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AMPK activation by metformin inhibits local innate immune responses in the isolated rat heart by suppression of TLR 4-related pathway
International Immunopharmacology 40 (2016) 501–507
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AMPK activation by metformin inhibits local innate immune responses in the isolated rat heart by suppression of TLR 4-related pathway Haleh Vaez a,b, Moslem Najafi a, Maryam Rameshrad a, Negisa Seyed Toutounchi b, Mehraveh Garjani c, Jaleh Barar d, Alireza Garjani a,e,⁎ a
Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran d Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran e Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 2 July 2016 Received in revised form 14 September 2016 Accepted 1 October 2016 Available online xxxx Keywords: Heart Metformin Toll-like receptor 4 AMPK NF-κB
a b s t r a c t Toll like receptors (TLRs) are key players in the innate immune responses. The energy sensing enzyme, AMPK, has been implicated in the modulation of immunity. The present study investigated whether AMPK activation by metformin could contribute to the regulation of immune responses in the isolated heart via suppression of TLR4 activity, independent of circulatory immunity. Isolated Wistar rat hearts were perfused with KrebsHenseleit buffer in the absence or presence of lipopolysaccharide (LPS; 0.2 μM), LPS + metformin (10 mM), and LPS + metformin + compound C (10 μM). Following measurement of hemodynamic parameters, TLR4activation related changes and TLR4 mRNA level in the heart was examined by western blotting and real-time PCR. The activation of AMPK was evaluated by measuring the ratio of p-AMPKα and p-ACC to their nonphosphorylated forms. The effluent and cardiac levels of TNF-α and IL6 were assayed by ELISA. LPS profoundly increased the levels of TLR4 mRNA, MyD88 (TLR4 adaptor protein), and NF-κB and also the release of TNF-α and IL6 from the heart. The enhancement in the TLR4 activity was associated with a significant depression of myocardial function. Metformin clearly augmented the phosphorylation of both AMPKα and ACC and in addition to improvement of cardiac performance, markedly suppressed the TLR4 activity. Antagonizing AMPK by compound C which is a selective inhibitor of AMPK pathway, considerably reversed the protective effects of metformin against the TLR4-related activity. The results of the study demonstrated the importance of TLR4-involved local immune responses in the LPS-induced myocardial dysfunction and indicated a clear link between AMPK and TLR4. © 2016 Published by Elsevier B.V.
1. Introduction The innate immune system has been implicated in a wide range of pathological conditions including sepsis and cardiovascular diseases. Toll-like receptors (TLRs) are a class of membrane bound receptors on eukaryotic cells that play a key role in the innate immunity. They are mainly expressed on sentinel cells such as macrophages and dendritic cells that recognize pathogen-associated molecular patterns (PAMPs). A classic example of pathogen-associated molecular pattern includes lipopolysaccharides (LPS) of gram-negative bacteria. Moreover, various endogenous ligands, called danger-associated molecular patterns (DAMPs), which are released in a variety of cardiovascular diseases such as sepsisinduced myocardial dysfunction, myocardial infarction, heart failure, ⁎ Corresponding author at: Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. E-mail address: [email protected] (A. Garjani).
http://dx.doi.org/10.1016/j.intimp.2016.10.002 1567-5769/© 2016 Published by Elsevier B.V.
and ischemia/reperfusion also activate TLRs [1]. Among all TLRs expressed in the heart, TLR2 and TLR4 are the most investigated. It has been demonstrated that activation of TLR2 and TLR4 by DAMPs and PAMPs leads to translocation of p65 subunit of nuclear factor kappa B (NF-κB) into the nucleus and hence induces the synthesis of TNF-α [2]. Myeloid differentiation factor 88 (MyD88) is the main adaptor protein employed in all TLR pathways except for TLR3. MyD88 recruits the interleukin (IL)-1 receptor associated kinases (IRAK), and in turn triggers the downstream of multiple signaling cascades. It activates the inhibitory κB kinase complex which directly phosphorylates IκBα, leading to nuclear translocation of the p65 component of the NF-κB complex and expression of target genes of proinflammatory cytokines (e.g. IL6 and TNF-α), cell adhesion molecules, and chemokines that recruit macrophages and neutrophils to the tissue [3]. Studies have shown that mice deficient of either TLR4 or interleukin (IL)-1 receptor associated kinase 1 (IRAK1) are preserved from the LPSinduced mortality and cardiac dysfunction [4,5]. Besides, TLR-mediated signaling contributes to myocardial damage and adverse cardiac
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remodeling following ischemia/reperfusion injury and myocardial infarction [6,7]. Recently, we published a study that claimed TLR4 is expressed in the intact and isolated heart tissues and when ligated by LPS, TNF-α release and myocardial dysfunction ensues [8,9]. The NF-κB-dependent pro-inflammatory cytokine release, following the TLR4 stimulation, has prominent role in the pathophysiology of cardiac function modification in sepsis. It is proposed that immunosuppressive and anti-inflammatory therapeutic strategies in cardiovascular disease, specifically in sepsisinduced myocardial dysfunction, can have beneficial effects on the outcomes. Inhibitors of specific inflammatory pathways are already either in use or under development; these include both classic drugs such as allopurinol, colchicine, and methotrexate and biologic therapies like TNF-α antibodies and IL-1β antagonists [10–12]. However, in contrast to experimental animal studies, targeting the innate immune system and specific inflammatory pathways for the treatment of cardiovascular diseases were not always promising in human [13]. Moreover, selective modulation of local cardiac immunity in the pathological conditions has not been studied yet. The existing challenges over utilization of anti-inflammatory therapies in cardiovascular disease make the search for the effective inflammatory modulators intriguing. AMP activated protein kinase (AMPK) is a multifaceted enzyme that acts as the sensor of intracellular energy; it can activate ATP-generating catabolic or inactivate ATP-consuming anabolic pathways. In other words, AMPK conserves energy in metabolic stresses and thus protects the cells in various pathological conditions. AMPK also regulates critical cellular processes including transcription and protein synthesis [14]. It is believed that AMPK is an important regulator of inflammatory responses in immune cells; and anti-inflammatory effects of some agents might actually be due to the activation of AMPK [15]. For instance, adiponectin inhibits LPS-activated TLR4 signaling pathways in hepatic Kupffer cells and in macrophages through mechanisms involving AMPK [16]. A study has demonstrated that AMPK antagonizes TLR-induced maturation of dendritic cells, which are the key regulators of innate and acquired immunity [17]. Our previous studies showed that both chronic and short term treatments with metformins which is a best known activator of AMPK, attenuated left ventricular dysfunction induced by myocardial infarction and alleviates myocardial injuries [18,19]. Clinical and experimental studies suggest that metformin, besides its