Cardioprotective effect of metformin in lipopolysaccharide-induced sepsis via suppression of toll-like receptor 4 (TLR4) in heart

Author’s Accepted Manuscript Cardioprotective effect of metformin in lipopolysaccharide-induced sepsis via suppression of toll-like receptor 4 (TLR4) in heart Haleh Vaez, Maryam Rameshrad, Moslem Najafi, Jaleh Barar, Abolfazl Barzegari, Alireza Garjani www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(15)30426-X http://dx.doi.org/10.1016/j.ejphar.2015.12.030 EJP70402

To appear in: European Journal of Pharmacology Received date: 8 November 2015 Revised date: 15 December 2015 Accepted date: 16 December 2015 Cite this article as: Haleh Vaez, Maryam Rameshrad, Moslem Najafi, Jaleh Barar, Abolfazl Barzegari and Alireza Garjani, Cardioprotective effect of metformin in lipopolysaccharide-induced sepsis via suppression of toll-like receptor 4 (TLR4) in heart, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.12.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cardioprotective effect of metformin in lipopolysaccharide-induced sepsis via suppression of toll-like receptor 4 (TLR4) in heart

Haleh Vaeza,b, Maryam Rameshrada,b, Moslem Najafia, Jaleh Bararc, Abolfazl Barzegaric, Alireza Garjania*

a

Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences,

Tabriz, Iran b

c

Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran

Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical

Sciences, Tabriz, Iran

*

Corresponding Author. Dr. Alireza Garjani Professor of Pharmacology Department of

Pharmacology Faculty of Pharmacy Tabriz University of Medical Sciences Tabriz – Iran Tel.: +98 411 3341315; +98 914 3014818 Fax: +98 411 3344798 E-mail: [email protected] ; [email protected]

Abstract Sepsis-induced myocardial dysfunction is a serious organ complication. In the present study, we investigated the effect of metformin on myocardial dysfunction and TLR4 activity in LPS-induced sepsis. Male Wistar rats were randomly divided into 3 groups (n=6): control received normal saline (0.5 ml), LPS group received lipopolysaccharide (0.5 mg/kg; i.p), and metformin treated group received LPS (0.5 mg/kg) + metformin (100 mg/kg; i.p). 9 h later

the hemodynamic parameters were recorded, blood samples were collected, and the hearts were removed and weighted. The concentration of TNF-α, content of MYD88, the phosphorylation of AMPK, and the rate of TLR4 expression in the heart were assessed. In the animals treated with metformin, the preservation of left ventricular function was associated with the reduction of myeloperoxidase activity (31%, P<0.01) in the heart and decrease of TNF-α level both in the serum and heart tissue (20%, P<0.01 and 42%, P<0.05, respectively). It was found that the level of phosphorylated AMPK in heart was significantly upregulated by 43% (P<0.001) in the metformin group while the content of TLRs adapter protein of MyD88 was reduced by 45% (P<0.05). This was associated with a remarkable decrease in the expression of myocardial TLR4. Furthermore, in a mice model of sepsis, coadministration of compound C (20 mg/kg) as an AMPK inhibitor reversed the suppressive effects of metformin on TLR4 expression and MYD88 protein level. These results suggest that metformin exhibits cardioprotective effects in sepsis by suppression of TLR4 activity, at least in part through pathways involving AMPK activation.

Keywords: Metformin, Sepsis, Toll-like receptor 4; Lipopolysaccharide; AMPK; Heart

1. Introduction

Heart is a vital organ that is usually affected by sepsis. Septic shock is the most severe complication of sepsis with mortality rate as high as 50% (Dombrovskiy et al., 2007). The presence of cardiovascular dysfunction in sepsis extensively increases the mortality rate from 20% in septic patients without cardiovascular complications to 70 - 90% (Parrillo et al., 1990). Various pharmacological and non-pharmacological approaches are employed in the treatment of sepsis-induced myocardial dysfunction. Most patients benefit from antibiotic

therapy and fluid replacement. The lack of an effective treatment for heart complications caused by sepsis make the research on alternative drugs necessary. The myocardial depression in sepsis seems to depend on the presence of cell wall receptor TLR4 (Baumgarten et al., 2006) and CD14 (Knuefermann et al., 2002b). Toll-like receptors (TLRs) are a family of receptors that play a critical role in the activation of innate immune response to lipopolysaccharide (Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2000). Alternatively, the activation of TLR4 signaling pathways results in the production of cytokines including tumor necrosis factor-alpha (TNF-α), which may cause cardiac impairment (Katsargyris et al., 2008; Maron et al., 2002; Zhou et al., 2000). Deletion of TLR4 in knockout mice has shown smaller infarction and demonstrated less inflammation in myocardial ischemia-reperfusion injury (Oyama et al., 2004). In addition, antagonizing TLR2 prior to reperfusion has been shown to reduce infarct size and improved cardiac function (Arslan et al., 2010). Recent studies have verified that activation of AMPK attenuates myocardial impairment and inflammatory responses associated with myocardial infarction (Soraya et al., 2014). Metformin is the solo member of biguanide class in the market which is primarily used in type 2 diabetes. It is well-known as an agent that improves peripheral insulin sensitivity and increases the peripheral uptake of glucose (Galuska et al., 1994; Hundal et al., 1992) and decreases hepatic glucose production (Hundal et al., 2000; Stumvoll et al., 1995). The activation of AMP-activated protein kinase (AMPK) constitutes the best-known mechanism of metformin action (Zhou et al., 2001) however, it is now clear that some of the biological responses of metformin mediated by AMPK-independent mechanisms (Saeedi et al., 2008; Towler and Hardie, 2007). The clinical and experimental outcomes suggest that metformin, apart from its hypoglycemic action, may alter both peripheral and central inflammation (Caballero et al., 2004; Dandona et al., 2004) and elicits anti-inflammatory

