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Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice
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PHAREP 286 1–7 Pharmacological Reports xxx (2015) xxx–xxx
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Original research article
Pretreatment with magnesium ameliorates lipopolysaccharideinduced liver injury in mice
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Q1 Dalia
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M. El-Tanbouly *, Rania M. Abdelsalam, Amina S. Attia, Mohamed T. Abdel-Aziz
Pharmacology & Toxicology Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 August 2014 Received in revised form 11 February 2015 Accepted 12 February 2015 Available online xxx
Background: Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, is involved in the pathogenesis of sepsis. LPS administration induces systemic inflammation that mimics many of the initial clinical features of sepsis and has deleterious effects on several organs including the liver and eventually leading to septic shock and death. The present study aimed to investigate the protective effect of magnesium (Mg), a well known cofactor in many enzymatic reactions and a critical component of the antioxidant system, on hepatic damage associated with LPS-induced endotoxima in mice. Methods: Mg (20 and 40 mg/kg, po) was administered for 7 consecutive days. Systemic inflammation was induced 1 h after the last dose of Mg by a single dose of LPS (2 mg/kg, ip) and 3 h thereafter plasma was separated, animals were sacrificed and their livers were isolated. Results: LPS-treated mice suffered from hepatic dysfunction revealed by histological observation, elevation in plasma transaminases activities, C-reactive protein content and caspase-3, a critical marker of apoptosis. Liver inflammation was evident by elevation in liver cytokines contents (TNF-a and IL-10) and MPO activity. Additionally, oxidative stress was manifested by increased liver lipoperoxidation, glutathione depletion, elevated total nitrate/nitrite (NOx) content and glutathione peroxidase (GPx) activity. Pretreatment with Mg largely mitigated these alternations. Conclusion: Pretreatment with Mg protects the liver from the acute injury which occurs shortly after septicemia. ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Q2 LPS Liver damage Magnesium Inflammation Septicemia Transaminases
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Introduction Q3
Excessive systemic inflammation in sepsis is the most common cause of death in intensive care units [1], ultimately resulting in multiple organ dysfunction syndrome [2,3]. The liver plays an important role in the pathogenesis of sepsis both as a source of inflammatory mediators as well as the target organ for the effects of these mediators [4,5]. Lipopolysaccharide (LPS) has been extensively studied as a major factor contributing to the pathogenesis of Gram-negative bacterial infection through eliciting a systemic inflammatory response which is characterized by liver failure, accompanied by severe hepatic injury [6].
* Corresponding author. E-mail address: [email protected] (D.M. El-Tanbouly).
Mg deficiency is associated with inflammatory responses [7] and aggravates endotoxin lethality [8], suggesting that inadequate Mg stores may exacerbate host inflammatory responses during infection. Similarly, deficiency of Mg exerts pro-oxidant effects on various tissues including cardiac tissue [9], brain, kidney [10], liver [11] and testis [12]. The deleterious effects of Mg deficiency are attributed to increased production of oxygen free radicals [13] and depletion of reduced glutathione [14]. Mg therapy has proven to be beneficial for numerous inflammatory conditions. For example, aerosolized magnesium sulfate is used for the clinical treatment of acute severe asthma [15], Mg supplementation in vivo preserves the integrity of the blood–brain barrier during experimental sepsis [16] and reduced the intrauterine growth restriction and suppresses inflammation in pregnant rats [17]. The present study was therefore, carried out to evaluate the hepatoprotective effect of Mg against liver injury in experimentally induced endotoxemia in mice.
http://dx.doi.org/10.1016/j.pharep.2015.02.004 1734-1140/ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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Materials and methods
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Animals
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Male albino Swiss mice weighing 25–30 g were purchased from the animal facility of Faculty of Pharmacy, Cairo University. Mice were housed under the appropriate conditions of controlled humidity, temperature and constant light cycle and allowed free access to a standard rodent chow diet and water. The investigation complies with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and was approved by the Ethics Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University (Permit Number: PT 1216).
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Chemicals
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Mg aspartate hydrochloride, lipopolysaccharide (LPS from E. coli serotype 0111:B4), all biochemical reagents and co-enzymes were obtained from Sigma–Aldrich Chemicals, St. Louis, MO, USA. Mg aspartate hydrochloride was obtained from Sekem pharmaceutical company, Egypt. All other chemicals were of analytical grade.
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Experimental design
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Mice were divided into four groups, 12 animals each. Group 1 and 2 received saline. Group 3 and 4 received Mg (20 mg/kg) and (40 mg/kg), respectively. Drug was freshly prepared in saline and orally administered for 7 consecutive days [18]. All mice received a single dose of LPS: (2 mg/kg; ip) [19] 1 h after the last dose of drug to ensure complete oral absorption except for group 1 which served as negative-control group.
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Sampling procedures
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3 h after LPS injection, the time of maximal liver injury as evident from a pilot study, blood samples were withdrawn in heparinized tubes from the retro-orbital sinus of all mice. Plasma was separated and divided into small aliquots that were stored at 80 8C to be used later for estimation of the chosen parameters. Livers were rapidly excised, washed with saline, weighed and homogenized in ice-cold saline to prepare 10% homogenates that were again divided into several aliquots and stored at 80 8C. Parts of the livers from each group were preserved in 10% formalin, prepared in saline, to be used for histopathological examination.
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Biochemical measurement
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Evaluation of liver injury The prepared plasma was used to for estimation of AST and ALT activities using commercially available kit (Quı´mica Clı´nica Aplicada S.A, Spain).
