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Human & Experimental Toxicology

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Research article

First published online June 18, 2015

Montelukast attenuates lipopolysaccharide-induced cardiac injury in rats

AE Khodir, HA Ghoneim, MA Rahim, and GM Suddek [email protected]View all authors and affiliations

https://doi.org/10.1177/0960327115591372

Abstract

This study investigates the possible protective effects of montelukast (MNT) against lipopolysaccharide (LPS)-induced cardiac injury, in comparison to dexamethasone (DEX), a standard anti-inflammatory. Male Sprague Dawley rats (160–180 g) were assigned to five groups (n = 8/group): (1) control; (2) LPS (10 mg/kg, intraperitoneal (i.p.)); (3) LPS + MNT (10 mg/kg, per os (p.o.)); (4) LPS + MNT (20 mg/kg, p.o.); and (5) LPS + DEX (1 mg/kg, i.p.). Twenty-four hours after LPS injection, heart/body weight (BW) ratio and percent survival of rats were determined. Serum total protein, creatine kinase muscle/brain (CK-MB), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) activities were measured. Heart samples were taken for histological assessment and for determination of malondialdehyde (MDA) and glutathione (GSH) contents. Cardiac tumor necrosis factor α (TNF-α) expression was evaluated immunohistochemically. LPS significantly increased heart/BW ratio, serum CK-MB, ALP, and LDH activities and decreased percent survival and serum total protein levels. MDA content increased in heart tissues with a concomitant reduction in GSH content. Immunohistochemical staining of heart specimens from LPS-treated rats revealed high expression of TNF-α. MNT significantly reduced percent mortality and suppressed the release of inflammatory and oxidative stress markers when compared with LPS group. Additionally, MNT effectively preserved tissue morphology as evidenced by histological evaluation. MNT (20 mg/kg) was more effective in alleviating LPS-induced heart injury when compared with both MNT (10 mg/kg) and DEX (1 mg/kg), as evidenced by decrease in positive staining by TNF-α immunohistochemically, decrease MDA, and increase GSH content in heart tissue. This study demonstrates that MNT might have cardioprotective effects against the inflammatory process during endotoxemia. This effect can be attributed to its antioxidant and/or anti-inflammatory properties.

Introduction

Sepsis, the systemic inflammatory response syndrome to infection, has high incidence and mortality rates around the world.^1,2 Lipopolysaccharide (LPS, the main component of gram-negative bacteria endotoxin) has been recognized as a principal component causing multiple organ injury. Myocardial dysfunction is a recognized manifestation of sepsis and septic shock, with myocardial depression occurring in almost 40–50% of patients.² Various mechanisms have been proposed for this dysfunction, including cardiac inflammation,³ mitochondrial dysfunction,^4,5 cardiac cell death by necrosis or apoptosis,⁶ impaired contractility secondary to the generation of tumor necrosis factor α (TNF-α),⁷ and induction of inducible nitric oxide synthase.^7,8 Agents that can downregulate the expression of inflammatory cytokines may have beneficial effects against LPS-induced multiple organ injury. In the recent years, reactive oxygen species (ROS) and reactive nitrogen species (RNS) have stimulated considerable interest as an important mechanism of LPS-induced organ injury.⁹ These ROS/RNS highly react with biological macromolecules, producing lipid peroxides, mutating DNA, and inactivating proteins.¹⁰ Certain antioxidants may alter the redox balance either by directly scavenging free radicals or by enhancing endogenous antioxidant defense system and hence can control the endotoxin-induced inflammation.¹¹

Montelukast (MNT) is an anti-inflammatory drug with antioxidant properties.¹² It is a selective and orally active leukotriene receptor antagonist that specifically inhibits the cysteinyl leukotriene (CysLT1) receptor and reduces the airway eosinophilic inflammation.¹³ CysLTs and the 5-lipoxygenase metabolites of arachidonic acid are proven to be potent inflammatory mediators that cause tissue injury.¹⁴ MNT has been shown to be effective in ameliorating oxidative damage in several rat models. Dengiz et al.¹² reported that MNT possesses a gastroprotective and antioxidant effects on indomethacin-induced gastric ulcer in rats. Additionally, Coskun et al.¹⁵ found that MNT can protect against oxidative injury in a rat model of cecal ligation and puncture. Therefore, the present study was designed to elucidate the potential protective effect of MNT on LPS-induced cardiac injury and dysfunction in rats.