antidiabetic effects, can exert systemic and local anti-inflammatory effects through mechanisms that are not clearly elucidated [20,21]. It was reported that metformin by decreasing inflammatory markers such as soluble intercellular adhesion molecule, vascular cell adhesion molecule-1, macrophage migration inhibitory factor, and C-reactive protein (CRP) can modulate inflammation [21–23]. Furthermore, it was demonstrated that metformin suppressed NF-κB activation in colitis-associated studies [24]. The best known explanation for the anti-inflammatory properties of metformin is AMPK activation [25]; however, AMPK-independent pathways are now considered to play a part too [26,27]. It has been hypothesized that AMPK activation could improve cardiac function following LPS-induced myocardial dysfunction through inhibiting TLR4 activity in the heart tissue independent of circulatory immune system. Therefore, in the present study, we investigated the role of AMPK activation by metformin on LPS-induced myocardial dysfunction and on TLR4 signaling pathway in the isolated rat hearts. Further, to confirm the effect of AMPK in this inflammatory cascade, we used compound C, which is a selective AMPK inhibitor. 2. Materials and Methods 2.1. Animals Male Wistar rats (260–290 g) were supplied by Laboratory Animal Center, Tabriz University of Medical Sciences, Iran. Animals 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 according to the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz, Iran which is in accordance with the National Institutes of Health guidelines (revised 2011) and was approved by the local authorities of Animal Ethics Committees (AEC references number: TBZMED. REC. 1394.721). 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, acetyl-CoA carboxylase (ACC), phospho-ACC and MyD88 were obtained from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibodies against glyceraldehyde phosphate dehydrogenase (GAPDH), NF-kB, β-actin, peroxidase-conjugated goat antirabbit and rabbit anti-mouse secondary antibodies were obtained from Abcam (Cambridge, MA). ELISA kit for TNF-α and IL-6 were from Bender MedSystems Inc. (Vienna, Austria). 2.3. Isolated Heart Perfusion The Langendorff technique of isolated heart perfusion has been described in detail in reference [8]. In short, animals were heparinized (1000 IU/kg) and anesthetized using a cocktail of ketamine (60 mg/kg), xylazine (10 mg/kg), and acepromazine (10 mg/kg) given intraperitoneally. When the rats no longer responded to external stimuli, their hearts were rapidly excised via bilateral thoracotomy and immersed in icecold modified Krebs-Henseleit buffer solution (KHBS). The harvested hearts were immediately cannulated to a langendorff apparatus (AD Instruments; Australia) and then were perfused through the ascending aorta at a constant flow (10 ml/min/g) with KHBS gassed with carbogen (5% CO2/95% O2). The pH was 7.38–7.56 at 37 °C. All hearts were initially rinsed with KHBS for 15 min in a non-recirculating mode before switching to recirculation (total volume of 100 ml). For monitoring coronary perfusion pressure (CPP), the aortic cannula was connected to a pressure transducer (MLT844 physiological pressure, AD Instruments; Australia). Left ventricular pressure was measured isovolumetrically by inserting a home-made latex balloon attached to a pressure transducer into the left ventricular cavity via the mitral valve after removing the atrial appendage. Left ventricular developed pressure (LVDP) was calculated as the difference between peak-systolic and end-diastolic pressure. The maximum and minimum rates of left ventricular pressure (dP/dtmax, dp/dtmin) as indices of left ventricular contractility and relaxation, and heart rate (HR) were monitored by PowerLab 8/35. 2.4. Experimental Protocol After the hearts had been stabilized, time was set to zero, and hearts were perfused in recirculating mode with 100 ml of assigned perfusates according to their experimental group for 180 min. Animals were allocated to six different experimental groups and the hearts of each group received a different treatment as below: (1) KHBS solution (control group), (2) LPS: KHBS solution containing LPS (0.2 μM), (3) LPS + Met: KHBS solution containing LPS (0.2 μM) + metformin (10 mM), (4) Met: KHBS solution containing metformin (10 mM), (5) CC: KHBS solution containing compound C (10 μM), (6) LPS + Met + CC: KHBS solution containing LPS (0.2 μM) + metformin (10 mM) + compound C (10 μM). Hemodynamic variables were monitored for the whole period of experiment. Perfusate samples were taken at the end of perfusion time
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and analyzed with enzymatic/colorimetric method by COBAS Integra 400 analyzer (Roche, Germany) to determine the lactate concentration [28]. 2.5. Quantitative Analysis of TLR-4 mRNA in Cardiac Tissue Total RNA was isolated from cryosections of the left ventricle 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 microgram of each extracted RNA sample was used for cDNA synthesis. Reverse transcriptase PCR was performed with random hexamer primer and M-Mel Reverse Transcriptase (Sialon, 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 (300 nM each primer), 10 μl 2× 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 s, annealing temperature (AT) for 30 s, and 72 °C for 25 s. For quantification, the target gene was normalized to the internal standard gene of β-actin. The PCR primers were designed as given below: For TLR4 (AT 60 °C): sense primer (5′-AAGTTATTGTGGTGGTGTCTAG-3′) antisense primer (5′-GAGGTAGGTGTTTCTGCTAAG-3′) For β-actin (AT 58 °C): sense primer (5′-GCT ACA GCT TCA CCA CCA CA-3′) antisense primer (5′-ATC GTA CTC CTG CTT GCT GA-3′) Interpretation of the result was performed using the Pfaffle Method [29]. 2.6. Western Blot Analysis Western blot analysis were performed according to assay described by Omar et al. [30] and Kewalramani et al. [31] 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 and the supernatant was collected, and the protein concentration was determined using the Bradford Protein Assay kit [32]. 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). The membrane was blocked in washing buffer with 5% nonfat milk for 2 h and incubated overnight with the corresponding primary antibodies at 4 °C. They were washed with a mixture of Tris-Buffered saline and Tween 20 solution (TBST) and incubated at rotator for 60 min with peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse secondary antibodies (1:5000 dilution). Subsequent to washing, the signals of detected proteins were visualized using the BM Chemiluminescence kit (Roche, Mannheim, Germany). The molecular weights of phosphoAMPKα, phospho-ACC, MyD88 and NF-κB 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 and ACC were normalized to non-phosphorylated AMPKα and ACC
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respectively. In the case of MyD88 the values were normalized by comparison to GAPDH and NF-κB were normalized to internal standard protein of β-actin. 2.7. Cytokine Assays The effluent and cardiac levels of TNF-α and IL-6 were assayed using rat enzyme-linked immunosorbent assay (ELISA) kits (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% TXT100 (v/v), and protease inhibitor cocktail (Roche, Mannheim, Germany). The samples were then centrifuged twice at 10,600 ×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 cytokines was expressed as pg/100 mg heart tissue or pg/ml of effluent.