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C-reactive protein assay Plasma level of C-reactive protein (CRP) was determined using mouse CRP-ELISA kit (DRG, Germany).
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Measurement of secreted cytokines Liver homogenate was used for estimation of tumor necrosis factor (TNF)-a and interleukin-10 (IL-10) using enzyme linked immunosorbent assay (ELISA) kit (R&D system, USA).
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Myeloperoxidase activity in the liver Liver meyloperoxidase (MPO) activity as an index of neutrophil infilteration was estimated according to the method described by
Bradley et al. [20]. The method is based on measuring the hydrogen peroxide-dependent oxidation of o-dianisidine, catalyzed by MPO. This results in the formation of a compound exhibiting an increased absorbance at 460 nm. One unit of MPO activity is defined as the amount of enzyme that degrades 1 mmol peroxide per min at 25 8C.
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NO metabolites measurement Liver NO was measured by quantification of NO metabolites nitrate/nitrite according to the method of Miranda et al. [21]. The assay determines NOx level based on the reduction of any nitrate to nitrite by vanadium followed by the detection of total nitrite by Griess reagent. The formed chromophoric azo derivative can be measured colorimetrically at 540 nm.
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Lipid peroxidation measurement Liver lipid peroxidation products were estimated by determination of the level of thiobarbituric acid reactive substances according to the described by Mihara and Uchiyama which depends on a colorimetric determination of a pink pigment product resulting from the reaction of TBARS with thiobarbituric acid in acidic medium, at high temperature. The resultant color product is extracted in n-butanol and measured at two wavelengths, namely 535 and 520 nm [22].
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Antioxidant enzyme activity assay Glutathione peroxidase (GPx) activity was measured in the liver homogenate using a specific kit obtained from OxisResearch (USA).
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Determination of liver GSH content The content of reduced glutathione (GSH) was determined in tissue homogenate according to the method of Beutler et al. [23]. The method depends on the fact that GSH (the most abundant non-protein thiol) reduces Ellman’s reagent [5,50 -dithiobis (2-nitrobenzoic acid)] (DTNB) to form a stable yellow product (5-mercapto-2-nitrobenzoic acid), which can be measured colorimetrically at 412 nm.
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Determination of caspase-3 activity The hepatic caspase-3 activity as a marker of apoptosis was carried out using ApoAlert caspase-3 colorimetric assay kit (USA). Caspase-3 activity was expressed as (nmol pNA/h/mg protein). Protein content was measured according to the method of Bradford [24].
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Histopathological evaluation Liver samples preserved for histopathology were fixed in 10% formalin and used to prepare paraffin blocks. Sections of 5 mm were obtained and stained with Hematoxylin and Eosin (H&E). Images were captured and processed using Adobe Photoshop (version 8).
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Statistical analysis
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Data were expressed as means standard error (SE). Results were analyzed using one-way-analysis of variance test (ANOVA) followed by Tukey Kramer multiple comparison’s test. For all statistical tests, the level of significance was set at p < 0.05. GraphPad Prism1 software package, version 5 (GraphPad Software, Inc., USA) was used to carry out all statistical tests.
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Results
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Assessment of liver function
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LPS injection significantly increased plasma AST (Fig. 1A) and ALT (Fig. 1B). Mg only in the dose of (40 mg/kg) significantly reduced the elevated plasma AST and ALT activity.
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Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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Fig. 1. Effect of Mg (20, 40 mg/kg, po) on plasma AST (A) and ALT (B). Each bar with vertical line represents means of 6–10 mice SE. *Significantly different from normal group at p < 0.05. @Significantly different from LPS-control group at p < 0.05.
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Liver histology
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The histological study revealed that liver section of saline treated mice showed normal hepatic structure that includes hepatic lobules consisting of a central vein surrounded by radiating hepatocytes. The liver of mice received LPS alone showed Kupffer cells activation and multiple focal hepatic necrosis associated with leucocytic cells infilteration. Liver sections of mice pretreated with (40 mg/kg) Mg showed apparently normal hepatocytes (Fig. 2).
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Pro and anti-inflammatory mediators
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Mice subjected to LPS injection showed significant elevation in liver TNF-a and IL-10 as well as plasma level of CRP. Mg only in the dose of (40 mg/kg) exhibited significant reductions in both plasma CRP and liver TNF-a content. Meanwhile, Mg (20, 40 mg/kg) did not show any significant change in liver IL-10 content (Table 1).
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Liver MPO activity
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LPS injection resulted in significant increase in liver MPO activity. Pretreatment with Mg (40 mg/kg) showed a significant reduction in liver MPO activity (Table 1).
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Oxidative stress biomarkers
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The previously mentioned state of inflammation in LPS group caused a significant increase in liver TBARS and GPx activity coupled with a significant reduction in liver GSH content. Both doses of Mg restored the reduced liver GPx activity, significantly
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reduced liver TBARS and produced a significant increase in liver GSH content reaching the normal values (Fig. 3A–C).
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Liver NO
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LPS injection to mice showed a significant increase in liver NOx content which correlated with the previous elevation in the liver TNF-a content and MPO activity. Mg (40 mg/kg) produced a significant reduction in liver NOx content (Fig. 4A).
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Caspase-3 activity
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Mice received LPS showed a significant increase in liver caspase-3 activity. Only the large dose of Mg significantly reduced the elevated liver caspase-3 activity (Fig. 4B).