Materials and methods

Experimental animals

Male Sprague Dawley rats aged 7–8 weeks and weighing 160–180 g were used in all of the experiments. They were obtained from Urology and Nephrology Center of Mansoura University, Mansoura, Egypt. The animals were maintained under standard conditions of temperature 24 ± 1°C and 55 ± 5% relative humidity with regular 12-h light/12-h dark cycles. Rats were acclimatized for 1 week before experiment. They were allowed free access to standard laboratory food and water. The experiments were conducted in accordance with the ethical guidelines for investigations in laboratory animals and were approved by the Ethical Committee of Faculty of Pharmacy, Mansoura University, Egypt.

Materials

MNT sodium (described chemically as [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl) ethenyl] phenyl]-3-[2-(1-hydroxy-1 methylethyl) phenyl] propyl] thio] methyl] cyclopropaneacetic acid, monosodium salt) was obtained as a generous gift from Merck & Co., Inc. (USA) and was suspended in carboxymethyl cellulose (CMC). LPS (Escherichia coli LPS, serotype 0111: B4) was purchased from Sigma Chemical Co. (St Louis, Missouri, USA). Diagnostic kits for determination of creatine kinase muscle/brain (CK-MB), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and total protein were purchased from BioMed Diagnostics, EGY-CHEM Co. (Egypt). TNF-α antibodies were purchased from Boster Biological Technology Co., Ltd (China). All of the remaining chemicals are of the highest commercially available grade.

Experimental design

Rats were divided into five groups (eight animals each) of the following schedule: control group received drug vehicle (0.5% CMC) 1 week before intraperitoneal (i.p.) administration of saline (0.9%) on the 7th day; LPS group received CMC for 7 days and then challenged with LPS (10 mg/kg, i.p.)¹⁶ on the 7th day; MNT + LPS groups received either MNT (10 mg/kg, per os (p.o.) using oral gavage)¹⁶ or MNT (20 mg/kg, p.o.)¹⁷ for 7 days with the last dose administered 2 h before LPS (10 mg/kg, i.p.) injection on the 7th day; and dexamethasone (DEX) + LPS group received DEX (1 mg/kg, i.p) 2 h before LPS (10 mg/kg, i.p.) injection.¹⁸

Normal rats under MNT or DEX treatment showed no relevant differences when compared with normal rats under vehicle saline group and thus, the results were excluded from tables and figures in order to facilitate data comparison and interpretation.

Twenty-four hours after vehicle or LPS challenge, all rats were weighed and the percent survival was recorded. All dead animals were replaced with other animals under the same treatment protocol to restore the groups with eight rats each. Blood samples were collected for determination of CK-MB, a marker of cardiac cell injury, ALP, and total protein levels. Serum LDH activity, as a marker of generalized tissue injury, was measured. Rats were killed by an overdose of ether and then heart samples were taken for histological assessment of the lesions and for the measurement of malondialdehyde (MDA, an end product of lipid peroxidation) and antioxidant-reduced glutathione (GSH) contents.

Determination of serum CK-MB activity

CK-MB activity was determined according to the method of Würzburg et al.¹⁹ The method is based on measuring CK activity in the presence of an antibody to the CK-M monomer. Reduced nicotinamide adenine dinucleotide (NADH+) is produced, and the rate of its formation, measured at 340 nm, is directly proportional to serum CK-B activity. CK-MB activity was calculated as units per liter.

Determination of serum LDH activity

LDH activity was assessed according to the method of Henry.²⁰ The method depends on the conversion of pyruvate to lactate by LDH-consuming NADH+, which absorbs light at 340 nm. Its consumption is directly proportional to serum LDH concentration. LDH activity was calculated as units per liter.

Determination of serum ALP activity

ALP activity was assessed according to Belfield and Goldberg.²¹ Hydrolysis of p-nitrophenyl phosphate gives p-nitrophenol and phosphate by ALP in alkaline medium. p-Nitrophenol has an intense yellow color in alkaline pH, and ALP activity in sample is determined by measuring the per time absorption increase at 405 nm.

Determination of serum total protein level

The estimation was done according to the standard procedures given along with the kits purchased. Protein reacts with copper ions (II) to produce a blue–violet color compound in alkaline medium. The color intensity is proportional to the concentration of total protein present in the sample.