actions, but its exact mechanism is unclear. It has been shown that metformin attenuates proinflammatory responses in endothelial cells (Isoda et al., 2006), diminishes human aortic smooth muscle cell proliferation (Li et al., 2005) and ameliorates macrophage activation (Mamputu et al., 2003). In the present study the effect of metformin on LPS-induced inflammation were assessed through measuring the immune-inflammatory markers of TLR4 mRNA and its critical adapter protein of MYD88 (myeloid differentiation primary response protein) in rat heart. Additionally, subsequent TLR-dependent inflammatory cytokine of TNF-α level was also evaluated. To assess the systemic and local rate of inflammation, the peripheral neutrophil count and tissue myeloperoxidase activity in the heart tissue were determined. To investigate whether metformin functions in an AMPK-dependent manner, we performed parallel experiments in mice using compound C as a pharmacological inhibitor of AMPK.

2.

Materials and methods

2.1.

Animals

Male Wistar rats (240-260 g) and male BALB/c mice (Abraham et al., 2000) (25-30 g, 8-10 weeks of age) were supplied by the Laboratory Animal Center, Medical Sciences University of Tabriz, Iran. Rats and mice were separately housed under specific conditions of 12 -12 h light to dark cycle in an air conditioned room at 23±2°C with 50±10% relative humidity. Food and water were supplied ad libitum. Animals were randomly allocated to different experimental groups. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz, Iran (National Institutes of Health Publication No. 85-23, revised 1985).

2.2.

Reagents

Metformin (1,1-dimethylbiguanide hydrochloride) was a generous gift from Osveh Pharmaceutical Inc (Tehran, Iran). LPS (lipopolysaccharide, Escherichia coli serotype K235) were purchased from Sigma (Missouri, USA). Compound C was from Sigma-Aldrich Chemie (Steinheim, Germany). Rabbit monoclonal antibodies against phospho-AMPKα (T172), AMPKα and MyD88 were obtained from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibodies against GAPDH, peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse secondary antibodies were obtained from Abcam (Cambridge, MA). ELISA kit for TNF was from Bender MedSystems Inc. (Vienna, Austria).

2.3.

Experimental protocol

2.3.1. Rat model of experiments

The animals were randomized into three groups of control, LPS and metformin (n=6 in each group). The control group received saline (0.5 ml, i.p.) 9 h prior to hemodynamic recording and heart resection. LPS and metformin groups both received LPS (0.5 mg/kg, i.p.) injection 9 h prior to removal of hearts. In the group treated with metformin, the animals received metformin (100 mg/kg, i.p.) 30 min prior to LPS injection. Metformin and LPS were dissolved in saline (0.5 ml). 9 h after LPS injection the hemodynamic parameters were recorded and following blood sampling the hearts were removed, weighted and maintained in -70 °C to perform the remaining experiments.

2.3.2. Mice model of experiments

The mice model of LPS-induced endotoxemia was used in this study as previously reported by Meng et al. (Meng et al., 1998) with minor modifications. After injection of LPS or drugs, time set zero and 9 h later the hearts were removed under anesthesia, rinsed in cold saline, snap-frozen in liquid nitrogen and were stored at -70°C for evaluating the level of MyD88 and TLR-4 expression by western blotting and Real-Time PCR, respectively. Five groups of male mice (n=6) were used: 1) Normal control group was given vehicle (normal saline, i.p.); 2) mice with sepsis were injected LPS (2mg/kg, i.p.); 3) Group 3 received compound C alone (20 mg/kg, i.p.) as an AMPK antagonist at time zero; 4) mice in group 4 were injected metfromin (100mg/kg) along with LPS and 5) Group 5 received compound C and metformin and LPS all together.

2.4.