2.8. Statistical Analysis Data are expressed as mean ± SEM. Differences between data sets were assessed by one-way analysis of variance followed by Tukey post-hoc test. For the real time PCR, Pair Wise Fixed Reallocation Randomization test using REST software was used to make comparisons between the groups. p b 0.05 was accepted as statistically significant. 3. Results 3.1. Cardiac Function and Hemodynamic Factors We evaluated cardiac function by continuous monitoring of hemodynamic parameters including coronary perfusion pressure (CPP), left ventricular developed pressure (LVDP), and left ventricular contractility (dP/dtmax) and relaxation (dp/dtmin) at baseline and every 30 min of perfusion period. There was no significant difference between the groups at stabilization time. To assess the possible effect of metformin and compound C alone on cardiac function, all hemodynamic parameters were also determined for these two groups. Considering the data comparison related to cardiac function including CPP, LVDP, dP/dtmax and dp/dtmin, there was no significant difference between metformin and compound C group compared to the control group (p b 0.05; data are not shown). LPS consistently caused a rise in CPP throughout the experiments; however, there was no significant difference between the mean values of CPP in groups perfused with LPS (Fig. 1A). Although left ventricular developed pressure was stable within the first 30 min of LPS perfusion, a sharp consistent decline of LVDP occurred at 60 min of perfusion and afterwards (p b 0.01 at 60 min, and p b 0.001 at 90, 120, 150 and 180 min; Fig. 1B). Metformin profoundly nullified the suppressive effects of LPS on LVDP at all-time points (p b 0.05 at 90 min, and p b 0.01 at 120, 150 and 180 min compared to LPS group; Fig. 1B). In the presence of compound C, as a selective AMPK inhibitor, the protective effects of metformin on LPS-induced LVDP depression were totally blocked (p b 0.05 at 90 and 120 min, p b 0.01 at 150, and p b 0.001 at 180 min compared to LPS + Met; Fig. 1B). After 90 min, myocardial contractility and relaxation (LV dP/dtmax and LV dP/dtmin) were also depressed in response to LPS perfusion (p b 0.05 at 90 and 120 min, and p b 0.01 at 150 and 180 min; Fig. 2). However, in the presence of metformin, the LPS-elicited decline in contractility and relaxation were markedly enhanced (p b 0.05 at 150 min). Compound C once more counteracted the protective effects of metformin on cardiac contractility and relaxation and reversed the LV dP/dtmax and LV dP/dtmin values close to the values of LPS group (p b 0.05 at 150 and 180 min; Fig. 2).
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3.2. Lactate Level in Cardiac Perfusate Lactate has long been considered as a waste by-product of metabolism in all body tissues; it particularly accumulates at sites of inflammation. In this experiment, we measured the lactate level of the recirculating perfusate at the end of 180 min perfusion time. While control hearts had low amounts of lactate (14.5 mM), LPS-treated hearts had higher levels of lactate by 71% increase compared to control (p b 0.001). Metformin suppressed LPS-mediated lactate release (24.3 ± 1.08 mM compared to 50.22 ± 2.74 mM, p b 0.001), while compound C markedly blocked the inhibitory effects of metformin on lactate production. There was a 49% increase in the lactate level when compound C was administered to LPS + Metformin group (p b 0.001; Fig. 3). 3.3. TLR4 mRNA Level in Cardiac Tissue When compared to the control group, quantification of TLR4 mRNA in the heart tissue showed a significant increase in the expression of the receptor after 180 min of exposure to LPS (7.64 fold, p b 0.001; Fig. 4). This amount was significantly reduced upon introduction of metformin (p b 0.01 compared to LPS); however, compound C substantially reversed the suppressive effects of metformin on LPS-elicited TLR4 expression (p b 0.01 compared to LPS + Met). Treatment with metformin or compound C alone had no significant effect on TLR4 mRNA level in the heart tissue (Fig. 4). Fig. 1. Coronary perfusion pressure (A) and left ventricular developed pressure (B) of different groups during 180 min perfusion period (the values were normalized to initial value). LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). **p b 0.01, ***p b 0.001 from control group; #p b 0.05, ##p b 0.01 from LPS group, + p b 0.05, ++ p b 0.01, +++ p b 0.001 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
3.4. Phosphorylation of AMPKα and ACC in the Heart Tissue In this experiment, to study the effects of metformin on AMPK pathway, we assessed the levels of phosphorylation of both α subunit of AMPK and its target site on acetyl-CoA carboxylase (ACC), Ser79. As shown in Fig. 5, phosphorylation of AMPK and its target site on ACC, Ser79, were not affected by LPS introduction. However, metformin significantly increased phosphorylation of AMPKα and ACC regardless of the presence or absence of LPS (p b 0.001; Fig. 5). However, there was a significant decline in the metformin-induced phosphorylation of AMPKα (p b 0.01, LPS + Met + CC compared to LPS + Met; Fig. 5A) as well as ACC (p b 0.001, LPS + Met + CC compared to LPS + Met; Fig. 5B) in the presense of compound C as an AMPK inhibitor. 3.5. MYD88 and NF-κB Protein Level in Cardiac Tissue To illuminate the intracellular downstream of LPS-induced TLR4 signaling pathway and investigate the effects of metformin on it, we
Fig. 2. Maximal rates of positive and negative changes in LV pressure (LV dP/dtmax; LV dP/dt min ) of different groups during 180 min perfusion period (the values were normalized to initial value). LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). *p b 0.05, **p b 0.01 from control group; # p b 0.01 from LPS group, + p b 0.05 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
Fig. 3. Lactate level in isolated heart perfusate of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎⁎⁎p b 0.001 from control group; ###p b 0.001 from LPS group, +++ p b 0.001 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
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Fig. 4. TLR4 mRNA expression level in isolated heart tissue of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ***p b 0.001 from control group; ##p b 0.01 from LPS group, ++ p b 0.01 from LPS + Met group using Pair Wise Fixed Reallocation Randomization test.
