The effect of progesterone on systemic inflammation and oxidative stress in the rat model of sepsis.

OBJECTIVES: To explore the protective effect of progesterone on inflammation and oxidative stress in a rat model of sepsis created by cecal ligation and puncture (CLP).
MATERIALS AND METHODS: Rats were randomly divided into 4 groups: Overiectomy group (OVX), sham operated (control), sepsis (CLP) group and progesterone-treated CLP group (CLP+ progesterone). The rats in CLP+ progesterone group received intraperitoneal progesterone (2 mg/kg). Cardiac blood samples were obtained for the measurement levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Tissue samples, including liver, kidney and uterus of rats were prepared to determine activities of myeloperoxidase (MPO), glutathione peroxidase (GPx) and levels of malondialdehyde (MDA).
RESULTS: Increased serum IL-6 and TNF-α levels were found in the CLP group in comparison with the control group (P = 0.01, P = 0.02; respectively). In CLP+ progesterone group, mean MDA concentration of kidney tissue was significantly lower than in CLP group (P = 0.003). Liver MDA concentration of the CLP+ progesterone group was not significantly different from that of the control group. While there were no significant differences among groups regarding liver MPO; in the CLP group, MPO activity in kidney (P = 0.02) and uterine tissues (P = 0.03) were found to be significantly higher compared to the control group. In CLP+ progesterone group, mean MPO activities of all tissues were not different than those of control group. The uterine tissue GPx activity in the CLP+ progesterone group was not statistically significantly different from control group.
CONCLUSIONS: We suggest that progesterone ameliorates sepsis syndrome by reduction of the inflammatory cytokines IL-6 and TNF-α, and by restoration of antioxidant enzyme activities in some tissues.

Introduction

Sepsis is defined as the systemic response to infection, and it has a complex pathophysiology.[1] While severe sepsis is manifested by organ dysfunction, septic shock is a type of severe sepsis marked by hypotension despite fluid resuscitation. However, although there are many different therapeutic modalities; severe sepsis and septic shock are major causes of mortality in intensive care units.[23]

There is inflammatory response in the pathogenesis of sepsis and associated with excessive production of cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). TNF-α plays a pivotal role in the pathogenesis of an early phase of shock.[45] The endothelial cells and neutrophils were activated with TNF-α to induce an inflammatory response. Additionally, expression of adhesion molecules increases on neutrophils. Activated neutrophils damage tissues by releasing oxygen-free radicals and proteases. In addition, TNF-α amplifies inflammatory cascades by activating macrophages/monocytes to secrete other pro-inflammatory cytokines. These cytokines cause tissue damage and contribute to apoptosis.[67]

While the effect of progesterone on cecal ligation and puncture (CLP)-induced tissue injury has not been studied, progesterone is reported to have a protective effect on cerebral or myocardial I/R injury in rats.[89] In one study that was conducted to examine the inhibitory effects of progesterone on inflammatory response, progesterone treatment decreased expression of inflammatory cytokines e.g., IL-1β and TNF-α in brain-injured rats.[8] Progesterone also may reduce oxidative stress by its membrane-stabilizing effect.[10]

The aim of this study was investigate whether progesterone affect systemic inflammation and tissue damage in female rat sepsis models of polymicrobial peritonitis caused by CLP.

Materials and Methods

Experimental Animals

This study was performed in accordance with the National Institute of Health's approved guidelines and the experimental protocol (protocol number: B.30.2.ATA.023.85-59) approved by the Animal Research Ethics Committee of Ataturk University, Medical Faculty, Erzurum, Turkey.

Twenty-eight adult female Sprague-Dawley rats (weighing 200-250 g) were purchased from the Ataturk University Experimental Animal Laboratory. All rats were kept in a light- and temperature-controlled room with 14:10-hour light-dark cycles and temperature of 22 ± 0.5°C, and were fed a standard pellet diet. Fifteen days before the experimental study, all rats were bilaterally ovariectomized to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels. Surgical anesthesia was provided intraperitoneally (i.p.) Thiopental dose of 25 mg/kg was administered and bilateral ovariectomy was performed by a midline dorsal skin incision. The peritoneal cavity was reached and ovaries were found. After the ligation of blood vessels, the connection fallopian tube and uterine horn was cut and ovaries were excised. Then the skin incision was closed using a 4/0 sterile synthetic absorbable suture. Surgical procedure was carried out under highly aseptic conditions.

