sham animals as described previously (5).
T-H procedure. A nonheparinized model of T-H, as previously described, was used in this study (17). Briefly, male Sprague-Dawley rats were fasted overnight before the experiment but allowed water ad libitum. Animals were anesthetized using isoflurane (Attane; Minrad, Bethlehem, PA) inhalation, followed by a 5-cm midline laparotomy to induce soft-tissue trauma. The abdomen was then closed in layers, and catheters were placed in both femoral arteries and the right femoral vein [polyethylene (PE)-50 tubing; Becton-Dickinson, Sparks, MD]. The animals were then restrained in a supine position, and the incision wounds were bathed with 1% lidocaine (Elkins-Sinn, Cherry Hill, NJ) throughout the surgical procedure to minimize postoperative pain. The rats were then allowed to awaken, following which they were bled rapidly to a mean arterial pressure (MAP) of 35–40 mmHg within 10 min. The time at which the animals could no longer maintain a MAP of 35–40 mmHg without infusing some fluid was defined as maximum bleed-out volume. The rats were maintained at that MAP until 40% of the shed blood volume was returned in the form of Ringer lactate. The animals were then resuscitated with four times the shed blood volume with Ringer lactate over 60 min. Adiol (1 mg/kg body wt iv) or vehicle was administered at the end of resuscitation. The catheters were then removed, the vessels were ligated, and the skin incisions were closed with sutures. Sham-operated animals underwent the same surgical procedure, which included the ligation of the femoral arteries and a femoral vein; however, neither hemorrhage nor resuscitation was performed. The animals were then returned to their cages and allowed food and water ad libitum. There was no mortality observed in this model of T-H shock.
Model of polymicrobial sepsis. At 20 h after the completion of fluid resuscitation or sham operation, the animals were again anesthetized with isoflurane, and polymicrobial sepsis was induced by cecal ligation and puncture (CLP) as described by Chaudry et al. (5). Briefly, following a 5-cm midline incision, the cecum was exposed and ligated just below the ileocecal valve. The cecum was then punctured twice with an 18-gauge needle and returned to the abdominal cavity. The abdominal cavity was then closed in layers, and the rats were resuscitated with saline solution subcutaneously (3 ml/100 g body wt). Animals were killed at 5 h following CLP to collect blood and tissue samples for further analysis. However, in the experiments in which survival was measured, the procedure of T-H and CLP was performed in a similar way, but 10 h after CLP, the gangrenous cecum was removed, the cavity was irrigated with warm saline, the abdominal incision was closed in layers, and mortality was recorded over a period of 10 days. The reason for this is to reduce the severity of the two-hit model; if we do not remove cecum, the model becomes very severe, and all animals will die within 24 h. No mortality is observed following T-H alone.
Plasma IL-6 and IL-10 levels. Plasma levels of IL-6 and IL-10 were determined using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
IL-6, IL-10, and iNOS mRNA expressions. Gene expressions of IL-6, IL-10, and iNOS were determined by quantitative real-time PCR as previously described (38). Total RNA was isolated from whole lung, heart, and liver samples using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The cDNA was generated from the total RNA samples by using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Each real-time PCR reaction was performed in a 10-µl reaction mixture containing 20 ng of cDNA, 2x PCR master mix (Applied Biosystems), and each probe and primer set. TaqMan gene expression assays (Applied Biosystems) for IL-6, IL-10, and iNOS, were purchased as probe and primer sets. The reaction mixture was denatured for one cycle of 2 min at 50°C and 10 min at 95°C and incubated for 40 cycles (denaturing for 15 sec at 95°C and annealing and extending for 1 min at 60°C) using the ABI Prism 7900HT (Applied Biosystems). All samples were tested in triplicate, and average values were used for quantification. rRNA (18S) was used as an endogenous control. The comparative threshold cycle (CT) method was used for quantification of gene expression. Analysis was performed using SDS v2.2 software (Applied Biosystems) according to the manufacturer's instruction. The normalized data was used and expressed as percent of the CLP + vehicle group, which was considered as 1.0.
Myeloperoxidase activity. MPO activity, an indicator of neutrophil cell accumulation, was determined from lung and heart samples collected at 5 h after CLP. Each sample was homogenized in a 10x volume of solution containing 0.5% (wt/vol) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and hydrogen peroxide (0.1 mM). The rate of change in absorbance was measured spectrophotometrically at 650 nm, and MPO activity was defined as units per gram of tissue protein. Protein concentration was determined using a Bio-Rad assay kit (Bio-Rad, Hercules, CA).
Cytokine-induced neutrophil chemoatractant-3 levels. Tissue levels of cytokine-induced neutrophil chemoatractant-3 (CINC-3) were determined using an ELISA kit (R&D Systems) according to the manufacturer's instructions. Tissue concentration of CINC-3 was defined as nanograms of CINC-3 per gram of tissue protein.
Alanine aminotransferase and lactate dehydrogenase assays. Plasma samples were examined for alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels. Measurements were performed spectrophotometrically by using diagnostic kits from Sigma according to the manufacturer's descriptions.
Statistical analysis. Values are means ± SE of 5–6 animals/group. For survival studies, 16 animals were used for each group. Statistical differences between groups were calculated either by one-way analysis of variance (ANOVA), followed by a Fisher LSD post hoc test, or by a Fisher exact test. Differences were considered significant if P < 0.05.