Preparation of tissue homogenates

Heart tissues were rinsed in chilled 1.15% potassium chloride (KCl; pH 7.4) and weighed quickly. Subsequently, the heart/BW ratio was determined. Homogenization was carried out in ice-cold KCl (1.15%, pH 7.4) to yield 10% w/v tissue homogenates using a variable speed homogenizer (OMNI international, Kennesaw, Georgia, USA). The homogenate was centrifuged at 1500g for 10 min. The resulting supernatants were used for assays of MDA and GSH.

Assay of lipid peroxidation

The content of MDA, the end product of lipid peroxidation, in the supernatants was measured according to the method described by Ohkawa et al.²² The level of MDA was derived from a standard curve prepared from serial dilutions of a 3.2-μM stock solution of 1,1,3,3-tetramethoxypropane that were treated in identical manner as the tissue homogenate samples. MDA was expressed as nanomoles per gram tissue.

Assay of reduced GSH

The concentration of GSH in the tissue homogenate was measured according to the method described by Ellman.²³ The concentration of GSH in the sample was derived by reference to a calibration curve of GSH prepared from serial dilutions of a 240-nM GSH stock solution that were treated in identical manner as the tissue homogenate samples. The result was expressed as micromoles per gram tissue.

Histopathological examination

Heart was rapidly dissected out and fixed by immersion in 10% neutral-buffered formalin solution (pH 7.4). After fixation, the tissues were embedded in paraffin for histopathological examination; 5-μm tissue sections were stained with hematoxylin and eosin and observed under a light microscope.

Immunohistochemical localization of TNF-α

Paraffin blocks from rat hearts were used for immunohistochemical analysis. Tissue sections were cut and placed on Superfrost^® plus microscope slides (Fisher Scientific, Waltham, Massachusetts, USA). Using the manual immunohistochemistry stainer, slides were stained by the following procedure. Detection was done using the histostain bulk kit (Invitrogen Lab-SA detection system, Carlsbad, California, USA). Sections were deparaffinized using EZ Prep solution. CC1 standard (citrate buffer, pH 6.0) was used for antigen retrieval. 1,4-Dideoxy-1,4-imino-d-arabinitol (DAB) inhibitor (3% hydrogen peroxide (H₂O₂) endogenous peroxidase) was blocked for 5 min at room temperature (RT). Sections were incubated with anti-TNF-α antibody (Boster Biological Technology Co., Ltd, dilution 1/100) for 40 min at RT, followed by incubation with the secondary antibody of Universal HRP Multimer for 8 min at 37°C. Slides were treated with DAB + H₂O₂ substrate for 8 min followed by hematoxylin and the bluing reagent counterstain at 37°C. Reaction buffer (phosphate-buffered saline) was used as washing solution. Staining intensity of positively stained cells was evaluated. Controls consisted of staining without primary antibody. Staining was documented with a digital camera (Olympus, Japan) mounted on a microscope (Leica, Wetzlar, Germany) at 200× or 400× magnification. Each specimen was examined by a senior pathologist.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance followed by Tukey’s Kramer test. The values are means ± SE for eight rats in each group. p value <0.05 was considered as significant. Chi-square test was used for the comparison of two proportions. Statistical calculations were carried out using INSTAT-2 computer program (GraphPad Software Inc. V2.04, San Diego, California, USA).

Results

Mortality

At the end of the experiment, rats treated with vehicle showed 100% survival. Rats treated with LPS showed a significant decrease in the percent survival (50%) compared with control nontreated rats. Administration of MNT (20 mg/kg, p.o.) for 7 days prior to LPS showed a significant increase in the percent survival of animals (87.5%) compared with LPS group, while MNT (10 mg/kg, p.o.) for 7 days prior to LPS produced a nonsignificant change in the percent survival of animals compared with LPS group. Administration of DEX (1 mg/kg, i.p.) 2 h prior to LPS injection induced a significant increase in the percent survival of animals (87.5% survival) compared with LPS group (Figure 1). The 20 mg/kg dose of MNT was more protective than the 10 mg/kg dose demonstrating similar pattern as in DEX-treated group.