Hemodynamic measurements

The rats were anesthetized by i.p. injection of ketamin (60 mg/kg), xylazin (10 mg/kg), and acepromazin (10 mg/kg) mixture. As the rats no longer responded to external stimuli a ventral midline skin incision was made from the lower mandible to the sternum (~3 cm). The left carotid artery was isolated using forceps and two silk ligatures were passed under the vessel. The artery was occluded toward the brain by tightening the loose ends of the upper ligature in a knot and the lower ligature was loosely tied about 1 cm from the upper tie. The artery was temporary occluded toward the heart using a Dieffenbach serrefines clamp (Elcon; Germany), and a tiny incision on the carotid artery was made using Vannas micro iris scissors (Medi Plus; USA), and a polyethylene cannula (Protex; OD 0.98 mm, ID 0.58 mm) connected to a pressure transducer (Powerlab system; AD Instruments, Australia) was

inserted into the artery for recording the systemic arterial blood pressure. To prevent the cannula letting out, the lower ligature was tightened after the cannula was passed into the artery. The mean arterial blood pressure was calculated from the systolic and diastolic blood pressure. To evaluate the cardiac left ventricular function, a Micro Tip catheter transducer (Millar Instruments, Inc., NC, USA) was advanced to the lumen of the left ventricle. By this means the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), the maximum rate of left ventricular (LV) pressure increase (LVdP/dtmax; contraction), the maximum rate of LV pressure decline (LVdP/dtmin; relaxation) were measured. All parameters were continuously recorded using a Powerlab system (AD Instruments, Australia) during 10 min (Soraya et al., 2012b).

2.5.

Blood sampling and tissue weights

Subsequent to the hemodynamic measurements, the blood sample was obtained from hepatic portal vein to assess neutrophil count. The heart was removed and weighed, then the wet heart weight to body weight ratio was calculated for evaluating the degree of myocardial weight change.

2.6.

Neutrophil counting in blood

Blood smears were provided by fresh blood samples taken just before heart isolation and after Gimsa coloring, the percent of neutrophils were counted at 100× zooming in optical microscope.

2.7.

Histopathological examination

For the histopathological examination, 9 h after LPS injections, the hearts were removed under anesthesia and serial 5µm-thick, transverse, 10% buffered formalin-fixed apex sections were embedded in paraffin and stained with hematoxylin and eosin (H&E) for histologic evaluation and Gomeri trichrome for distinguishing connective tissue, muscle and collagen fibers. Myocardial leucocytes were counted according to the methods described by Lancel et. al. (Lancel et al., 2004). Briefly, myocardial leucocytes were counted on five random fields on each slide with a magnification of 100×. The infiltration of myocardial leucocytes was expressed as the average number of leucocytes per field.

2.8.

Myeloperoxidase assay

Myeloperoxidase (MPO) was measured in the heart tissue for quantifying the activity of myocardial neutrophils, as previously described by Mullane et al. (Mullane et al., 1985). In brief, the tissue samples were homogenized (IKA Hemogenizer, Staufen, Germany) in phosphate buffer (50 mM, pH = 6) containing 0.5% hexa-decyltrimethyl-ammonium bromide (HTAB) for 45 s repeated 5 times with 1 min intervals at 7600 g. The homogenates intermittently were sonicated for 20 s and frozen- thawed 3 times, and then centrifuged at 2100 g, in 4°C for 20 min. An aliquot of the supernatant (0.1 ml) or standard (Sigma, Germany) was then allowed to react with 2.9 ml of phosphate buffer (50 mM; pH=6) containing 0.167 mg/ml of O-dianisidin dihydrochloride and 0.0005% hydrogen peroxide. After 5 min, the reaction was stopped by adding 0.1 ml of 1.2 M hydrochloric acid. The absorbance was measured spectrophotometrically (Cecil 9000, Cambridge, UK) at 460 nm. The concentrations were calculated using calibration curve and were expressed as mille units of MPO activity per gram weight of wet tissue (mU/g).

2.9.

Detection of the TLR-4mRNA in cardiac tissue by quantitative real-time PCR

Total RNA was isolated from tissues using RNX- Plus Solution (SinaClon, Iran) according to the manufacturer’s instructions. The integrity of the extracted RNA was evaluated by agarose electrophoresis and purity of RNA was determined by optical density measurement (A260/A280 Ratio) using nanodrop instrument (ND 1000, Wilmington, USA). One μg of each extracted RNA sample was used for cDNA synthesis. Retro transcription was performed with random hexamer primer and M-MuLV Reverse Transcriptase (SinaClon, Iran). All reactions were performed in a total volume of 20 μl using the iQ5 optical system (Bio-Rad laboratories, Inc, Hercules, CA). This 20 μl reaction mixture contained: 1 μl cDNA, 0.6 μl primer (300nM each primer), 10 μl 2X qPCR Green-Master Mix (EvaGreen, Jena Bioscience, Germany), and up to 20 μl PCR-grade water. All reactions were performed in triplicates and with a negative control along with NTC included in each experiment. The thermocycling conditions were as follow: 1 cycle at 94 °C for 10 min, 40 cycles at 95 °C for 15 sec, 59 °C (annealing temperature) for 30 sec and 72 °C for 25 sec. For quantification, the target gene was normalized to the internal standard gene of GAPDH (glyceraldehyd-3phosphate dehydrogenase). The primers were designed for detection of the TLR4 gene expressions, as given below: For TLR4, Forward: 5′-AAGTTATTGTGGTGGTGTCTAG-3′; Reverse: 5′-GAGGTAGGTGTTTCTGCTAAG-3′. For GAPDH, Forward: 5′-TTGTCAAGCTCATTTCCTGGTATG-3′; Reverse: 5′- GGATAGGGCCTCTCTTGCTCA-3′.