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Discussion
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Septic shock and tissue injury from endotoxins during bacteremia remains an important clinical concern. The liver participates in host defense and tissue repair by controlling the coagulation and inflammatory process, thus hepatic injury is common in septicemia [6]. In the present study, the possible hepatoprotective effect of Mg was investigated against the hepatic response to LPS injection as a model that simulates septicemia in animals and humans. In the liver, LPS interacts with Kuffer cells leading to their activation and the release of cytotoxic agents including TNF-a. It has been proven that TNF-a is the key mediator in the destruction of hepatocyte in human liver diseases [25] and [26]. The finding that LPS-induced elevation in IL-10 is in harmony with Wang et al. who reported that the levels of TNF-a and IL-10 in serum and liver homogenate of septic mice increased with passage of time [27]. As with many other acute phase proteins, C-reactive protein (CRP) is predominantly synthesized by the liver, mainly in response to interleukin 6 (IL-6) [29]. TNFa and IL-1b are also regulatory mediators of CRP synthesis [28]. The elevated liver MPO activity reported in this study is in harmony with several studies that observed endotoxin-induced increase in MPO activity in the liver [29] and [30] and pulmonary tissue [31]. Owing to its rich vasculature, the liver is at immediate risk of neutrophil-mediated damage in Gram-negative sepsis [6,32,33] through excessive leukocytes extravasation which is itself damaging to blood vessel. Altered oxidative stress biomarkers observed in the present study was manifested as an increase in TBARS and GPx accompanied with a decrease in GSH content. Lots of studies have shown that excessive production of ROS and other free radicals in sepsis, such as H2O2, superoxide and NO, which are associated with inflammation, lead to a condition of oxidative stress and contribute to high mortality rates [34,35,36]. Increased free radicals that caused peroxidation of membrane lipids lower the GSH content of the cell [37]. During endotoxemia, liver serves as the main source of plasma GSH and exhibited enhanced sinusoidal glutathione efflux [38,39]. A large number of studies demonstrate the protective effect of antioxidant enzymes in various model of endotoxic shock [40,41]. Therefore, enhanced tissue antioxidant enzymes during endotoxemia may be a preventive measure of the host to handle the ROS load after bacterial LPS administration [42]. Remarked increase in hepatic NOx content was observed in this study. It was reported that endogenously produced NO causes hepatocellular damage by stimulating the expression of TNF-a [43]. NO may also participate in the development of oxidative stress associated with endotoxemia. The loss of a single electron generates the highly reactive nitrosyl cation (NO+) [44]. In addition the reaction of NO with superoxide generates peroxynitrite
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Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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Fig. 2. Photomicrograph of sections of liver tissue of (A) a normal mouse showing the normal histological structure of hepatic lobule formed of cords of hepatocytes radiating from the central vein (H & E 400). LPS treated-mice (B) showing karyomegaly of some nuclei (small arrow) and portal infiltration with leucocytes (large arrow) (H & E 400). (C) Showing Kupffer cells activation (small arrow) and multiple focal hepatic necrosis associated with leucocytic cells infilteration (large arrows) (H & E 400). (D) Apoptosis of hepatocytes (small arrows) and portal edema associated with leucocytic infilteration (large arrow) (H & E 400). (E) A mouse pretreated with (20 mg/kg) Mg showing multiple focal hepatic necrosis associated with inflammatory cells infiltration (large arrows) (H & E 400). (F) A mouse pretreated with (40 mg/kg) Mg showing apparent normal hepatocytes with Kupffer cells activation (large arrow) (H & E 400).
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(ONOO). Both NO+ and ONOO are potent oxidants react with protein, lipids, DNA and scavenged by GSH [44]. Generation of these reactive RNS could explain the loss of GSH and the appearance of markers of oxidative stress. There is also a growing evidence indicating that NO is able to induce the release of cytochrome C from the mitochondria to the cytosol, followed by caspase-3 activation and apoptosis [45]. Histopathological changes and Increased plasma transaminases (ALT & AST) activities, in the present investigation, is a conventional indicator of liver injury associated with LPS injection in mice, indicating loss of functional integrity of hepatic membrane and cellular leakage of enzymes normally located in the cytosol [46]. The current investigation showed that Mg (40 mg/kg) pretreatment for 7 consecutive days mitigated hepatic injury associated
with LPS injection, as evidenced by the lowered plasma transaminases activities and the marked improvement in liver histology. Previous study reported the hepatoprotective effect of Mg against lead-induced hepatic damage in rats [47]. Several investigators revealed the inhibitory effect of magnesium sulphate on endotoxin-stimulated inflammatory cytokine production [48–50]. Furthermore, Lin et al. showed that magnesium sulphate reduced the release of IL-1b, TNF-a and IL-6 from a murine macrophage-like cell line [51]. Mg may exert antiinflammatory properties via its inhibitory effect on calcium (Ca) influx into cells [50] as it is a potent inhibitor of L-type Ca channel, the major Ca channel associated with increased Ca influx [52] which is crucial in regulating signaling pathways expression and subsequent inflammatory molecules production during endotoxemia [53,54].