Figure 1. Effect of LPS (10 mg/kg, i.p.) and MNT (10 or 20 mg/kg, p.o.) on percent survival of rats. *Significantly different from control group using χ² test. #Significantly different from LPS-treated group using χ² test. $Significantly different from LPS/MNT (10 mg/kg)-treated group using χ² test. LPS: lipopolysaccharide; MNT: montelukast; i.p.: intraperitoneal; p.o.: per os.

Serum CK-MB, ALP, LDH, and total protein levels

As shown in Table 1, LPS significantly increased serum levels of CK-MB, ALP, and LDH while decreased serum total protein levels.

Table 1. Effect of LPS (10 mg/kg, i.p.) and/or MNT (10 or 20 mg/kg, p.o.) on serum biochemical markers.^a

Treatment	CK-MB (U/L)	ALP (U/L)	LDH (U/L)	Total protein (g/dL)
Control	635.3 ± 47.88	372.9 ± 38.51	1350 ± 169.5	7.44 ± 0.27
LPS	1497 ± 194.9^b	821.3 ± 72.99^b	4409 ± 170.9^b	5.32 ± 0.14^b
LPS/MNT (10 mg/kg)	1068 ± 33.27^b,c	521.5 ± 21.22^c	1675 ± 68.94^c	6.20 ± 0.15^b
LPS/MNT (20 mg/kg)	853 ± 112.6^c	491.3 ± 47.66^c	1355 ± 68.94^c	7.15 ± 0.06^c,d
LPS/DEX	879.3 ± 47.21^c	514.4 ± 45.92^c	1417 ± 108.8^c	7.13 ± 0.34^c

ANOVA: analysis of variance; LPS: lipopolysaccharide; DEX: dexamethasone; MNT: montelukast; CK-MB: creatine kinase muscle/brain; ALP: alkaline phosphatase; LDH: lactate dehydrogenase; i.p.: intraperitoneal; p.o.: per os.

^aData are expressed as mean ± SEM, n = 8.

^bSignificantly different from control group using one-way ANOVA followed by Tukey–Kramer multiple comparisons test (p < 0.05).

^cSignificantly different from LPS-treated group using one-way ANOVA followed by Tukey–Kramer multiple comparisons test (p < 0.05).

^dSignificantly different from LPS/MNT (10 mg/kg)-treated group using one-way ANOVA followed by Tukey–Kramer multiple comparisons test (p < 0.05).

In LPS group, CK-MB level was significantly increased when compared with control group. Administration of MNT (20 mg/kg) and DEX produced a significant decrease in serum CK-MB compared with LPS group showing a nonsignificant difference in comparison to control group. Administration of MNT (10 mg/kg) produced a significant decrease in serum CK-MB compared with LPS-grouped animals while the value was still significantly different from the control group.

LPS injection significantly increased ALP level to 821.3 ± 72.99 U/L, and both doses of MNT and DEX reversed the increase in ALP levels to 521.5 ± 21.22, 491.3 ± 47.66, and 514.4 ± 45.92 U/L, respectively (Table 1).

Serum LDH activity was found to be higher in LPS group compared with the control group and was completely reversed by treatment with MNT or DEX (Table 1).

As shown in Table 1, LPS caused a significant decrease in serum total protein level 24 h after LPS exposure, compared with control rats (p < 0.05). The protein content in the LPS + MNT groups was significantly higher than the LPS group (p < 0.05). In both LPS/MNT (20 mg/kg) and DEX groups, serum total protein was significantly higher than that in the LPS/MNT (10 mg/kg) group.

Heart/BW ratio

Heart/BW ratio of the rats was found to be higher in LPS group compared with the control group (reflecting the inflammation and edema in cardiac tissues induced by LPS) and was completely reversed by treatment with MNT or DEX (Table 2).