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MyD88 expression in LPS-treated hearts was greatly reversed (p b 0.05, LPS + Met + CC compared to LPS + Met; Fig. 6A). NF-κB-p65 is a subunit of NF-κB that plays a crucial role in inflammatory responses; NF-κB inhibitor also interacts with NF-κB through p65. To evaluate the involvement of NF-κB in TLR4 signaling pathway, we assessed the expression levels of NF-κB using anti-NF-κB p65 antibodies. As shown in Fig. 6B, LPS treatment significantly increased NF-κB expression about 5.5 fold, which was assessed by the measurement of p65 protein expression (p b 0.001). Western-blot analysis showed that the LPS-stimulated increase of NF-κB p65 level was approximately reduced to the control level in the presence of metformin (p b 0.001 compared to LPS group). The effect of metformin in an individual group also was assessed and there was no significant difference in the p65 protein level compared to the control group. The p65 protein level in the group received compound C alone was slightly increased in comparison to the control group (p b 0.05) indicating that inhibition of intrinsic cellular AMPK can induce NF-κB expression. Co-administration compound C with metformin significantly (p b 0.01) abolished suppressive effect of metformin on LPS-induced increase of p65 subunit of NF-κB expression in LPS + Met + CC group (Fig. 6B). 3.6. The Effluent and Cardiac Levels of TNF-α and IL-6
measured the tissue levels of MYD88 in isolated rat hearts after 180 min of LPS perfusion. MyD88 values in metformin or compound C alone treated groups were similar to that of the control group. After LPS perfusion, a 5 fold increase was detected in the myocardial MyD88 level (p b 0.001, LPS compared to control; Fig. 6A). However, LPS-induced MyD88 expression was markedly blocked by administration of metformin (p b 0.001, Met + LPS compared to LPS; Fig. 6A). Interestingly, in the presence of compound C, the metformin-elicited suppression of
Pro-inflammatory cytokines such as TNF-α and IL-6 are the endproducts of TLR4 signaling pathway. To examine the effects of metformin on TLR signaling pathway, we measured the values of TNF-α and IL-6 both in the cardiac effluent and tissue after 180 min. A profound amount of TNF-α and IL-6 was detectable in both cardiac effluent and tissue in response to LPS perfusion (p b 0.001; Fig. 7). Metformin caused a drop in effluent and myocardial TNF-α level, consistent with decreased level of IL-6 (p b 0.01). As expected, the metformin-induced
Fig. 5. AMPKα (A) and ACC (B) phosphorylation level in isolated heart tissue of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎⁎⁎ p b 0.001 from control group; ## p b 0. 01, ### p b 0.001 from LPS group, ++ p b 0. 01, +++ p b 0.001 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
Fig. 6. MYD88 (A) and NF-κB p65 protein (B) levels in isolated heart tissue of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎p b 0.05, ⁎⁎⁎p b 0.001 from control group; ##p b 0.01, ###p b 0.001 from LPS group, + p b 0.05, ++ p b 0.01 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
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decline in cytokine levels was reversed by compound C both in the heart tissue and cardiac effluent (p b 0.01 and p b 0.05 respectively; Fig. 7). 4. Discussion In the present study, we noticed that LPS causes a marked increase in the mRNA level of TLR4 in the isolated rat hearts, despite the elimination of systemic and circulatory immune system. The increase in the tissue level of TLR4 mRNA was associated with a notable myocardial dysfunction, manifested by a reduction in the left ventricular developed pressure and left ventricular contractility. Our data also provided the first evidence that metformin has significant beneficial effects on the left ventricular function through AMPK activation, which is confirmed by the increase in the phosphorylation of α subunit of AMPK and elevation of P-ACC content in the heart tissue. The AMPK activation by metformin suppressed TLR4 downstream by reduction in MyD88, NF-κB, TNFα, and IL6 levels in the isolated heart tissue. Furthermore, the fact that inhibition of AMPK by compound C reversed the cardioprotective and anti-inflammatory effects of metformin, along with the augmentation of TLR4 expression, suggests that metformin suppresses TLR4 expression through AMPK-dependent pathway. Recent in vivo studies of rats and mice, including those investigated in our lab, have shown that metformin exhibits its cardioprotective effects in sepsis through suppression of TLR4 activity [33]. Our previous studies have also demonstrated that an isolated rat heart, independent of systemic and circulatory immune system, is capable of producing TNF-α through TLR4 and MyD88 activation, suggesting a local immune response in the isolated heart [8]. However, the connection between the cardioprotective effects of metformin and AMPK activation was not clearly known. As demonstrated in our study, metformin provoked phosporylation of α subunit of AMPK on Thr172 and hence increased phosphorylated Ser79 on ACC, which is the prototypical AMPK target site on ACC. Since metformin had very little effect on phosphorylation of ACC in the presence of compound C as an AMPK antagonist, we can
Fig. 7. TNF-α (A) and IL-6 (B) level in cardiac effluent and tissue of isolated heart of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎⁎⁎ p b 0.001 from control group; ## p b 0.01 from LPS group, + p b 0.05, ++ p b 0.01 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
conclude that phosphorylation of ACC is primarily mediated by activated AMPK. In this study, we used the isolated rat heart model to eliminate the effects of confounding, pro-inflammatory factors such as circulatory monocytes on TRL4 signaling pathway in the heart. The principal ligand for TLR4 is LPS. The interaction of LPS with TLR4 can induce an immune response either through MyD88-dependent or MyD88-independent pathway. The MyD88 adaptor connects to IRAK and then triggers a critical signaling pathway which culminates in the translocation of NF-κB to the nucleus and transcription of many genes involved in the immune response, including the genes of proinflammatory cytokines such as TNF-α and IL-6 [34]. In this study, we revealed that metformin exerts its anti-inflammatory properties by interfering in all critical steps of LPS-induced innate immunity response, including TLR4 ligation, MyD88 and NF-κB protein expression, and cytokine synthesis. As demonstrated, AMPK activation is indispensable for preserving the anti-inflammatory effects of metformin. Therefore, we can conclude that AMPK serves a critical role in the regulation of myocardial immunity in inflammatory conditions such as sepsis and myocardial infarction. The negative inotropic effect of LPS was evident within ≈90 min of perfusion and it progressed steadily until the end of the experiment, while CPP remained unaffected. The most sensible explanation for this outcome, which is also demonstrated in other studies, is that LPS provokes both the release of thromboxane and prostacyclin; the vasodilatory effects of prostacyclin might antagonize the vasoconstrictor effects of TxA2 [35]. We know that both endothelial and smooth muscle cells of the coronary vasculature and cardiomyocytes are capable of synthesizing cytokines [36]; moreover, LPS depresses contractility of isolated rat hearts by inducing TNF-a synthesis and subsequent activation of the sphingomyelinase pathway [37]. As a consequence, we can assume that LPS exerts its negative inotropic effects by local cardiac synthesis of cytokines; and metformin in part contributes to its cardioprotective effects through impeding cytokine synthesis. Over the inflammatory conditions such as LPS perfusion, while the contractile activity of the heart declines, inhibition of protein synthesis by AMPK preserves ATP for vital cellular functions such as ion