Table 1 Effect of Mg on LPS-induced change in plasma level of C-reactive protein, liver contents of TNF-a, IL-10 and liver MPO activity. Groups
CRP (ng/ml)
TNF-a (pg/g wet tissue)
IL-10 (pg/g wet tissue)
MPO (U/g wet tissue)
Saline Saline/LPS Mg 20/LPS Mg 40/LPS
28.74 2.43 103.73 5.18* 106.85 5.36* 53.1 2.33*,a
27.31 2.72 96 4.9* 79.99 5.82* 35.99 7.42a
138.29 15.26 391.66 20.58* 341.63 33.33* 338.33 16.99*
0.15 0.008 0.45 0.025* 0.38 0.009* 0.25 0.023*,a
Values are means of 6–10 mice SE. * Significantly different from normal group at p < 0.05. a Significantly different from LPS-control group at p < 0.05.
Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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Fig. 3. Effect of Mg (20, 40 mg/kg, po) on liver content of TBARS (A), liver GPx activity (B) and GSH content of the liver (C). Each bar with vertical line represents means of 6–10 mice SE. *Significantly different from normal group at p < 0.05. @Significantly different from LPS-control group at p < 0.05.
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Mg reduced liver MPO activity, this effect is in accordance with the previous studies. Lee et al. [50] demonstrated that magnesium sulphate has a protective effect in acute lung injury in endotoxemic rats by inhibiting MPO activity. In addition, Magnesium sulphate
Fig. 4. Effect of Mg (20, 40 mg/kg, po) on liver content of NOx (A) and liver caspase-3 activity (B). Each bar with vertical line represents means of 6–10 mice SE. *Significantly different from normal group at p < 0.05. @Significantly different from LPS-control group at p < 0.05.
decreased MPO activity in injured spinal cord [55] and fetal skin tissue after intrauterine ischemia/reperfusion injury [56]. Lee et al. demonstrated that magnesium sulphate lowered lung NO content following LPS-induced endotoxemia in rats [50]. Several studies reported that Mg effectively prevented excessive NO production in Shumiya cataract rat lens [57,58]. Moreover, Yokoyama et al. reported that Mg deficiency enhanced NO production by iNOS in alveolar macrophage isolated from rats [59]. Several studies revealed that magnesium sulphate may exert its anti-inflammatory properties by inhibiting endotoxin-induced NF-kB activation [48,49]. Furthermore, Lin et al. [51] revealed that the inhibitory effect of Mg on NF-kB activation may involve its ability to antagonize LPS-induced increased Ca influx that was previously reported to promote NF-kB activation [54]. Mg is not only a direct scavenger of free radicals, but can also inhibit NADPH oxidase, which in turn lowers the production of superoxide radical [60]. In vitro, Bussiere et al. [61] reported that free radicals production was reduced in the present of high extracellular Mg. Mg neuronal protection is believed to be through the reduction of intracellular Ca and consequently alleviation of oxygen free radicals generation [62]. Mg was reported to mitigate the elevated TBARS in liver and kidney induced by cadmium toxicity [63] as well as lung content of TBARS in response to endotoxin [50]. In the current study, the increase in liver GSH content is in harmony with Hans et al. who revealed that GSH in plasma and liver of alloxanic diabetic rats were significantly elevated by Mg supplementation [64]. Liver caspase-3 activity was reduced after pretreatment of mice with Mg (40 mg/kg). Similarly, Solaroglu et al. [65] revealed that Mg treatment decreased caspase-3 activity in rat spinal cord subjected to injury. Moreover, Mg deficiency induced apoptosis in isolated rat hepatocytes [66] probably as a result of an oxidative stress-related mechanism [67].
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Conclusions
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The present results offer a special recommendation regarding the use of Mg in the prevention of liver injury associated with
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Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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septicemia. The hepatoprotective effect of Mg might be due to its anti-inflammatory, antioxidant and antiapoptotic properties. Clinical investigations are necessary to investigate the protective effect of Mg against different organs dysfunction in various systemic inflammatory responses.
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Funding
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The authors declare that they have not received any financial support for this research.
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Conflict of interest
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The authors declare that there is no conflict of interest.
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Acknowledgements
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The authors are grateful to Dr. Kawkab Abdel Aziz Ahmed, Department of Histology, Faculty of veterinary Medicine, Cairo University, for her efforts in performing and explaining histological examinations.
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References
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J Ethnopharmacol 1998;63(3):181–6. [47] Todorovic T, Vujanovic D. The influence of magnesium on the activity of some enzymes (AST, ALT, ALP) and lead content in some tissues. Magnes Res 2002;15(3–4):173–7. [48] Makhlouf MA, Simhan HN. Effect of tocolytics on interleukin-8 production by human amniotic and decidual cells. J Reprod Immunol 2006;69(1):1–7. [49] Rochelson B, Dowling O, Schwartz N, Metz CN. Magnesium sulfate suppresses inflammatory responses by human umbilical vein endothelial cells (HuVECs) through the NFkappaB pathway. J Reprod Immunol 2007;73(2): 101–7.