Interpretation of the result was performed using the Pfaffle Method (Pfaffl, 2001).

2.10. Western blot analysis

Western blot analyses were performed according to assay described by Omar et al. (Omar et al., 2012) and Kewalramani et al (Kewalramani et al., 2007) with minor modifications. Briefly, ventricular section of heart tissue was homogenized in ice-cold solution containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM Sodium Pyrophosphate (NaPPi), 50 mM NaF, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.1% SDS (w/v), 1% TXT-100 (v/v), and protease inhibitor cocktail (Roche, Mannheim, Germany). Subsequently, the tissue homogenate was centrifuged at 10600 g for 10 min at 4 °C. The supernatant was aliquoted and stored at −80 °C for further analysis. The protein content of the supernatant was quantified using a Bradford Protein Assay kit (Bradford, 1976). The samples were mixed with a sample loading buffer, and 50 μg of the homogenate protein was subjected to SDSPolyacrylamide gel electrophoresis using Bio-Rad mini protean tetra system (Hercules, CA). After separation in polyacrylamide gel, the aliquots were transferred to an Immobilon-P membrane (Millipore, Billerica, MA). Nonspecific antibody binding was inhibited by incubation in TBST [20 mM Tris-buffered saline (pH 7.5) with 0.1% Tween 20] containing 5% non-fat dried milk for 1 h at rotator. The membranes were then probed using a range of primary antibodies raised against phospho-AMPKα (Thr172), AMPKα, MyD88, as well as GAPDH (1:1000 dilution). The membranes were incubated overnight with the antibodies at 4°C. They were washed with TBST and incubated at rotator for 60 min with peroxidaseconjugated goat anti-rabbit and rabbit anti-mouse secondary antibodies (1:5000 dilution). Subsequent to washing, antibodies were visualized using the BM Chemiluminescence kit (Roche, Mannheim, Germany). The molecular weights of phospho-AMPKα and MyD88

were confirmed according to their protein markers (PageRuler Unstained Protein Ladder, Fermentas, Lithuania). Densitometric analysis of the immunoblots was performed using image j software (Wayne Rasband, National Institute of Health, USA). The densitometric values for phosphorylated AMPKα were normalized to AMPKα and in the case of MyD88 the values were normalized to GAPDH.

2.11. Cytokine assays

The serum and cardiac levels of TNF-α were assayed using rat enzyme-linked immunosorbent assay (ELISA) kits (Rat TNF-α, Bender Med Systems, Vienna, Austria) according to manufacturer’s recommendations. Briefly, the samples were in an ice-cold solution containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM Sodium Pyrophosphate (NaPPi), 50 mM NaF, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.1% SDS (w/v), 1% TXT-100 (v/v), and protease inhibitor cocktail (Roche, Mannheim, Germany). The samples were then centrifuged twice at 10600 g for 10 min at 4 °C. The resulting supernatants were used for the assay. The optical density of each well was measured at 450 nm using a microplate reader. The concentration of the cytokine was expressed as pg/100 mg heart tissue or pg/ml of serum.

2.12. Statistical analysis

The results were expressed as mean±S.E.M. The data were statistically analyzed using one-way ANOVA followed by LSD post-hoc test to compare the mean values between the treatment groups and the control. Differences were considered significant at P<0.05.

3.

Results

3.1.

The effects of metformin on hemodynamic responses

As demonstrated in Table 1, 9 h after LPS injection the mean arterial blood pressure (MAP) was significantly increased from 101±4.2 mmHg in the control group to 127±3.6 mmHg in the LPS-received group (P<0.01). In addition, LPS also significantly increased the heart rate from 252±5 bpm to 322±8 (P<0.001). The increased MAP and heart rate were reduced close to the normal values of 113±4.3 mmHg (P<0.05) and 281±5 bpm (P<0.01), respectively by metformin. LPS had no significant effect on the left ventricular developed pressure calculated by subtracting LVDP from LVSP. However, metformin enhanced the left ventricular function by increasing the developed pressure from 116±2.9 mmHg in LPS group to 127±3 mmHg (P<0.01). The left ventricular contraction and relaxation indexed as LV dP/dtmax and LV dP/dtmin were suppressed by LPS but did not attain a significant level. In this regard, both the contractility and relaxation were profoundly improved by metformin, whereas LV dP/dtmax was significantly increased to 5351±206 mmHg/sec (P<0.01) and LV dP/dtmin was significantly decreased to -4980±179 mmHg/sec (P<0.01).