transportation. As demonstrated in our study, metformin augmented phosphorylation of ACC which in turn increases mitochondrial beta-oxidation and thereby increases ATP production, contributing to metabolic effects of metformin. The immune system is a complex network of cells and tissues that protect the body against invasive pathogens; it is typically divided into innate and acquired immunity. Cardiac injury activates the innate immunity which in turn triggers an organized inflammatory reaction that results in the healing process and ventricular remodeling. Although cardiac remodeling may initially have some short-term benefits, if left unchecked, it can become maladaptive and lead to heart failure. Recently there has been a great interest in developing therapeutic strategies which aim at reducing the inflammation after myocardial injury. Anti-inflammatory therapies need to be delicately balanced, since either broad suppression of inflammatory response - for example by glucocorticoids- or unbridled inflammation may impair the reparative process. Therefore, we need to develop therapies which regulate specific inflammatory pathways. Accordingly, selectively modulating AMPK-dependent pathway in TRL4 signaling downstream which both suppresses inflammation and preserves energy seems to offer a safe and efficient approach for the treatment of myocardial injuries. Multiple studies have demonstrated that circulatory immune cells like monocytes and neutrophils play a great part in orchestration of inflammatory responses after a cardiac injury; however, the possible role of local innate immune system alone is not clearly illuminated. The results of the present study indicate that TRL4 signaling pathway of local cardiac immunity plays a prominent role in the development of inflammatory reactions after a cardiac insult. Furthermore, metforminmediated AMPK activation bestows cardioprotective effects through blocking TRL4 pathway in inflammatory conditions. Our study is novel since it is the first to demonstrate that metformin exerts its
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cardiprotective effects through AMPK-dependent mechanism in TLR4 signaling pathway in isolated rat heart model. In the present study our focus was mainly on the cardioprotective effects of metformin in septic conditions; however, its pharmacologic use can extend beyond and into the protection of other solid organs (i.e. liver, kidney) against septic shock injuries. Conflict of interests The authors declare no conflict of interests. Acknowledgment This study was supported by a grant from the Research Vice Chancellors of Tabriz University of Medical Sciences, Tabriz, Iran. The authors are very grateful to Research Center for Pharmaceutical Nanotechnology (RCPN) at Tabriz University of Medical Sciences, for providing RT-PCR instruments in qPCR analysis. This article was written based on a data set of Haleh Vaez Ph.D. thesis registered in Tabriz University of Medical Sciences (NO. 90). References [1] D.J. Marchant, J.H. Boyd, D.C. Lin, D.J. Granville, F.S. Garmaroudi, B.M. McManus, Inflammation in myocardial diseases, Circ. Res. 110 (2012) 126–144. [2] J.H. Boyd, S. Mathur, Y. Wang, R.M. Bateman, K.R. Walley, Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response, Cardiovasc. Res. 72 (2006) 384–393. [3] A. Linde, D. Mosier, F. Blecha, T. Melgarejo, Innate immunity and inflammation–new frontiers in comparative cardiovascular pathology, Cardiovasc. Res. 73 (2007) 26–36. [4] S. Nemoto, J.G. Vallejo, P. Knuefermann, A. Misra, G. Defreitas, B.A. Carabello, et al., Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart, Am. J. Physiol. Heart Circ. Physiol. 282 (2002) H2316–H2323. [5] J.A. Thomas, S.B. Haudek, T. Koroglu, M.F. Tsen, D.D. Bryant, D.J. White, et al., IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction, Am. J. Physiol. Heart Circ. Physiol. 285 (2003) H597–H606. [6] A.J. Chong, A. Shimamoto, C.R. Hampton, H. Takayama, D.J. Spring, C.L. Rothnie, et al., Toll-like receptor 4 mediates ischemia/reperfusion injury of the heart, J. Thorac. Cardiovasc. Surg. 128 (2004) 170–179. [7] S.C. Kim, A. Ghanem, H. Stapel, K. Tiemann, P. Knuefermann, A. Hoeft, et al., Toll-like receptor 4 deficiency: smaller infarcts, but no gain in function, BMC Physiol. 7 (2007) 5. [8] M. Rameshrad, N. Maleki-Dizaji, H. Vaez, H. Soraya, A. Nakhlband, A. Garjani, Lipopolysaccharide induced activation of toll like receptor 4 in isolated rat heart suggests a local immune response in myocardium, Iran. J. Immunol. 12 (2015) 104–116. [9] M. Rameshrad, H. Soraya, N. Maleki-Dizaji, H. Vaez, A. Garjani, A769662, a direct AMPK activator, attenuates lipopolysaccharideinduced acute heart and lung inflammation in rats, Mol. Med. Rep. (2016). [10] A. Kelkar, A. Kuo, W.H. Frishman, Allopurinol as a cardiovascular drug, Cardiol. Rev. 19 (2011) 265–271. [11] S. Deftereos, G. Giannopoulos, N. Papoutsidakis, V. Panagopoulou, C. Kossyvakis, K. Raisakis, et al., Colchicine and the heart: pushing the envelope, J. Am. Coll. Cardiol. 62 (2013) 1817–1825. [12] P.M. Ridker, T.F. Luscher, Anti-inflammatory therapies for cardiovascular disease, Eur. Heart J. 35 (2014) 1782–1791. [13] P.M. Ridker, Targeting inflammatory pathways for the treatment of cardiovascular disease, Eur. Heart J. 35 (2014) 540–543. [14] D.A. Escobar, A.M. Botero-Quintero, B.C. Kautza, J. Luciano, P. Loughran, S. Darwiche, et al., Adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation, J. Surg. Res. 194 (2015) 262–272.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
AMPK activation by metformin inhibits local innate immune responses in the isolated rat heart by suppression of TLR 4-related pathway Haleh Vaez a,b, Moslem Najafi a, Maryam Rameshrad a, Negisa Seyed Toutounchi b, Mehraveh Garjani c, Jaleh Barar d, Alireza Garjani a,e,⁎ a
Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran d Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran e Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c
a r t i c l e
i n f o
Article history: Received 2 July 2016 Received in revised form 14 September 2016 Accepted 1 October 2016 Available online xxxx Keywords: Heart Metformin Toll-like receptor 4 AMPK NF-κB
a b s t r a c t Toll like receptors (TLRs) are key players in the innate immune responses. The energy sensing enzyme, AMPK, has been implicated in the modulation of immunity. The present study investigated whether AMPK activation by metformin could contribute to the regulation of immune responses in the isolated heart via suppression of TLR4 activity, independent of circulatory immunity. Isolated Wistar rat hearts were perfused with KrebsHenseleit buffer in the absence or presence of lipopolysaccharide (LPS; 0.2 μM), LPS + metformin (10 mM), and LPS + metformin + compound C (10 μM). Following measurement of hemodynamic parameters, TLR4activation related changes and TLR4 mRNA level in the heart was examined by western blotting and real-time PCR. The activation of AMPK was evaluated by measuring the ratio of p-AMPKα and p-ACC to their nonphosphorylated forms. The effluent and cardiac levels of TNF-α and IL6 were assayed by ELISA. LPS profoundly increased the levels of TLR4 mRNA, MyD88 (TLR4 adaptor protein), and NF-κB and also the release of TNF-α and IL6 from the heart. The enhancement in the TLR4 activity was associated with a significant depression of myocardial function. Metformin clearly augmented the phosphorylation of both AMPKα and ACC and in addition to improvement of cardiac performance, markedly suppressed the TLR4 activity. Antagonizing AMPK by compound C which is a selective inhibitor of AMPK pathway, considerably reversed the protective effects of metformin against the TLR4-related activity. The results of the study demonstrated the importance of TLR4-involved local immune responses in the LPS-induced myocardial dysfunction and indicated a clear link between AMPK and TLR4. © 2016 Published by Elsevier B.V.