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[59] Yokoyama T, Oono H, Miyamoto A, Ishiguro S, Nishio A. Magnesium-deficient medium enhances NO production in alveolar macrophages isolated from rats. Life Sci 2003;72(11):1247–57. [60] Afanas’ev IB, Suslova TB, Cheremisina ZP, Abramova NE, Korkina LG. Study of antioxidant properties of metal aspartates. Analyst 1995;120(3): 859–62. [61] Bussiere FI, Gueux E, Rock E, Girardeau JP, Tridon A, Mazur A, et al. Increased phagocytosis and production of reactive oxygen species by neutrophils during magnesium deficiency in rats and inhibition by high magnesium concentration. Br J Nutr 2002;87(2):107–13. [62] Lang-Lazdunski L, Heurteaux C, Dupont H, Widmann C, Lazdunski M. Prevention of ischemic spinal cord injury: comparative effects of magnesium sulfate and riluzole. J Vasc Surg 2000;32(1):179–89. [63] Boujelben M, Ghorbel F, Vincent C, Makni-Ayadi F, Guermazi F, Croute F, et al. Lipid peroxidation and HSP72/73 expression in rat following cadmium chloride administration: interactions of magnesium supplementation. Exp Toxicol Pathol 2006;57(5–6):437–43. [64] Hans CP, Chaudhary DP, Bansal DD. Effect of magnesium supplementation on oxidative stress in alloxanic diabetic rats. Magnes Res 2003;16(1):13–9. [65] Solaroglu I, Kaptanoglu E, Okutan O, Beskonakli E, Attar A, Kilinc K. Magnesium sulfate treatment decreases caspase-3 activity after experimental spinal cord injury in rats. Surg Neurol 2005;64(Suppl. 2):S17–21. [66] Martin H, Richert L, Berthelot A. Magnesium deficiency induces apoptosis in primary cultures of rat hepatocytes. J Nutr 2003;133(8):2505–11. [67] Martin H, Uring-Lambert B, Adrian M, Lahlou A, Bonet A, Demougeot C, et al. Effects of long-term dietary intake of magnesium on oxidative stress, apoptosis and ageing in rat liver. Magnes Res 2008;21(2):124–30.
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Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep 1 2
Original research article
Pretreatment with magnesium ameliorates lipopolysaccharideinduced liver injury in mice
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Q1 Dalia
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M. El-Tanbouly *, Rania M. Abdelsalam, Amina S. Attia, Mohamed T. Abdel-Aziz
Pharmacology & Toxicology Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 August 2014 Received in revised form 11 February 2015 Accepted 12 February 2015 Available online xxx
Background: Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, is involved in the pathogenesis of sepsis. LPS administration induces systemic inflammation that mimics many of the initial clinical features of sepsis and has deleterious effects on several organs including the liver and eventually leading to septic shock and death. The present study aimed to investigate the protective effect of magnesium (Mg), a well known cofactor in many enzymatic reactions and a critical component of the antioxidant system, on hepatic damage associated with LPS-induced endotoxima in mice. Methods: Mg (20 and 40 mg/kg, po) was administered for 7 consecutive days. Systemic inflammation was induced 1 h after the last dose of Mg by a single dose of LPS (2 mg/kg, ip) and 3 h thereafter plasma was separated, animals were sacrificed and their livers were isolated. Results: LPS-treated mice suffered from hepatic dysfunction revealed by histological observation, elevation in plasma transaminases activities, C-reactive protein content and caspase-3, a critical marker of apoptosis. Liver inflammation was evident by elevation in liver cytokines contents (TNF-a and IL-10) and MPO activity. Additionally, oxidative stress was manifested by increased liver lipoperoxidation, glutathione depletion, elevated total nitrate/nitrite (NOx) content and glutathione peroxidase (GPx) activity. Pretreatment with Mg largely mitigated these alternations. Conclusion: Pretreatment with Mg protects the liver from the acute injury which occurs shortly after septicemia. ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Q2 LPS Liver damage Magnesium Inflammation Septicemia Transaminases
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Introduction Q3
Excessive systemic inflammation in sepsis is the most common cause of death in intensive care units [1], ultimately resulting in multiple organ dysfunction syndrome [2,3]. The liver plays an important role in the pathogenesis of sepsis both as a source of inflammatory mediators as well as the target organ for the effects of these mediators [4,5]. Lipopolysaccharide (LPS) has been extensively studied as a major factor contributing to the pathogenesis of Gram-negative bacterial infection through eliciting a systemic inflammatory response which is characterized by liver failure, accompanied by severe hepatic injury [6].
* Corresponding author. E-mail address: [email protected] (D.M. El-Tanbouly).
Mg deficiency is associated with inflammatory responses [7] and aggravates endotoxin lethality [8], suggesting that inadequate Mg stores may exacerbate host inflammatory responses during infection. Similarly, deficiency of Mg exerts pro-oxidant effects on various tissues including cardiac tissue [9], brain, kidney [10], liver [11] and testis [12]. The deleterious effects of Mg deficiency are attributed to increased production of oxygen free radicals [13] and depletion of reduced glutathione [14]. Mg therapy has proven to be beneficial for numerous inflammatory conditions. For example, aerosolized magnesium sulfate is used for the clinical treatment of acute severe asthma [15], Mg supplementation in vivo preserves the integrity of the blood–brain barrier during experimental sepsis [16] and reduced the intrauterine growth restriction and suppresses inflammation in pregnant rats [17]. The present study was therefore, carried out to evaluate the hepatoprotective effect of Mg against liver injury in experimentally induced endotoxemia in mice.
http://dx.doi.org/10.1016/j.pharep.2015.02.004 1734-1140/ß 2015 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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Materials and methods
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Animals
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Male albino Swiss mice weighing 25–30 g were purchased from the animal facility of Faculty of Pharmacy, Cairo University. Mice were housed under the appropriate conditions of controlled humidity, temperature and constant light cycle and allowed free access to a standard rodent chow diet and water. The investigation complies with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and was approved by the Ethics Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University (Permit Number: PT 1216).