3.2.

The effects of metformin on body weight and on heart to body weight ratio

The body weight changes were determined as a marker to assess the effect of LPS and metformin plus LPS on the animals' activity and appetite. As shown in Fig. 1A, LPS alone caused a significant (P<0.001) body weight loss compared to the control group (∆BW = -2.3g in control group, ∆BW = -14.3g in LPS group). Despite metformin inhibited the LPS-induced

weight loss compared to LPS group (∆BW = -14.3g vs. ∆BW = -11.6g), however the inhibition did not reached a significant level by co-administration of metformin (Fig. 1A). The heart weight to body weight (HW/BW) ratio also was determined to assess the extent of edematous developed by LPS and to answer this question whether treatment with metformin may protect the heart from the LPS-induced damage (Fig. 1B). It was observed that LPS induced a significant increase in HW/BW ratio (3.02±0.03 g/kg vs. 2.57±0.02 g/kg; P<0.001). Treatment with metformin was found to significantly (P<0.001) protect the heart from the LPS-induced edematous (Fig. 1B).

3.3.

The effects of metformin on the peripheral neutrophil count and tissue

myeloperoxidase activity

LPS injection resulted in a significant (P<0.001 vs. control) increase in neutrophil infiltration into the heart tissue. Accordingly, the control group demonstrated MPO activity levels of 2043±125 mU/g of wet tissue, whereas a marked (P<0.001) increase was detected in LPS-injected group (3535±176 mU/g). Metformin treatment led to a significant decrease in myocardial MPO activity as compared to rats of the LPS-received group (P<0.01). This reduction in MPO activity by metformin was associated with a similar decline in the percentage of peripheral neutrophil, which was significantly (P<0.01) decreased from 88.72±2% in LPS group to 63.42±5.8% in metformin receiving group (Fig. 2).

3.4

. The effects of metformin on histology of heart tissue

Histological evaluation by light microscopy demonstrated that the myofibrils were contiguously aligned in the control rats and the morphology and structure of the nuclei and

cells were normal (Fig. 3A). However, LPS administration significantly increased focal hemorrhages and leucocyte infiltration in myocardial cell interludes and the myofibrils displayed a disrupted and disordered arrangement (Fig. 3B). Following treatment with metformin, the myocardial fibers were contiguously and more neatly arranged. Furthermore, metformin decreased the hyperemia of blood vesselsand erythrocyte extravasation compared to LPS group. In addition, less accumulation of polymorphonucleocytes (PMN) in the border zone of vessels was observed in metformin treated groups (Fig. 3C). There was no apparent difference of cardiac morphology between groups in gomori trichrome staining (figures are not shown). LPS administration significantly increased leucocyte infiltration into the cardiac interstitium. As shown in Fig. 3D, LPS-increased cardiac leucocyte infiltration by 72% when compared to control group (P<0.001). However, LPS-induced cardiac leucocyte infiltration was significantly decreased by 61.71% in metformin receiving group compared to LPS group (P<0.001).

3.5.

Detection of the TLR-4 mRNA in cardiac tissue by quantitative real-time PCR

Figure 4 shows the levels of TLR4 mRNA expression under the various experimental conditions. LPS markedly increased the expression of TLR4 in cardiac tissue approximately 2.5 fold as compared to the control group (P<0.01). The mRNA level of TLR4 was significantly reduced (P<0.05) upon metformin treatment in comparison with LPS group (Fig. 4).

3.6.

The effects of metformin on AMPKα phosphorylation in myocardium

We further investigated whether the dose of metformin used in this study could activate AMPK in the myocardium. The rat heart tissue was used to assess the phosphorylation status and the rate of AMPK activation was expressed as the ratio of phosphorylated form of AMPK to non-phosphorylated AMPK. Results demonstrated a slight and non-significant increase (+5%) in AMPK activation in LPS group compared to control. However, a significant (P<0.001 vs. LPS) increase of 43% in the phosphorylation of AMPK at threonine residue 172 and activation of AMPK was observed in the group treated with metformin (Fig. 5).

3.7.

The effects of metformin on MyD88 protein expression in myocardium

Stimulation of TLR4 through LPS can lead to a MyD88-dependent activation of several intracellular mediators resulting in NF-κB transcription factor and increasing the systemic and tissue level of cytokines like TNFα. Thereby, the level of MyD88 protein in the myocardium was assessed in the present study. As expressed in Fig. 6, MyD88 protein content in the LPS group was upregulated when compared to control group (+59%, P<0.01). Metformin treated rats displayed a significant (P<0.05) reduction in the expression of MyD88 by 45% in comparison with LPS group (Fig. 6).

3.8.