Animal Groups and Study Design

The rats were randomly divided into four groups of seven individuals: The overiectomy group (OVX), the sham operated (control) group, the sepsis (CLP) group and the progesterone-treated CLP group (CLP+ progesterone). A CLP method of polymicrobial sepsis was applied to the rats in CLP and CLP+ progesterone groups. Polymicrobial sepsis was induced by cecal ligation and two-hole puncture, as described by Wichterman et al.,[11] with minor modifications. Thiopental (25 mg/kg) was used to provide surgical anesthesia in rats. After shaving the rats’ abdomen, a midline laparotomy was performed and the cecum was ligated just below the ileocecal valve using 3-0 silk ligatures. Using a 12-gauge needle, the cecum was perforated through the cecum distal to the point of ligation at two locations 1 cm apart. The cecum was then returned to the abdomen and the abdominal incision was closed using a 4/0 sterile synthetic absorbable suture. The wound was bathed in a 1% lidocaine solution to provide analgesia. Laparotomy was performed on the control group, and the cecum was manipulated, although not ligated or perforated. All animals were resuscitated with normal saline (2 mL/100 g body weight) injected subcutaneously at the time of surgery and also at 6 hours postoperatively. Rats of CLP+ progesterone group received i.p. injections of 2 mg/kg progesterone in peanut oil at 2 hours after sepsis induction. Sham-operated rats served as controls. Peanut oil was used as a vehicle and administered by i.p. route to the control group. Postoperatively, the rats were deprived of food, although they had free access to water until they were sacrificed. Sixteen hours after the surgery, all rats were sacrificed with an overdose of a general anesthetic (thiopental sodium, 50 mg/kg).

Cardiac blood samples were obtained for measurement of the levels of TNF-α and IL-6. Serum samples were aliqouted and stored at –80°C until the analysis date. The liver, uterus and kidney tissues of all rats were rapidly removed and washed in ice-cold saline. The organs were labeled and stored at –80°C until the biochemical analyses were conducted. Activities of myeloperoxidase (MPO), indicating neutrophil infiltration, and glutathione peroxidase (GPx), which is an antioxidant enzyme, and concentration of malondialdehyde (MDA), a marker of lipid peroxidation, were measured in tissue samples.

Preparation of Tissues

A portion of tissue was homogenized in a potassium phosphate buffer (pH: 6) containing 5% hexadecyltrimethyl ammonium bromide solution (w/v) for MPO activity measurement; the rest of the tissue was homogenized using a solution of 1.15% KCl for GPx and MDA measurements. The homogenates were centrifuged at 8000xg, +4°C for 15 minutes. Supernatants were then used for biochemical analysis.

Serum TNF-α and IL-6 levels

Serum TNF-α and IL-6 levels were measured using rat TNF-α ELISA kit (Invitrogen, Carlsbad, CA, USA; catalog number: KRC3011, lot number: 776995A) and rat IL-6 Platinum ELISA kit (eBioscience, San Diego, CA, USA; catalog number: BMS625, lot number: 60405021) according to the manufacturer's instructions. Intra-assay coefficiency of variation (CV) was 5.8% for TNF-α and 5% for IL-6. Inter-assay CV was 8.3% for TNF-α and 9% for IL-6. Results were expressed as pg/ml.

MPO activity

MPO activity was measured according to Bradley et al.'s modified method.[12] It was determined by adding 100 μL of the supernatant to 1.9 mL of 10 mmol/L phosphate buffer (PH = 6.0) and 1 mL of 1.5 mmol/L o-dianisidine hydrochloride containing 0.0005% (wt/vol) hydrogen peroxide. The changes in each sample's absorbance at 450 nm were recorded on a UV–vis spectrophotometer. MPO activity was expressed as U/g protein.

GPx activity

GPx activity was measured according to the method described by Paglia and Valentine.[13] GPx in tissue catalyzed the oxidation of glutathione by hydrogen hydroperoxide. Then, in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate, the oxidized glutathione was converted to its reduced form with a concomitant oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate. The decrease in absorbance was measured at 340 nm. GPx activity was expressed as U/g protein.