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Chemicals
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Mg aspartate hydrochloride, lipopolysaccharide (LPS from E. coli serotype 0111:B4), all biochemical reagents and co-enzymes were obtained from Sigma–Aldrich Chemicals, St. Louis, MO, USA. Mg aspartate hydrochloride was obtained from Sekem pharmaceutical company, Egypt. All other chemicals were of analytical grade.
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Experimental design
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Mice were divided into four groups, 12 animals each. Group 1 and 2 received saline. Group 3 and 4 received Mg (20 mg/kg) and (40 mg/kg), respectively. Drug was freshly prepared in saline and orally administered for 7 consecutive days [18]. All mice received a single dose of LPS: (2 mg/kg; ip) [19] 1 h after the last dose of drug to ensure complete oral absorption except for group 1 which served as negative-control group.
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Sampling procedures
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3 h after LPS injection, the time of maximal liver injury as evident from a pilot study, blood samples were withdrawn in heparinized tubes from the retro-orbital sinus of all mice. Plasma was separated and divided into small aliquots that were stored at 80 8C to be used later for estimation of the chosen parameters. Livers were rapidly excised, washed with saline, weighed and homogenized in ice-cold saline to prepare 10% homogenates that were again divided into several aliquots and stored at 80 8C. Parts of the livers from each group were preserved in 10% formalin, prepared in saline, to be used for histopathological examination.
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Biochemical measurement
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Evaluation of liver injury The prepared plasma was used to for estimation of AST and ALT activities using commercially available kit (Quı´mica Clı´nica Aplicada S.A, Spain).
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C-reactive protein assay Plasma level of C-reactive protein (CRP) was determined using mouse CRP-ELISA kit (DRG, Germany).
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Measurement of secreted cytokines Liver homogenate was used for estimation of tumor necrosis factor (TNF)-a and interleukin-10 (IL-10) using enzyme linked immunosorbent assay (ELISA) kit (R&D system, USA).
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Myeloperoxidase activity in the liver Liver meyloperoxidase (MPO) activity as an index of neutrophil infilteration was estimated according to the method described by
Bradley et al. [20]. The method is based on measuring the hydrogen peroxide-dependent oxidation of o-dianisidine, catalyzed by MPO. This results in the formation of a compound exhibiting an increased absorbance at 460 nm. One unit of MPO activity is defined as the amount of enzyme that degrades 1 mmol peroxide per min at 25 8C.
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NO metabolites measurement Liver NO was measured by quantification of NO metabolites nitrate/nitrite according to the method of Miranda et al. [21]. The assay determines NOx level based on the reduction of any nitrate to nitrite by vanadium followed by the detection of total nitrite by Griess reagent. The formed chromophoric azo derivative can be measured colorimetrically at 540 nm.
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Lipid peroxidation measurement Liver lipid peroxidation products were estimated by determination of the level of thiobarbituric acid reactive substances according to the described by Mihara and Uchiyama which depends on a colorimetric determination of a pink pigment product resulting from the reaction of TBARS with thiobarbituric acid in acidic medium, at high temperature. The resultant color product is extracted in n-butanol and measured at two wavelengths, namely 535 and 520 nm [22].
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Antioxidant enzyme activity assay Glutathione peroxidase (GPx) activity was measured in the liver homogenate using a specific kit obtained from OxisResearch (USA).
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Determination of liver GSH content The content of reduced glutathione (GSH) was determined in tissue homogenate according to the method of Beutler et al. [23]. The method depends on the fact that GSH (the most abundant non-protein thiol) reduces Ellman’s reagent [5,50 -dithiobis (2-nitrobenzoic acid)] (DTNB) to form a stable yellow product (5-mercapto-2-nitrobenzoic acid), which can be measured colorimetrically at 412 nm.
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Determination of caspase-3 activity The hepatic caspase-3 activity as a marker of apoptosis was carried out using ApoAlert caspase-3 colorimetric assay kit (USA). Caspase-3 activity was expressed as (nmol pNA/h/mg protein). Protein content was measured according to the method of Bradford [24].
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Histopathological evaluation Liver samples preserved for histopathology were fixed in 10% formalin and used to prepare paraffin blocks. Sections of 5 mm were obtained and stained with Hematoxylin and Eosin (H&E). Images were captured and processed using Adobe Photoshop (version 8).
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Statistical analysis
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Data were expressed as means standard error (SE). Results were analyzed using one-way-analysis of variance test (ANOVA) followed by Tukey Kramer multiple comparison’s test. For all statistical tests, the level of significance was set at p < 0.05. GraphPad Prism1 software package, version 5 (GraphPad Software, Inc., USA) was used to carry out all statistical tests.
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Results
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Assessment of liver function
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LPS injection significantly increased plasma AST (Fig. 1A) and ALT (Fig. 1B). Mg only in the dose of (40 mg/kg) significantly reduced the elevated plasma AST and ALT activity.
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Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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Fig. 1. Effect of Mg (20, 40 mg/kg, po) on plasma AST (A) and ALT (B). Each bar with vertical line represents means of 6–10 mice SE. *Significantly different from normal group at p < 0.05. @Significantly different from LPS-control group at p < 0.05.
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Liver histology
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The histological study revealed that liver section of saline treated mice showed normal hepatic structure that includes hepatic lobules consisting of a central vein surrounded by radiating hepatocytes. The liver of mice received LPS alone showed Kupffer cells activation and multiple focal hepatic necrosis associated with leucocytic cells infilteration. Liver sections of mice pretreated with (40 mg/kg) Mg showed apparently normal hepatocytes (Fig. 2).