The effects of metformin on LPS-induced increase in TNF-α level in serum and heart

tissue

To further explore the protection against LPS-induced TLR activation by metformin, the potential effect of metformin on the level of TNFα, which is a key pro-inflammatory cytokine released following TLRs activation, was examined in the present study. As

expected, LPS stimulated the TNF-α production and a high amount of the cytokine was measured both in serum and heart tissue of the rats injected with LPS. Compared with the level of TNF-α in the control group (75 pg/ml in serum and 472 pg/100 mg wet tissue), LPS profoundly increased the level of TNF-α (222 pg/ml in serum and 825 pg/100 mg wet tissue; P<0.001) both in serum and myocardium. Metformin significantly reduced the level of TNFα to 128 pg/ml in serum (Fig. 7A; P<0.01) and 658 pg/100 mg in the wet tissue (Fig. 7B; P<0.05).

3.9.

Detection of the TLR-4 mRNA in mice cardiac tissue by quantitative real-time PCR

Figure 8 shows the levels of TLR4 mRNA expression under the various experimental conditions. LPS upregulated the expression of TLR4 in cardiac tissue approximately 2.25 fold as compared to the control group (P<0.001). To exclude any probable effect of compound C on TLR4 expression, a separate group of receiving only compound C was designed and it was shown that compound C had no per se effect on TLR4 mRNA level. The group treated with metformin displayed a significant (P<0.05) reduction of TLR4 expression compared with LPS group. Antagonizing AMPK by co-administration of compound C with LPS+Metformin significantly (P<0.05) reversed the suppressive effects of metformin on TLR4 expression (Fig. 8).

3.10

. The effects of metformin on MyD88 protein content in mice myocardium

As expressed in Fig. 9, MyD88 protein content in the LPS group was markedly increased when compared to control group (+62%, P<0.001). The MyD88 expression level was significantly reduced (-43%, P<0.05) upon metformin treatment in comparison with LPS

group. Compound C reversed the lowering action of metformin on LPS induced MyD88 expression in the heart tissue however; the effect did not attain a significant level (Fig. 9).

4.

DISCUSSION

The results of the present study clearly demonstrated that: 1) a single dose of metformin, preserved myocardial function in rats with LPS-induced sepsis, 2) treatment with metformin reduced the serum and heart tissue levels of TNF-α and tissue infiltration of neutrophils which were elevated by LPS, 3) the beneficial effects of treatment with single dose of metformin in sepsis were associated with reductions in TLR4 signaling as indicated by depression of myocardial levels of TLR4 mRNA and MyD88 protein, and 4) the protective effects of metformin were accompanied with AMPK activation. Furthermore, we found that the effects of metformin on TLR4 signaling pathway were AMPK-dependent. AMPK-dependency was defined as the of reversal or overcoming the influence of metformin by use of pharmacological inhibitor of AMPK (compound C) on given parameters. One of the major clinical concerns in patients with septic shock is myocardial dysfunction which if not controlled, usually leads to death. Most of the cardiovascular complications in sepsis are driven from bacterial LPS. Cardiac effects of LPS in septic patients have an utmost importance and many of the cardio-depressive effects of LPS in sepsis are induced by TLR4 mediated production of cytokines especially TNF-α (Grandel et al., 2000). The results of different studies suggest that metformin, in addition to its efficacy as an insulin sensitizer, may have therapeutic potential to reduce cardiovascular complications in diabetic patients (Caballero et al., 2004; De Jager et al., 2005) and to treat inflammatory diseases (Hirsch et al., 2013; Isoda et al., 2006; Koh et al., 2014). However, the relevance of

these diabetes-based actions and the exact molecular mechanisms of metformin beyond its glycemic control still remain poorly understood. In the present study, we attempted to evaluate the effects of metformin on LPSstimulated TLR4 activation pathway in heart. The results showed that metformin in addition to improvement of myocardial dysfunction essentially reduced TLR4 gene expression and subsequently reduced the protein level of MYD88 and TNF-α level in the heart. The beneficial effect of metformin against LPS-induced myocardial depression was also associated with suppression of neutrophil infiltration into the myocardium. This is the first report which demonstrates acute administration of metformin can reduce early sepsis-induced inflammation response in the heart which is confirmed by gene expression data. Furthermore, cardiac enlargment index of heart to body weight ratio was decreased. All hemodynamic factors of mean arterial blood pressure, heart rate, left ventricular developed pressure and maximum and minimum rates of left ventricular (LV) pressure changes were improved by metformin. To assess the systemic and local rate of inflammation, the peripheral neutrophil count and the myeloperoxidase activity in the heart tissue were evaluated and it was resulted that metformin reduced both of these parameters. The AMPK activity in the myocardium, as expected, was increased by metformin. Thus, our results provide strong evidence that the anti-inflammatory effect of metformin is mediated, at least in part, via attenuation of the TLR4 pathway. It is reported that inhibiting AMPK activation by compound C could reverse metformin-induced down-regulation of ICAM-1 (Liu et al., 2014). ICAM-1 (Intercellular Adhesion Molecule 1) is a type of intercellular adhesion molecule which its signaling seems to produce a recruitment of inflammatory immune cells such as macrophages and granulocytes. ICAM-1 expression can be regulated by the nuclear transcriptional factor NFκB (Tak and Firestein, 2001). On the other hand, studies have illustrated the potency of