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RESULTS |
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Similar to the cytokine release, iNOS expression in liver, heart, and lung was also significantly upregulated following T-H and subsequent CLP. Adiol administration following T-H significantly prevented the increase of iNOS mRNA expression in the liver, heart, and lung (Fig. 3, A–C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The results indicate that MPO activity in the heart and lung increased significantly in the combined model of T-H and sepsis, indicating neutrophil activation under those conditions. In contrast, the MPO activity was unaffected in the heart as well as in the lung at 5 h after the onset of sepsis. However, since we did not use normal rats for comparison, it could be argued that the MPO was indeed increased with sepsis alone. Although this may be so, the fact remains that adiol had no effect on heart or lung MPO in the model of sepsis alone, thereby suggesting that 5 h may be too early for sepsis in itself to produce neutrophil activation. Further support for the notion that 5 h after the onset of sepsis without prior T-H did not produce any significant adverse effects comes from the finding that liver LDH and ALT levels were also not increased at that interval without or with adiol administration. These results are in accordance with our previous results that showed that the liver enzymes were not increased at 5 h after CLP (35). Furthermore, CINC-3 was not affected significantly at 5 h after CLP, and although CINC-3 levels were detected in the liver and lung, the levels were not affected by adiol, again suggesting that these are normal levels of CINC-3 in the liver and lung. Altogether, the results as summarized in this manuscript clearly suggest that adiol prevents both the inflammatory response and organ dysfunction following a combined insult of T-H and CLP.
A number of studies have also shown that IL-10 is capable of inhibiting the production of macrophage-derived proinflammatory cytokines. Since ablation of Kupffer cells by gadolinium chloride also decreased circulating IL-10 levels following T-H, it indicates that Kupffer cells also represent the major cellular source of elevated circulating IL-10 levels under those conditions (26). In view of these results, it can be suggested that adiol-induced salutary effects are mediated, at least in part, by a decreased cytokine production by Kupffer cells. Although the precise mechanism responsible for the salutary effects of adiol on cytokine production remains unknown, it is possible that adiol mediates its salutary effects via the estrogen receptor(s). In this regard, studies have indicated that estrogen-like activity of adiol is observed at physiological concentration in breast cancer cells (20). Furthermore, adiol causes an increase in estrogen receptor-dependent -galactosidase activity in yeast (20). Previous studies from our laboratory have shown that proestrus female mice, which have high circulating levels of estrogen and progesterone, have a significantly lower mortality following T-H and the induction of subsequent sepsis (9) or sepsis alone (40) than male counterparts. Moreover, the salutary effects of DHEA (a parent compound of adiol) on organ functions are abolished if the estrogen receptor antagonist ICI 182,780 is administered with DHEA (12). This suggests that in addition to adiol, DHEA also mediates its effects via the estrogen receptor(s) (12).
Another potential mechanism of the salutary effect of adiol could be the activation of peroxisomes. Waxman (36) has demonstrated that adiol can activate peroxisome proliferator gamma (PPAR-
). Activated PPAR- could reduce organ injury in T-H shock and systemic inflammation in polymicrobial sepsis (1, 41). Our recent study indicated that adiol treatment following T-H activated PPAR-, and treatment with PPAR- antagonist prevented the salutary effects of adiol under those conditions (unpublished observations). Thus it is likely that adiol produces its salutary effects via PPAR-; however, more studies are needed to delineate the precise mechanism of the salutary effects of this compound following T-H and/or sepsis.
Previous studies have shown that CLP alone induces an inflammatory response at a time point later than 5 h, causes organ dysfunction, and potentiates the inflammatory response following T-H (9, 13, 16, 35). However, CLP-induced inflammatory response in the absence of T-H was not observed at an early time point (i.e., 5 h after CLP). The primary reason to perform CLP in this study was not to determine the effect of CLP on the inflammatory response or organ function, rather its potential effect on the post-T-H inflammatory response and organ function. Since at 5 h there was no effect of CLP on inflammatory response, we selected this time point to be optimum to differentiate between the responses induced by CLP and T-H alone as well as the responses induced in the event that the two, CLP and T-H, are combined. Thus measurement at 5 h after CLP has allowed us to determine the effect of CLP on the T-H-induced inflammatory response and organ function and whether those potential effects are prevented in adiol-treated animals.
Our studies suggest that T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil infiltration in lung and heart, and caused liver injury. These alterations in inflammatory mediators accompanied with an increase in mortality following T-H and sepsis. Adiol administration suppresses proinflammatory cytokines and decreases mortality rates following T-H and sepsis. The improved survival from sepsis by adiol treatment following T-H appears to be due to decreased organ damage and improved organ functions. We found that these improvements are accompanied with a reduction in IL-6 and/or iNOS production. Collectively, these findings suggest a relationship between increased inflammatory mediators and mortality following T-H and sepsis. Nevertheless, more studies are required to delineate the mechanism by which the decrease in inflammatory response results in an increase in antimicrobial defense. Furthermore, whether adiol modulation of global inflammatory response or any particular chemokine or cytokine is responsible for the salutary effects remains to be established. In summary, although the precise mechanism of the salutary effects of adiol requires further investigations, our studies suggest that adjuvant use of adiol following T-H appears to be a novel approach for reducing the mortality rates following T-H and the occurrence of subsequent sepsis.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* L. Szalay and T. Shimizu contributed equally to this work. 
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REFERENCES |
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ligand 15-deoxy
12,14 prostaglandin J2 reduces the organ injury in hemorrhagic shock. Shock 22: 555–561, 2004.[CrossRef][ISI][Medline] is a new therapeutic target in sepsis and inflammation. Shock 23: 393–399, 2005.[CrossRef][ISI][Medline]This article has been cited by other articles:
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