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Pro and anti-inflammatory mediators
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Mice subjected to LPS injection showed significant elevation in liver TNF-a and IL-10 as well as plasma level of CRP. Mg only in the dose of (40 mg/kg) exhibited significant reductions in both plasma CRP and liver TNF-a content. Meanwhile, Mg (20, 40 mg/kg) did not show any significant change in liver IL-10 content (Table 1).
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Liver MPO activity
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LPS injection resulted in significant increase in liver MPO activity. Pretreatment with Mg (40 mg/kg) showed a significant reduction in liver MPO activity (Table 1).
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Oxidative stress biomarkers
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The previously mentioned state of inflammation in LPS group caused a significant increase in liver TBARS and GPx activity coupled with a significant reduction in liver GSH content. Both doses of Mg restored the reduced liver GPx activity, significantly
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reduced liver TBARS and produced a significant increase in liver GSH content reaching the normal values (Fig. 3A–C).
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Liver NO
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LPS injection to mice showed a significant increase in liver NOx content which correlated with the previous elevation in the liver TNF-a content and MPO activity. Mg (40 mg/kg) produced a significant reduction in liver NOx content (Fig. 4A).
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Caspase-3 activity
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Mice received LPS showed a significant increase in liver caspase-3 activity. Only the large dose of Mg significantly reduced the elevated liver caspase-3 activity (Fig. 4B).
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Discussion
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Septic shock and tissue injury from endotoxins during bacteremia remains an important clinical concern. The liver participates in host defense and tissue repair by controlling the coagulation and inflammatory process, thus hepatic injury is common in septicemia [6]. In the present study, the possible hepatoprotective effect of Mg was investigated against the hepatic response to LPS injection as a model that simulates septicemia in animals and humans. In the liver, LPS interacts with Kuffer cells leading to their activation and the release of cytotoxic agents including TNF-a. It has been proven that TNF-a is the key mediator in the destruction of hepatocyte in human liver diseases [25] and [26]. The finding that LPS-induced elevation in IL-10 is in harmony with Wang et al. who reported that the levels of TNF-a and IL-10 in serum and liver homogenate of septic mice increased with passage of time [27]. As with many other acute phase proteins, C-reactive protein (CRP) is predominantly synthesized by the liver, mainly in response to interleukin 6 (IL-6) [29]. TNFa and IL-1b are also regulatory mediators of CRP synthesis [28]. The elevated liver MPO activity reported in this study is in harmony with several studies that observed endotoxin-induced increase in MPO activity in the liver [29] and [30] and pulmonary tissue [31]. Owing to its rich vasculature, the liver is at immediate risk of neutrophil-mediated damage in Gram-negative sepsis [6,32,33] through excessive leukocytes extravasation which is itself damaging to blood vessel. Altered oxidative stress biomarkers observed in the present study was manifested as an increase in TBARS and GPx accompanied with a decrease in GSH content. Lots of studies have shown that excessive production of ROS and other free radicals in sepsis, such as H2O2, superoxide and NO, which are associated with inflammation, lead to a condition of oxidative stress and contribute to high mortality rates [34,35,36]. Increased free radicals that caused peroxidation of membrane lipids lower the GSH content of the cell [37]. During endotoxemia, liver serves as the main source of plasma GSH and exhibited enhanced sinusoidal glutathione efflux [38,39]. A large number of studies demonstrate the protective effect of antioxidant enzymes in various model of endotoxic shock [40,41]. Therefore, enhanced tissue antioxidant enzymes during endotoxemia may be a preventive measure of the host to handle the ROS load after bacterial LPS administration [42]. Remarked increase in hepatic NOx content was observed in this study. It was reported that endogenously produced NO causes hepatocellular damage by stimulating the expression of TNF-a [43]. NO may also participate in the development of oxidative stress associated with endotoxemia. The loss of a single electron generates the highly reactive nitrosyl cation (NO+) [44]. In addition the reaction of NO with superoxide generates peroxynitrite
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Please cite this article in press as: El-Tanbouly DM, et al. Pretreatment with magnesium ameliorates lipopolysaccharide-induced liver injury in mice. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.02.004
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Fig. 2. Photomicrograph of sections of liver tissue of (A) a normal mouse showing the normal histological structure of hepatic lobule formed of cords of hepatocytes radiating from the central vein (H & E 400). LPS treated-mice (B) showing karyomegaly of some nuclei (small arrow) and portal infiltration with leucocytes (large arrow) (H & E 400). (C) Showing Kupffer cells activation (small arrow) and multiple focal hepatic necrosis associated with leucocytic cells infilteration (large arrows) (H & E 400). (D) Apoptosis of hepatocytes (small arrows) and portal edema associated with leucocytic infilteration (large arrow) (H & E 400). (E) A mouse pretreated with (20 mg/kg) Mg showing multiple focal hepatic necrosis associated with inflammatory cells infiltration (large arrows) (H & E 400). (F) A mouse pretreated with (40 mg/kg) Mg showing apparent normal hepatocytes with Kupffer cells activation (large arrow) (H & E 400).