metformin on blocking the NF-κB signaling and decreasing the TNF-α-induced ICAM-1 (Hattori et al., 2006; Isoda et al., 2006). It has been demonstrated that increased expression of adhesion molecules, such as VCAM-1 and ICAM-1, will be a key step for the infiltration of neutrophils and leucocytes into the myocardium (Raeburn et al., 2002). We observed that LPS administration significantly increased leucocyte infiltration and metformin reversely suppressed transmigration and infiltration of leucocytes into the myocardium. It can be concluded that metformin exerts its protective effect in reducing neutrophil count and the myeloperoxidase activity and improving cardiac histology partly through AMPK activation and diminished TNF-α production. There was no apparent difference of cardiac morphology between groups in gomori trichrome staining at the end of the 9 h of experiment duration. As this staining method is specific for connective tissue and collagens and since the alteration process of these components of myocardium is time dependent, it is supposed that in our study there was not enough time to cell degeneration or fibrosis formation. LPS administration induces NF-κB activation (Lee et al., 2015) leading to robust myocardial cytokines expression such as TNF-α and therefore myocardial dysfunction (Baumgarten et al., 2001; Knuefermann et al., 2002a). LPS is also fond to upregulate TLR4 and the mice deficient in TLR4, CD14, and IRAK-1 had improved cardiac function and were protected from endotoxin induced myocardial inflammation (Knuefermann et al., 2002a; Thomas et al., 2003). It has been reported that AMPK activation by metformin and the subsequent suppression of TLRs activity could be considered as a target in protecting the infarcted heart (Soraya et al., 2012a). Consistent with these results, in all LPS received rats of our study, the myocardial and serum level of TNF-α was increased and reversely decreased by metformin therapy.

Reportedly, LPS exhibited reduced sarcomere shortening and Ca2+ transients in cardio myocytes leading to cardiac dysfunction (Tavener et al., 2004). On the other hand, it has been demonstrate that independent of insulin sensitizing action, metformin provides cardio protection against high glucose induced abnormalities in myocyte relaxation, presumably through tyrosine kinase-dependent changes in intracellular Ca2+ handling (Ren et al., 1999). Therefore metformin may act via this mechanism in reducing cardiac dysfunction arised from LPS administration. Our results provide additional data about AMPK-dependent mechanisms by which metformin may modulate the inflammatory response in myocardium. It was revealed that metformin-induced TLR4 expression decrease to be mediated by AMPK activation. The decreased TLR expression after metformin exposure was reversed by simultaneous presence of the AMPK inhibitor compound C. In case of MYD88, although not significant but there was also increase in protein expression in use of compound C. It was demonstrated that in endotoxemic model of inflammation, MyD88 and Trif (two TLR4 associated signaling cascade adapter proteins) play an equally important role in mediating inflammation (IL-1β, IL-6, and TNFα) and cardiac depression (Feng et al., 2011). Since in the present study, only the MYd88 dependent signaling is assessed, the effect of metformin on the other pathways should also be considered.

5.

Conclusion

The results of the present study demonstrate that metformin preserves cardiac function in sepsis by suppressing LPS-induced inflammatory responses possibly via AMPK activation and attenuation of TLR4 activity. Since metformin is a widely used safe drug with few adverse effects, using this long-established drug with new applications in human health

may be a promising way to develop an effective therapy especially in immune-inflammatory disease. The exact mechanism of metformin and the relevance of AMPK activation and TLR4 suppression require further investigation.

Acknowledgment The present study was supported by a grant from the Research Vice Chancellors of Tabriz University of Medical Sciences, Tabriz, Iran.

Conflicts of interest There are no conflicts of interest.

Refereences

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Fig. 1. Effect of metformin on body weight changes and heart weight (HW) to body weight (BW) ratio in rats treated with LPS alone or LPS plus metformin. LPS (0.5 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) was injected 30 min before LPS injection. 1A: The animals were weighed immediately before injection of drugs and at the end of the experiment (9 h). 1B: The hearts were weighed immediately after resection. LPS: Lipopolysaccharide; Met: Metformin. Results are presented as mean±S.E.M (n=6). ***P<0.001 from the control group; ## P<0.01 from LPS group using one way ANOVA with LSD post-hoc test.

Fig. 2. Effects of metformin on the peripheral neutrophil count (line graph) and myeloperoxidase activity (bar graphs). LPS (0.5 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) was injected 30 min before LPS injection. LPS: Lipopolysaccharide; Met: Metformin. Results are presented as mean±S.E.M (n=6).