1. Introduction The innate immune system has been implicated in a wide range of pathological conditions including sepsis and cardiovascular diseases. Toll-like receptors (TLRs) are a class of membrane bound receptors on eukaryotic cells that play a key role in the innate immunity. They are mainly expressed on sentinel cells such as macrophages and dendritic cells that recognize pathogen-associated molecular patterns (PAMPs). A classic example of pathogen-associated molecular pattern includes lipopolysaccharides (LPS) of gram-negative bacteria. Moreover, various endogenous ligands, called danger-associated molecular patterns (DAMPs), which are released in a variety of cardiovascular diseases such as sepsisinduced myocardial dysfunction, myocardial infarction, heart failure, ⁎ Corresponding author at: Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. E-mail address: [email protected] (A. Garjani).
http://dx.doi.org/10.1016/j.intimp.2016.10.002 1567-5769/© 2016 Published by Elsevier B.V.
and ischemia/reperfusion also activate TLRs [1]. Among all TLRs expressed in the heart, TLR2 and TLR4 are the most investigated. It has been demonstrated that activation of TLR2 and TLR4 by DAMPs and PAMPs leads to translocation of p65 subunit of nuclear factor kappa B (NF-κB) into the nucleus and hence induces the synthesis of TNF-α [2]. Myeloid differentiation factor 88 (MyD88) is the main adaptor protein employed in all TLR pathways except for TLR3. MyD88 recruits the interleukin (IL)-1 receptor associated kinases (IRAK), and in turn triggers the downstream of multiple signaling cascades. It activates the inhibitory κB kinase complex which directly phosphorylates IκBα, leading to nuclear translocation of the p65 component of the NF-κB complex and expression of target genes of proinflammatory cytokines (e.g. IL6 and TNF-α), cell adhesion molecules, and chemokines that recruit macrophages and neutrophils to the tissue [3]. Studies have shown that mice deficient of either TLR4 or interleukin (IL)-1 receptor associated kinase 1 (IRAK1) are preserved from the LPSinduced mortality and cardiac dysfunction [4,5]. Besides, TLR-mediated signaling contributes to myocardial damage and adverse cardiac
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remodeling following ischemia/reperfusion injury and myocardial infarction [6,7]. Recently, we published a study that claimed TLR4 is expressed in the intact and isolated heart tissues and when ligated by LPS, TNF-α release and myocardial dysfunction ensues [8,9]. The NF-κB-dependent pro-inflammatory cytokine release, following the TLR4 stimulation, has prominent role in the pathophysiology of cardiac function modification in sepsis. It is proposed that immunosuppressive and anti-inflammatory therapeutic strategies in cardiovascular disease, specifically in sepsisinduced myocardial dysfunction, can have beneficial effects on the outcomes. Inhibitors of specific inflammatory pathways are already either in use or under development; these include both classic drugs such as allopurinol, colchicine, and methotrexate and biologic therapies like TNF-α antibodies and IL-1β antagonists [10–12]. However, in contrast to experimental animal studies, targeting the innate immune system and specific inflammatory pathways for the treatment of cardiovascular diseases were not always promising in human [13]. Moreover, selective modulation of local cardiac immunity in the pathological conditions has not been studied yet. The existing challenges over utilization of anti-inflammatory therapies in cardiovascular disease make the search for the effective inflammatory modulators intriguing. AMP activated protein kinase (AMPK) is a multifaceted enzyme that acts as the sensor of intracellular energy; it can activate ATP-generating catabolic or inactivate ATP-consuming anabolic pathways. In other words, AMPK conserves energy in metabolic stresses and thus protects the cells in various pathological conditions. AMPK also regulates critical cellular processes including transcription and protein synthesis [14]. It is believed that AMPK is an important regulator of inflammatory responses in immune cells; and anti-inflammatory effects of some agents might actually be due to the activation of AMPK [15]. For instance, adiponectin inhibits LPS-activated TLR4 signaling pathways in hepatic Kupffer cells and in macrophages through mechanisms involving AMPK [16]. A study has demonstrated that AMPK antagonizes TLR-induced maturation of dendritic cells, which are the key regulators of innate and acquired immunity [17]. Our previous studies showed that both chronic and short term treatments with metformins which is a best known activator of AMPK, attenuated left ventricular dysfunction induced by myocardial infarction and alleviates myocardial injuries [18,19]. Clinical and experimental studies suggest that metformin, besides its antidiabetic effects, can exert systemic and local anti-inflammatory effects through mechanisms that are not clearly elucidated [20,21]. It was reported that metformin by decreasing inflammatory markers such as soluble intercellular adhesion molecule, vascular cell adhesion molecule-1, macrophage migration inhibitory factor, and C-reactive protein (CRP) can modulate inflammation [21–23]. Furthermore, it was demonstrated that metformin suppressed NF-κB activation in colitis-associated studies [24]. The best known explanation for the anti-inflammatory properties of metformin is AMPK activation [25]; however, AMPK-independent pathways are now considered to play a part too [26,27]. It has been hypothesized that AMPK activation could improve cardiac function following LPS-induced myocardial dysfunction through inhibiting TLR4 activity in the heart tissue independent of circulatory immune system. Therefore, in the present study, we investigated the role of AMPK activation by metformin on LPS-induced myocardial dysfunction and on TLR4 signaling pathway in the isolated rat hearts. Further, to confirm the effect of AMPK in this inflammatory cascade, we used compound C, which is a selective AMPK inhibitor. 2. Materials and Methods 2.1. Animals Male Wistar rats (260–290 g) were supplied by Laboratory Animal Center, Tabriz University of Medical Sciences, Iran. Animals 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 according to the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz, Iran which is in accordance with the National Institutes of Health guidelines (revised 2011) and was approved by the local authorities of Animal Ethics Committees (AEC references number: TBZMED. REC. 1394.721). 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, acetyl-CoA carboxylase (ACC), phospho-ACC and MyD88 were obtained from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibodies against glyceraldehyde phosphate dehydrogenase (GAPDH), NF-kB, β-actin, peroxidase-conjugated goat antirabbit and rabbit anti-mouse secondary antibodies were obtained from Abcam (Cambridge, MA). ELISA kit for TNF-α and IL-6 were from Bender MedSystems Inc. (Vienna, Austria). 