MDA levels

Tissue MDA levels were determined spectrophotometrically according to the method described by Ohkawa et al.[14] Briefly, supernatant (0.5 ml) was added to a solution containing 0.2 mL of 80 g/L sodium lauryl sulfate, 1.5 mL of 200 g/L acetic acid, 1.5 mL of 8 g/L 2-thiobarbiturate and 0.3 mL distilled water. The mixture was incubated at boiled water for 1 h. Upon cooling, 5 mL of n-butanol: Pyridine (15:l) was added. The mixture was vortexed and centrifuged. The absorbance of the supernatant was measured at 532 nm. The standard curve was obtained by using 1,1,3,3-tetramethoxypropane. Results were expressed as μmol/g protein.

Statistical Analysis

Statistical analysis was carried out with a statistical software package (SPSS 17.0). Results were given as mean ± SD. Differences among groups were tested using the ANOVA post hoc LSD test. Significance was considered as P < 0.05.

Results

Increased plasma IL-6 and TNF-α levels (pg/ml) were found in the CLP group (36.17 ± 3.41 and 15.56 ± 4.46) in comparison with the control group (26.85 ± 2.74 and 11.5 ± 1.72) (P = 0.01, P = 0.02; respectively). However in CLP+ progesterone group, the serum levels of IL-6 and TNF-α (31.79 ± 7.89 and 12.99 ± 3.42) were similar to the control group [Figure 1]. MPO, GPx activities and MDA levels in liver, kidney and uterine tissues of study groups are presented in Table 1. In the OVX group, while liver and uterine MDA concentrations (μmol/g protein) were similar to control, the kidney MDA concentration was higher than those of the control group (P = 0.02). In the CLP group, kidney MDA concentration was significantly higher than in the control (P = 0.0001) and OVX groups (P = 0.02). In CLP+ progesterone group, mean MDA concentration of kidney tissue was significantly lower than in CLP group (P = 0.003). MDA concentrations in the liver and uterine tissues were significantly elevated in the CLP group compared to the control group (P = 0.01 for both). Liver MDA concentration of the CLP+ progesterone group was not significantly different from that of control group. While there were no significant differences among groups regarding to liver MPO (U/g protein); in the CLP group, MPO activity in kidney (P = 0.02) and uterine tissue (P = 0.03) were found to be significantly higher compared to the control group. In CLP+ progesterone group, mean MPO activities of all tissues were not different than those of control group. While the GPx activity (U/g protein) in uterine tissue of the CLP group was lower than those of the control group (P = 0.02), the GPx activity in the CLP+ progesterone group was not statistically significantly different from control group. In the kidney and liver tissues, there were no significant differences regarding GPx activity among groups.

Figure 1

Comparison of groups according to serum TNF alpha and IL-6 levels. *P = 0.01 and **P = 0.02 compared to control group

Table 1

MPO and GPˣ activities and MDA levels in liver, kidney and uterine tissues of control, CLP, OVX and CLP+Prog groups

Discussion

Oxidative stress is important in sepsis.[15] Excessive production of cytokines (e.g., IL-6, TNF-α) is reported in a part of sepsis pathogenesis.[1617] The mortality and morbidity of sepsis are high despite advances in new therapeutic agents. Considering that sepsis is a very significant disease that leads to death in intensive care units, there is a great need to better understand the pathogenesis of this disease and develop effective treatment modalities. The purpose of this study was to examine the effects of progesterone on systemic inflammation and tissue damage of ovariectomized rats in a CLP-induced sepsis model.