***P<0.001 from control group; ##P<0.01, ###P<0.001 from LPS group using one way ANOVA with LSD post-hoc test.

Fig. 3. Histological appearance of heart sections stained with Haematoxylin and Eosin with original magnification of 100× by light microscopy. LPS (0.5 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) was injected 30 min before LPS injection. A: Control group showing normal morphology of myocardium with arranged myocardial bundles B: LPS group showing disrupted arrangement of myofibrils and erythrocyte extravasation (E). The hyperemia of blood vessels and accumulation of PMN inside the vessel and infiltration in between myocardial bundles are shown (PMN). C: Metformin group showing contiguously and more neatly arranged myocardial fibers with less erythrocytes leakage. D: leucocyte infiltration examination. LPS: Lipopolysaccharide; Met: Metformin. Results are presented as mean±S.E.M (n=6).***P<0.001 from control group; ###P<0.001 from LPS group using one way ANOVA with LSD post-hoc test.

Fig. 4. Effects of metformin on the levels of TLR4 mRNA expression in cardiac tissue. LPS (0.5 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) was injected 30 min before LPS injection. LPS: Lipopolysaccharide; Met: Metformin. Results are presented as mean±S.E.M (n=6). **P<0. 01 from control group; #P<0.05 from LPS group using one way ANOVA with LSD post-hoc test.

Fig. 5. Effects of metformin on phosphorylation at threonine 172 residue of AMPKα in myocardium. LPS (0.5 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) was injected 30 min before LPS injection. LPS: Lipopolysaccharide; Met:

Metformin. Results are presented as mean±S.E.M (n=6). ###P<0.001 from LPS group using one way ANOVA with LSD post-hoc test.

Fig. 6. Effects of metformin on MyD88 protein expression in rat myocardium. LPS (0.5 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) was injected 30 min before LPS injection. LPS: Lipopolysaccharide; Met: Metformin. Results are presented as mean±S.E.M (n=6). **P<0.01 from control group; # P<0.05 from LPS group using one way ANOVA with LSD post-hoc test.

Fig. 7. Effects of metformin on the LPS-induced increase in the level of TNF-α in serum (A) and myocardium (B). Rats were given LPS (0.5 mg/kg, i.p.) 9 h prior to heart resection and metformin (100 mg/kg, i.p.) was injected 30 min before LPS injection. LPS: Lipopolysaccharide; Met: Metformin. Results are presented as mean±S.E.M (n=6). ** P<0.01 from control group; # P<0.05 from LPS group using one way ANOVA with LSD post-hoc test.

Fig. 8. Effects of compound C on the level of TLR4 mRNA expression in mice cardiac tissue. LPS (2 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) and/or compound C (20 mg/kg, i.p.) were injected 30 min prior to LPS injection. LPS: Lipopolysaccharide; CC: Compound C; Met: Metformin. Results are presented as mean±S.E.M (n=6). ***P<0. 001 from control group; #P<0.05 from LPS group; +P<0.05 from LPS+Met group using one way ANOVA with LSD post-hoc test.

Fig. 9. Effects of compound C on MyD88 protein expression in mice cardiac tissue. LPS (2 mg/kg, i.p.) was given 9 h prior to heart resection and metformin (100 mg/kg, i.p.) and/or

compound C (20 mg/kg, i.p.) were injected 30 min prior to LPS injection. LPS: Lipopolysaccharide; CC: Compound C; Met: Metformin. Results are presented as mean±S.E.M (n=6). ***P<0. 001 from control group; #P<0.05 from LPS group using one way ANOVA with LSD post-hoc test.

Table. 1. The Effects of metformin (100 mg/kg, i.p.) on hemodynamic variables and left ventricular functions in rats received LPS (0.5 mg/kg, i.p). Groups

MAP

Heart rate

LV Dev P

LV dP/dt max

LV dP/dt min

(mmHg)

(bpm)

(mmHg)

(mmHg/sec)

(mmHg/sec)

Control

101±4.2

252±5

118±4.3

5082±172

-4667±192

LPS

127±3.6 a

322±8 b

116±2.9

4712±200

-4257±232

Metformin+LPS

113±4.3 c

281±5 d

127±3 c

5351±206 c

-4980±179 c

n=6

Data are presented as mean±S.E.M (n=6). MAP: mean arterial pressure; bpm: beat per minute; LV Dev P: left ventricular developed pressure; LV dp/dtmax: maximal rate of Left ventricular pressure increase (contractility); LV dp/dtmin: maximal rate of Left ventricular pressure decline (relaxation). a P<0.01; b P<0.001 vs control group; c P<0.05, d P<0.01 as compared with LPS treated group using one way ANOVA with LSD post-hoc test.

Fig 1

Fig 2

Fig 3

Fig 4

Fig 5

Control

LPS

LPS+Metformin

Fig 6

Control

LPS

LPS+Metformin

Fig 7

Fig 8

Fig 9