2.3. Isolated Heart Perfusion The Langendorff technique of isolated heart perfusion has been described in detail in reference [8]. In short, animals were heparinized (1000 IU/kg) and anesthetized using a cocktail of ketamine (60 mg/kg), xylazine (10 mg/kg), and acepromazine (10 mg/kg) given intraperitoneally. When the rats no longer responded to external stimuli, their hearts were rapidly excised via bilateral thoracotomy and immersed in icecold modified Krebs-Henseleit buffer solution (KHBS). The harvested hearts were immediately cannulated to a langendorff apparatus (AD Instruments; Australia) and then were perfused through the ascending aorta at a constant flow (10 ml/min/g) with KHBS gassed with carbogen (5% CO2/95% O2). The pH was 7.38–7.56 at 37 °C. All hearts were initially rinsed with KHBS for 15 min in a non-recirculating mode before switching to recirculation (total volume of 100 ml). For monitoring coronary perfusion pressure (CPP), the aortic cannula was connected to a pressure transducer (MLT844 physiological pressure, AD Instruments; Australia). Left ventricular pressure was measured isovolumetrically by inserting a home-made latex balloon attached to a pressure transducer into the left ventricular cavity via the mitral valve after removing the atrial appendage. Left ventricular developed pressure (LVDP) was calculated as the difference between peak-systolic and end-diastolic pressure. The maximum and minimum rates of left ventricular pressure (dP/dtmax, dp/dtmin) as indices of left ventricular contractility and relaxation, and heart rate (HR) were monitored by PowerLab 8/35. 2.4. Experimental Protocol After the hearts had been stabilized, time was set to zero, and hearts were perfused in recirculating mode with 100 ml of assigned perfusates according to their experimental group for 180 min. Animals were allocated to six different experimental groups and the hearts of each group received a different treatment as below: (1) KHBS solution (control group), (2) LPS: KHBS solution containing LPS (0.2 μM), (3) LPS + Met: KHBS solution containing LPS (0.2 μM) + metformin (10 mM), (4) Met: KHBS solution containing metformin (10 mM), (5) CC: KHBS solution containing compound C (10 μM), (6) LPS + Met + CC: KHBS solution containing LPS (0.2 μM) + metformin (10 mM) + compound C (10 μM). Hemodynamic variables were monitored for the whole period of experiment. Perfusate samples were taken at the end of perfusion time
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and analyzed with enzymatic/colorimetric method by COBAS Integra 400 analyzer (Roche, Germany) to determine the lactate concentration [28]. 2.5. Quantitative Analysis of TLR-4 mRNA in Cardiac Tissue Total RNA was isolated from cryosections of the left ventricle 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 microgram of each extracted RNA sample was used for cDNA synthesis. Reverse transcriptase PCR was performed with random hexamer primer and M-Mel Reverse Transcriptase (Sialon, 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 (300 nM each primer), 10 μl 2× 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 s, annealing temperature (AT) for 30 s, and 72 °C for 25 s. For quantification, the target gene was normalized to the internal standard gene of β-actin. The PCR primers were designed as given below: For TLR4 (AT 60 °C): sense primer (5′-AAGTTATTGTGGTGGTGTCTAG-3′) antisense primer (5′-GAGGTAGGTGTTTCTGCTAAG-3′) For β-actin (AT 58 °C): sense primer (5′-GCT ACA GCT TCA CCA CCA CA-3′) antisense primer (5′-ATC GTA CTC CTG CTT GCT GA-3′) Interpretation of the result was performed using the Pfaffle Method [29]. 2.6. Western Blot Analysis Western blot analysis were performed according to assay described by Omar et al. [30] and Kewalramani et al. [31] 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 and the supernatant was collected, and the protein concentration was determined using the Bradford Protein Assay kit [32]. 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). The membrane was blocked in washing buffer with 5% nonfat milk for 2 h and incubated overnight with the corresponding primary antibodies at 4 °C. They were washed with a mixture of Tris-Buffered saline and Tween 20 solution (TBST) and incubated at rotator for 60 min with peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse secondary antibodies (1:5000 dilution). Subsequent to washing, the signals of detected proteins were visualized using the BM Chemiluminescence kit (Roche, Mannheim, Germany). The molecular weights of phosphoAMPKα, phospho-ACC, MyD88 and NF-κB 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 and ACC were normalized to non-phosphorylated AMPKα and ACC
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respectively. In the case of MyD88 the values were normalized by comparison to GAPDH and NF-κB were normalized to internal standard protein of β-actin. 2.7. Cytokine Assays The effluent and cardiac levels of TNF-α and IL-6 were assayed using rat enzyme-linked immunosorbent assay (ELISA) kits (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% TXT100 (v/v), and protease inhibitor cocktail (Roche, Mannheim, Germany). The samples were then centrifuged twice at 10,600 ×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 cytokines was expressed as pg/100 mg heart tissue or pg/ml of effluent.
2.8. Statistical Analysis Data are expressed as mean ± SEM. Differences between data sets were assessed by one-way analysis of variance followed by Tukey post-hoc test. For the real time PCR, Pair Wise Fixed Reallocation Randomization test using REST software was used to make comparisons between the groups. p b 0.05 was accepted as statistically significant. 3. Results 3.1. Cardiac Function and Hemodynamic Factors We evaluated cardiac function by continuous monitoring of hemodynamic parameters including coronary perfusion pressure (CPP), left ventricular developed pressure (LVDP), and left ventricular contractility (dP/dtmax) and relaxation (dp/dtmin) at baseline and every 30 min of perfusion period. There was no significant difference between the groups at stabilization time. To assess the possible effect of metformin and compound C alone on cardiac function, all hemodynamic parameters were also determined for these two groups. Considering the data comparison related to cardiac function including CPP, LVDP, dP/dtmax and dp/dtmin, there was no significant difference between metformin and compound C group compared to the control group (p b 0.05; data are not shown). LPS consistently caused a rise in CPP throughout the experiments; however, there was no significant difference between the mean values of CPP in groups perfused with LPS (Fig. 1A). Although left ventricular developed pressure was stable within the first 30 min of LPS perfusion, a sharp consistent decline of LVDP occurred at 60 min of perfusion and afterwards (p b 0.01 at 60 min, and p b 0.001 at 90, 120, 150 and 180 min; Fig. 1B). Metformin profoundly nullified the suppressive effects of LPS on LVDP at all-time points (p b 0.05 at 90 min, and p b 0.01 at 120, 150 and 180 min compared to LPS group; Fig. 1B). In the presence of compound C, as a selective AMPK inhibitor, the protective effects of metformin on LPS-induced LVDP depression were totally blocked (p b 0.05 at 90 and 120 min, p b 0.01 at 150, and p b 0.001 at 180 min compared to LPS + Met; Fig. 1B). After 90 min, myocardial contractility and relaxation (LV dP/dtmax and LV dP/dtmin) were also depressed in response to LPS perfusion (p b 0.05 at 90 and 120 min, and p b 0.01 at 150 and 180 min; Fig. 2). However, in the presence of metformin, the LPS-elicited decline in contractility and relaxation were markedly enhanced (p b 0.05 at 150 min). Compound C once more counteracted the protective effects of metformin on cardiac contractility and relaxation and reversed the LV dP/dtmax and LV dP/dtmin values close to the values of LPS group (p b 0.05 at 150 and 180 min; Fig. 2).