Progesterone, a sex steroid hormone has been reported to suppress the oxidative damage in cardiac and brain tissues,[89] decrease oxidative damage in the colonic mucosa[18] and reduce cell apoptosis in brain tissue.[19] However, the potential role of progesterone in sepsis has not been studied. In our study, increased serum levels of IL-6 and TNF-α reduced after progesterone treatment in a rat model created sepsis with CLP. Also, CLP increased the MPO activity in kidney and uterine tissues, but similar values to the control group were observed after treatment with progesterone. The key finding in our study was that exogenously given progesterone in sepsis resulted in a significant decrease in systemic inflammation and MPO activity, as a leukocyte activation marker, compared to rats that did not receive it. Emerging evidence suggests that progesterone has the potential to influence inflammation.[2021] In a study, expression of TNF-α reduced in progesterone-treated rats compared to controls and authors suggested that progesterone administration is beneficial for cerebral trauma and infarction by inhibiting inflammatory reaction.[8] Additionally, in Dhote et al.'s study,[9] they investigated the effect of progesterone on oxidative stress and myocardial ischemia markers in the myocardial ischemia-reperfusion model. At the end of the study, MPO activity was reduced after progesterone treatment in female rats. They concluded that the administration of progesterone during myocardial injury reduces inflammatory reactivity and provides better cardioprotection in female rats compared to male and they suggested that progesterone therapy may be useful in myocardial injury due to a diminished inflammatory response.

There is no consensus among researchers regarding the influence of gender on survival in patients with sepsis. For example, Esper et al.[22] found no gender differences, Adrie et al.[23] reported higher risk in men and Dombrovskiy et al.[24] declared higher risk in women in terms of sepsis survival. On the other hand, Schroder et al.[25] suggested that sex hormones estrogen and progesterone may be effective in providing protection against sepsis in women. Before beginning the experimental study, we removed the ovaries of all rats to eliminate endogenous progesterone production and to reduce systemic ovarian hormone levels. Our results demonstrated that progesterone attenuates the sepsis-induced elevations of IL-6 and TNF- α. We applied 2 mg/kg dose of progesterone immediately after CLP. In the brain injury model, it was suggested that progesterone in inhibiting the proinflammatory cytokines is time-dependent.[26] Progesterone effectively reduced the gene expression of IL-1β and TNF-α at 3 hours post-injury. After 8 and 12 hours of continued administration of progesterone, cytokine elevations were not observed. Therefore, we selected the progesterone delivery time according to this data. It has been demonstrated that progesterone is also neuroprotective in cerebral ischemia and traumatic brain injury.[1026] Zhao et al.[27] researched the effects of progesterone on the intestinal pathophysiologic changes following subarachnoid hemorrhage in male rats. After progesterone administration (2 mg/kg i.p.), the concentrations of IL-1β, TNF-α and IL-6 were found to be significantly lower in rat ileum tissue. They found that progesterone administration modulates intestinal inflammatory response. Used dose of progesterone in this current study was selected based on this study.[27]

Researchers have examined the effect of different agents in the treatment of sepsis.[161728] Sahin et al.[28] examined the effect of carnosine in an experimental septic shock model. They concluded that carnosine may be an effective treatment for oxidative damage in cases of septic shock. According to our literature data, this present study is the first investigating the efficacy of progesterone on sepsis in a rat model created sepsis using CLP.

MDA is a lipid peroxidation product and a marker for tissue damage.[29] In a recent study, Karatepe et al.[18] researched the effects of progesterone on an experimental colitis model. They reported both lower blood and colonic tissue levels of MDA, IL-6 and TNF-α in rats treated with progesterone (subcutaneously with dose of 2 mg/kg) compared to the rats that only created colitis by intrarectal administration of 5 mg trinitrobenzene sulfonic acid. In another study,[30] investigating possible neuroprotective effects of progesterone in rats subjected to global cerebral ischemia, progesterone administration ameliorated ischemia-induced decrease in glutathione (major endogenous antioxidant) and increase in MDA levels in hippocampus, striatum and cortex. They evaluated the remarkable neuroprotective effect of progesterone reducing oxidative stress. In our study, the MDA concentration in tissues of uterine, liver and kidney and plasma IL-6 and TNF-α level increased in rats after created CLP, but the values after progesterone treatment were similar to the values in the control group. Also while increased uterine tissue GPx activity (an enzymatic antioxidant) was found in rats to create sepsis, similar uterine GPx activity to the control group was observed after progesterone treatment in this current study.

Conclusion

It may be suggested that progesterone ameliorates sepsis syndrome by reduction of the inflammatory cytokines, IL-6 and TNF-α, and by restoration of antioxidant defense system. Progesterone therefore may be beneficial in controlling inflammation in sepsis. Further prospective and randomized clinical controlled trials are required to investigate the therapeutic role of progesterone in tissue damage resulting from sepsis.