Table 2. Effect of LPS (10 mg/kg, i.p.) and/or MNT (10 or 20 mg/kg, p.o.) on heart/BW ratio and heart antioxidant status.^a

Treatment	Heart/BW ratio (×10⁻³)	MDA (nmol/g tissue)	GSH (μmol/g tissue)
Control	3.6 ± 0.02	0.38 ± 0.017	0.46 ± 0.01
LPS	4.2 ± 0.1^b	1.12 ± 0.17^b	0.36 ± 0.009^b
LPS/MNT (10 mg/kg)	3.8 ± 0.04^c	0.87 ± 0.09^b	0.39 ± 0.007^b
LPS/MNT (20 mg/kg)	3.6 ± 0.04^c	0.39 ± 0.016^c,d,e	0.46 ± 0.009^c,d,e
LPS/DEX	3.7 ± 0.1^c	0.82 ± 0.06^b	0.4 ± 0.02^b

ANOVA: analysis of variance; LPS: lipopolysaccharide; DEX: dexamethasone; BW: body weight; MNT: montelukast; MDA: malondialdehyde; GSH: glutathione; p.o.: per os; i.p.: intraperitoneal.

^aData are expressed as mean ± SEM, n = 8.

^bSignificantly different from control group using one-way ANOVA followed by Tukey–Kramer multiple comparisons test (p < 0.05).

^cSignificantly different from LPS-treated group using one-way ANOVA followed by Tukey–Kramer multiple comparisons test (p < 0.05).

^dSignificantly different from LPS/MNT (10 mg/kg)-treated group using one-way ANOVA followed by Tukey–Kramer multiple comparisons test (p < 0.05).

^eSignificantly different from DEX-treated group using one-way ANOVA followed by Tukey–Kramer multiple comparisons test (p < 0.05).

Tissue MDA and GSH levels

As shown in Table 2, LPS resulted in a significant increase in cardiac MDA levels to 1.12 ± 0.17 nmol/g tissue. These changes were significantly decreased by MNT (20 mg/kg) to the normal control levels (0.39 ± 0.016 nmol/g tissue). Both 10 mg/kg MNT and DEX did not significantly affect cardiac MDA levels when compared with LPS-treated rats.

LPS induced a significant decrease in heart GSH content compared with control nontreated animals (Table 2). The protection evoked by MNT (20 mg/kg) against LPS-induced changes in cardiac content of GSH was superior to that of MNT (10 mg/kg) and the standard DEX.

Histopathological examination

As shown in Figure 2(a), rats of the control group showed normal histology while heart of rats treated with LPS showed marked inflammation of heart wall (Figure 2(b)). These histological changes were absent in MNT or DEX-LPS-treated groups (Figure 2(c) to (e)).

Figure 2. Light microscopy photomicrographs of representative using hematoxylin and eosin-stained histological sections of the heart of (a) control group showing normal heart wall. (b) LPS group showing marked inflammation of the cardiac wall (arrow). (c), (d), and (e) LPS/MNT (10 mg/kg); LPS/MNT (20); and LPS/DEX group, respectively, showing normal heart wall. Original magnification ×200. LPS: lipopolysaccharide; DEX: dexamethasone; MNT: montelukast.

TNF-α expression

TNF-α expression in the cardiac tissues was evaluated by immunohistochemical detection. No positive staining for TNF-α was observed in the cardiac tissues from control rats (Figure 3(b)). On the contrary, immunostained sections obtained from LPS animals at 24 h revealed dense granules and some TNF-α-immunostained areas in the myocardiocytes reflecting severe inflammatory response (Figure 3(c)).

Treatment of LPS rats with MNT (10 mg/kg) administered orally 7 days prior to LPS revealed scattered immunostained granules by TNF-α in some myocardiocytes reflecting mild cardiac affection (Figure 3(d)), while MNT (20 mg/kg) administered to LPS-treated rats showed no evidence of positive staining by TNF-α, reflecting absence of inflammatory response (Figure 3(e)). LPS/DEX group showed dense intra-myocardiocyte immunostained granules and small foci by TNF-α reflecting moderate myocardial affection (Figure 3(f)).

Discussion

In this study, we observed that LPS injection resulted in a significant oxidative damage in cardiac tissues, as evidenced by increased lipid peroxidation with a concomitant decrease in endogenous antioxidant level. Moreover, generalized tissue injury was observed as evidenced by high serum LDH and ALP levels. The oxidative damage and tissue inflammation caused by LPS were abolished by MNT. In accordance with these biochemical changes, the morphological evaluation of the tissues revealed that MNT also was effective in protecting the heart tissues from LPS-induced degenerative changes. In addition to its beneficial effects, MNT treatment caused a dramatic reduction in proinflammatory cytokine TNF-α expression in heart tissues.