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(ONOO). Both NO+ and ONOO are potent oxidants react with protein, lipids, DNA and scavenged by GSH [44]. Generation of these reactive RNS could explain the loss of GSH and the appearance of markers of oxidative stress. There is also a growing evidence indicating that NO is able to induce the release of cytochrome C from the mitochondria to the cytosol, followed by caspase-3 activation and apoptosis [45]. Histopathological changes and Increased plasma transaminases (ALT & AST) activities, in the present investigation, is a conventional indicator of liver injury associated with LPS injection in mice, indicating loss of functional integrity of hepatic membrane and cellular leakage of enzymes normally located in the cytosol [46]. The current investigation showed that Mg (40 mg/kg) pretreatment for 7 consecutive days mitigated hepatic injury associated
with LPS injection, as evidenced by the lowered plasma transaminases activities and the marked improvement in liver histology. Previous study reported the hepatoprotective effect of Mg against lead-induced hepatic damage in rats [47]. Several investigators revealed the inhibitory effect of magnesium sulphate on endotoxin-stimulated inflammatory cytokine production [48–50]. Furthermore, Lin et al. showed that magnesium sulphate reduced the release of IL-1b, TNF-a and IL-6 from a murine macrophage-like cell line [51]. Mg may exert antiinflammatory properties via its inhibitory effect on calcium (Ca) influx into cells [50] as it is a potent inhibitor of L-type Ca channel, the major Ca channel associated with increased Ca influx [52] which is crucial in regulating signaling pathways expression and subsequent inflammatory molecules production during endotoxemia [53,54].
Table 1 Effect of Mg on LPS-induced change in plasma level of C-reactive protein, liver contents of TNF-a, IL-10 and liver MPO activity. Groups
CRP (ng/ml)
TNF-a (pg/g wet tissue)
IL-10 (pg/g wet tissue)
MPO (U/g wet tissue)
Saline Saline/LPS Mg 20/LPS Mg 40/LPS
28.74 2.43 103.73 5.18* 106.85 5.36* 53.1 2.33*,a
27.31 2.72 96 4.9* 79.99 5.82* 35.99 7.42a
138.29 15.26 391.66 20.58* 341.63 33.33* 338.33 16.99*
0.15 0.008 0.45 0.025* 0.38 0.009* 0.25 0.023*,a
Values are means of 6–10 mice SE. * Significantly different from normal group at p < 0.05. a Significantly different from LPS-control group at p < 0.05.
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Fig. 3. Effect of Mg (20, 40 mg/kg, po) on liver content of TBARS (A), liver GPx activity (B) and GSH content of the liver (C). Each bar with vertical line represents means of 6–10 mice SE. *Significantly different from normal group at p < 0.05. @Significantly different from LPS-control group at p < 0.05.
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Mg reduced liver MPO activity, this effect is in accordance with the previous studies. Lee et al. [50] demonstrated that magnesium sulphate has a protective effect in acute lung injury in endotoxemic rats by inhibiting MPO activity. In addition, Magnesium sulphate
Fig. 4. Effect of Mg (20, 40 mg/kg, po) on liver content of NOx (A) and liver caspase-3 activity (B). Each bar with vertical line represents means of 6–10 mice SE. *Significantly different from normal group at p < 0.05. @Significantly different from LPS-control group at p < 0.05.
decreased MPO activity in injured spinal cord [55] and fetal skin tissue after intrauterine ischemia/reperfusion injury [56]. Lee et al. demonstrated that magnesium sulphate lowered lung NO content following LPS-induced endotoxemia in rats [50]. Several studies reported that Mg effectively prevented excessive NO production in Shumiya cataract rat lens [57,58]. Moreover, Yokoyama et al. reported that Mg deficiency enhanced NO production by iNOS in alveolar macrophage isolated from rats [59]. Several studies revealed that magnesium sulphate may exert its anti-inflammatory properties by inhibiting endotoxin-induced NF-kB activation [48,49]. Furthermore, Lin et al. [51] revealed that the inhibitory effect of Mg on NF-kB activation may involve its ability to antagonize LPS-induced increased Ca influx that was previously reported to promote NF-kB activation [54]. Mg is not only a direct scavenger of free radicals, but can also inhibit NADPH oxidase, which in turn lowers the production of superoxide radical [60]. In vitro, Bussiere et al. [61] reported that free radicals production was reduced in the present of high extracellular Mg. Mg neuronal protection is believed to be through the reduction of intracellular Ca and consequently alleviation of oxygen free radicals generation [62]. Mg was reported to mitigate the elevated TBARS in liver and kidney induced by cadmium toxicity [63] as well as lung content of TBARS in response to endotoxin [50]. In the current study, the increase in liver GSH content is in harmony with Hans et al. who revealed that GSH in plasma and liver of alloxanic diabetic rats were significantly elevated by Mg supplementation [64]. Liver caspase-3 activity was reduced after pretreatment of mice with Mg (40 mg/kg). Similarly, Solaroglu et al. [65] revealed that Mg treatment decreased caspase-3 activity in rat spinal cord subjected to injury. Moreover, Mg deficiency induced apoptosis in isolated rat hepatocytes [66] probably as a result of an oxidative stress-related mechanism [67].
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Conclusions
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The present results offer a special recommendation regarding the use of Mg in the prevention of liver injury associated with
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septicemia. The hepatoprotective effect of Mg might be due to its anti-inflammatory, antioxidant and antiapoptotic properties. Clinical investigations are necessary to investigate the protective effect of Mg against different organs dysfunction in various systemic inflammatory responses.
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Funding
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The authors declare that they have not received any financial support for this research.
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Conflict of interest
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The authors declare that there is no conflict of interest.
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Acknowledgements
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The authors are grateful to Dr. Kawkab Abdel Aziz Ahmed, Department of Histology, Faculty of veterinary Medicine, Cairo University, for her efforts in performing and explaining histological examinations.
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References
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