LPS, a bacterial cell wall component, is known to induce the production of several inflammatory cytokines, dysfunction of the endothelial cell layer, tissue edema, and injury.²⁴ Fujimura et al.²⁵ defined a period of over 16 h as the late phase of sepsis. In the current study, we defined the late phase of sepsis at the 24th hour. Mortality was high in this model. In the current study, the mortality rate was 50% survival (4/8) at 24 h. This result is in agreement with the study of Sun et al.²⁶ who reported that this mortality from endotoxemia may be due to oxidative stress developed and reactive oxygen species. In the present study, MNT decreased the rate of mortality to be 37.5% (3/8) and 12.5% (1/8) in the 10-mg/kg and 20-mg/kg MNT-treated LPS groups, respectively. Similar to 20-mg/kg MNT-treated group, administration of DEX (1 mg/kg, i.p.) 2 h before LPS decreased the rate of mortality to 12.5% (1/8). Furthermore, there was no mortality in the control group; a result that is in agreement with Coskun et al.¹⁵ The effect of MNT on LPS-induced changes in heart/body ratio was evaluated. LPS induced a significant increase in this ratio, which may reflect the inflammation and edema in heart tissues induced by LPS. MNT-treated groups showed a significant decrease in heart/BW ratio compared with LPS group confirming the anti-inflammatory effect of MNT.

In the present study, administration of LPS induced a significant increase in serum ALP and LDH as indicators of generalized tissue damage. MNT treatment caused a dramatic reduction in the serum LDH and ALP activity. This result is in agreement with the investigation reported by Sener et al.²⁷ In the study of Mohamadin et al.,¹⁶ the hepatic injury following LPS administration is well established by the elevated levels of serum LDH and ALP indicating cellular leakage and loss of functional integrity of hepatic membrane, and administration of MNT abrogated LPS-induced hepatotoxicity, by the reverted activities of ALP and LDH to their near normal levels in LPS-treated rats.

Myocardial dysfunction, a common complication after sepsis, significantly contributes to the death of patients with septic shock. In this study, administration of LPS (10 mg/kg) induced a significant increase in serum CK-MB. This result is in agreement with the investigation reported by Peek et al.²⁸ Additionally, Zhang et al.²⁹ reported that LPS plays a pivotal role in myocardial anomalies in sepsis. In the current work, MNT could protect heart against LPS-induced cardiac injury as manifested by decreased release of serum enzyme CK-MB. The protective effect of MNT was dose dependently evidenced in the serum since elevated levels of CK-MB, ALP, and LDH were markedly lower than those that were elicited by LPS.

Administration of LPS induced a significant decrease in serum total protein. The protective effect of MNT was dose dependently evidenced in the serum since low levels of total protein were markedly higher than those that were elicited by LPS. This finding is in agreement with the study of Mohamadin et al.¹⁶ in which pretreatment with MNT (10 mg/kg) significantly attenuated the severity of hepatic injury induced by LPS as evidenced by effective increase in the total protein and albumin levels.

In the current study, LPS produced oxidative stress in the heart, as it was evident from the significant increase in cardiac MDA contents along with a depletion of tissue GSH level indicating the enhancement of lipid peroxidation as a result of impaired antioxidant defense mechanism. Tissue MDA is a marker of lipid peroxidation that can cause changes in membrane fluidity and permeability, and thus increase the rate of protein degradation, which will eventually lead to cell lysis.¹⁵ The 20 mg/kg dose of MNT was more effective than the 10 mg/kg dose and DEX against LPS-induced changes in heart content of MDA. Similar results related to the effects of MNT on lipid peroxidation have been previously reported.^15,30

GSH acts as a major cellular antioxidant defense system by scavenging free radicals and other reactive oxygen species. The LPS-induced oxidative stress can lead to depletion of GSH,³¹ and the reduction of GSH may, in turn, aggravate the LPS toxicity probably via diminution of the antioxidant defense. It has been proposed that antioxidants that maintain the concentration of reduced GSH may restore the cellular defense mechanisms, block lipid peroxidation, and thus protect against the oxidative tissue damage. In accordance with these findings, our results verify that MNT maintained GSH levels. The protection evoked by MNT (20 mg/kg) against LPS-induced changes in heart content of GSH was superior to that of MNT (10 mg/kg) and the standard DEX. MNT had also been demonstrated to increase the level of GSH in rats with indomethacin-induced gastric ulcer,¹² gentamicin-induced nephrotoxicity and oxidative damage,³² and those with cisplatin-induced nephrotoxicity.³³

Our results confirm that the inhibition of tissue lipid peroxidation along with replenishment of GSH content by MNT implies that the compound is beneficial in maintaining oxidant–antioxidant balance. Although, MNT does not have a known effect to stimulate GSH biosynthesis, it seems plausible that it can improve the antioxidant defense system by inhibiting the lipid peroxidation reaction, thus, mitigating the consumption of GSH. This result is consistent with other reports that demonstrated that MNT could abrogate LPS-induced markers of liver injury and suppresses the release of inflammatory and oxidative stress markers via its antioxidant properties and enhancement of enzymatic antioxidant activities.¹⁶ Ozkan et al.³⁴ suggested that MNT with its anti-inflammatory and antioxidant properties may be of potential therapeutic value in protecting the liver against oxidative injury due to ischemia–reperfusion.

LPS is known to induce the production of several inflammatory and chemotactic cytokines. TNF-α is considered as an important mediator for sepsis and concomitant cardiac injury. TNF-α activates the inflammatory cascade by inciting the production of several cytokines and chemokines, and by enhancing endothelial adhesion molecules expression on vascular endothelial cells that promote neutrophil adherence to these cells. There is good evidence that overproduction of TNF-α is involved in the development of LPS-induced organ dysfunction.³⁵ In the present study, LPS caused the appearance of some TNF-α-immunstained areas in the myocardiocytes reflecting severe inflammatory response. MNT (20 mg/kg) treatment resulted in absence of positive staining by TNF-α, reflecting the absence of inflammatory response, while treatment of LPS rats with MNT (10 mg/kg) revealed a mild cardiac affection. Both effects are superior to that obtained by DEX treatment in which dense intra-myocardiocyte immunostained granules and small foci by TNF-α were present reflecting moderate myocardial affection.

These data suggest that the ability of MNT given to rats to produce less inflammatory cytokines in response to LPS-induced sepsis may, in part, account for a decrease in cytokine-related cardiac injury. It seems likely that the anti-inflammatory effect of MNT in LPS-induced sepsis involves the suppression of the proinflammatory mediator, TNF-α produced by the leukocytes and macrophages.

In the present study, cardiac histoarchitecture of LPS-treated rats resulted in marked diffuse inflammatory cell infiltration, which basically supported the alterations observed in biochemical assays. It might be due to the formation of highly reactive radicals because of oxidative threat induced by LPS.³⁶ MNT group had showed a remarkable improvement in pathological damage compared with LPS-induced cardiac injury. Coskun et al.¹⁵ reported that MNT has protective effect on antioxidant enzymes and proinflammatory cytokines on the heart in a rat model of cecal ligation and puncture-induced sepsis. Also, Chen et al.³⁷ suggested that MNT has cardioprotective role during myocardial injury by halting the leukotriene-induced inflammatory response in a rat model of isoproterenol-induced myocardial ischemia and necrosis. This may represent an approach to the treatment of myocardial ischemia with leukotriene antagonists. The present study found that the protective effect of MNT may be related to its ability to enhance antioxidant status and regulate proinflammatory cytokine production. MNT (20 mg/kg) is generally more effective than DEX. With lower risk of side effects, MNT might be preferred than DEX. Further clinical studies are required in order to elucidate the potential effectiveness of MNT in septic patients.

Conflict of interest

The authors declared no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References

1. Fernandes CJ Jr, Cesar de Assuncao MS. Myocardial dysfunction in sepsis: a large, unsolved puzzle. Crit Care Res Pract 2012; 2012: 896430.

Abstract

Introduction

Materials and methods

Experimental animals

Materials

Experimental design

Determination of serum CK-MB activity

Determination of serum LDH activity

Determination of serum ALP activity

Determination of serum total protein level

Preparation of tissue homogenates

Assay of lipid peroxidation

Assay of reduced GSH

Histopathological examination

Immunohistochemical localization of TNF-α

Statistical analysis

Results

Mortality

Serum CK-MB, ALP, LDH, and total protein levels

Heart/BW ratio

Tissue MDA and GSH levels

Histopathological examination

TNF-α expression

Discussion

Conflict of interest

Funding

References

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