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3.2. Lactate Level in Cardiac Perfusate Lactate has long been considered as a waste by-product of metabolism in all body tissues; it particularly accumulates at sites of inflammation. In this experiment, we measured the lactate level of the recirculating perfusate at the end of 180 min perfusion time. While control hearts had low amounts of lactate (14.5 mM), LPS-treated hearts had higher levels of lactate by 71% increase compared to control (p b 0.001). Metformin suppressed LPS-mediated lactate release (24.3 ± 1.08 mM compared to 50.22 ± 2.74 mM, p b 0.001), while compound C markedly blocked the inhibitory effects of metformin on lactate production. There was a 49% increase in the lactate level when compound C was administered to LPS + Metformin group (p b 0.001; Fig. 3). 3.3. TLR4 mRNA Level in Cardiac Tissue When compared to the control group, quantification of TLR4 mRNA in the heart tissue showed a significant increase in the expression of the receptor after 180 min of exposure to LPS (7.64 fold, p b 0.001; Fig. 4). This amount was significantly reduced upon introduction of metformin (p b 0.01 compared to LPS); however, compound C substantially reversed the suppressive effects of metformin on LPS-elicited TLR4 expression (p b 0.01 compared to LPS + Met). Treatment with metformin or compound C alone had no significant effect on TLR4 mRNA level in the heart tissue (Fig. 4). Fig. 1. Coronary perfusion pressure (A) and left ventricular developed pressure (B) of different groups during 180 min perfusion period (the values were normalized to initial value). LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). **p b 0.01, ***p b 0.001 from control group; #p b 0.05, ##p b 0.01 from LPS group, + p b 0.05, ++ p b 0.01, +++ p b 0.001 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
3.4. Phosphorylation of AMPKα and ACC in the Heart Tissue In this experiment, to study the effects of metformin on AMPK pathway, we assessed the levels of phosphorylation of both α subunit of AMPK and its target site on acetyl-CoA carboxylase (ACC), Ser79. As shown in Fig. 5, phosphorylation of AMPK and its target site on ACC, Ser79, were not affected by LPS introduction. However, metformin significantly increased phosphorylation of AMPKα and ACC regardless of the presence or absence of LPS (p b 0.001; Fig. 5). However, there was a significant decline in the metformin-induced phosphorylation of AMPKα (p b 0.01, LPS + Met + CC compared to LPS + Met; Fig. 5A) as well as ACC (p b 0.001, LPS + Met + CC compared to LPS + Met; Fig. 5B) in the presense of compound C as an AMPK inhibitor. 3.5. MYD88 and NF-κB Protein Level in Cardiac Tissue To illuminate the intracellular downstream of LPS-induced TLR4 signaling pathway and investigate the effects of metformin on it, we
Fig. 2. Maximal rates of positive and negative changes in LV pressure (LV dP/dtmax; LV dP/dt min ) of different groups during 180 min perfusion period (the values were normalized to initial value). LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). *p b 0.05, **p b 0.01 from control group; # p b 0.01 from LPS group, + p b 0.05 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
Fig. 3. Lactate level in isolated heart perfusate of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎⁎⁎p b 0.001 from control group; ###p b 0.001 from LPS group, +++ p b 0.001 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
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Fig. 4. TLR4 mRNA expression level in isolated heart tissue of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ***p b 0.001 from control group; ##p b 0.01 from LPS group, ++ p b 0.01 from LPS + Met group using Pair Wise Fixed Reallocation Randomization test.
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MyD88 expression in LPS-treated hearts was greatly reversed (p b 0.05, LPS + Met + CC compared to LPS + Met; Fig. 6A). NF-κB-p65 is a subunit of NF-κB that plays a crucial role in inflammatory responses; NF-κB inhibitor also interacts with NF-κB through p65. To evaluate the involvement of NF-κB in TLR4 signaling pathway, we assessed the expression levels of NF-κB using anti-NF-κB p65 antibodies. As shown in Fig. 6B, LPS treatment significantly increased NF-κB expression about 5.5 fold, which was assessed by the measurement of p65 protein expression (p b 0.001). Western-blot analysis showed that the LPS-stimulated increase of NF-κB p65 level was approximately reduced to the control level in the presence of metformin (p b 0.001 compared to LPS group). The effect of metformin in an individual group also was assessed and there was no significant difference in the p65 protein level compared to the control group. The p65 protein level in the group received compound C alone was slightly increased in comparison to the control group (p b 0.05) indicating that inhibition of intrinsic cellular AMPK can induce NF-κB expression. Co-administration compound C with metformin significantly (p b 0.01) abolished suppressive effect of metformin on LPS-induced increase of p65 subunit of NF-κB expression in LPS + Met + CC group (Fig. 6B). 3.6. The Effluent and Cardiac Levels of TNF-α and IL-6
measured the tissue levels of MYD88 in isolated rat hearts after 180 min of LPS perfusion. MyD88 values in metformin or compound C alone treated groups were similar to that of the control group. After LPS perfusion, a 5 fold increase was detected in the myocardial MyD88 level (p b 0.001, LPS compared to control; Fig. 6A). However, LPS-induced MyD88 expression was markedly blocked by administration of metformin (p b 0.001, Met + LPS compared to LPS; Fig. 6A). Interestingly, in the presence of compound C, the metformin-elicited suppression of
Pro-inflammatory cytokines such as TNF-α and IL-6 are the endproducts of TLR4 signaling pathway. To examine the effects of metformin on TLR signaling pathway, we measured the values of TNF-α and IL-6 both in the cardiac effluent and tissue after 180 min. A profound amount of TNF-α and IL-6 was detectable in both cardiac effluent and tissue in response to LPS perfusion (p b 0.001; Fig. 7). Metformin caused a drop in effluent and myocardial TNF-α level, consistent with decreased level of IL-6 (p b 0.01). As expected, the metformin-induced
Fig. 5. AMPKα (A) and ACC (B) phosphorylation level in isolated heart tissue of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎⁎⁎ p b 0.001 from control group; ## p b 0. 01, ### p b 0.001 from LPS group, ++ p b 0. 01, +++ p b 0.001 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
Fig. 6. MYD88 (A) and NF-κB p65 protein (B) levels in isolated heart tissue of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎p b 0.05, ⁎⁎⁎p b 0.001 from control group; ##p b 0.01, ###p b 0.001 from LPS group, + p b 0.05, ++ p b 0.01 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
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decline in cytokine levels was reversed by compound C both in the heart tissue and cardiac effluent (p b 0.01 and p b 0.05 respectively; Fig. 7). 4. Discussion In the present study, we noticed that LPS causes a marked increase in the mRNA level of TLR4 in the isolated rat hearts, despite the elimination of systemic and circulatory immune system. The increase in the tissue level of TLR4 mRNA was associated with a notable myocardial dysfunction, manifested by a reduction in the left ventricular developed pressure and left ventricular contractility. Our data also provided the first evidence that metformin has significant beneficial effects on the left ventricular function through AMPK activation, which is confirmed by the increase in the phosphorylation of α subunit of AMPK and elevation of P-ACC content in the heart tissue. The AMPK activation by metformin suppressed TLR4 downstream by reduction in MyD88, NF-κB, TNFα, and IL6 levels in the isolated heart tissue. Furthermore, the fact that inhibition of AMPK by compound C reversed the cardioprotective and anti-inflammatory effects of metformin, along with the augmentation of TLR4 expression, suggests that metformin suppresses TLR4 expression through AMPK-dependent pathway. Recent in vivo studies of rats and mice, including those investigated in our lab, have shown that metformin exhibits its cardioprotective effects in sepsis through suppression of TLR4 activity [33]. Our previous studies have also demonstrated that an isolated rat heart, independent of systemic and circulatory immune system, is capable of producing TNF-α through TLR4 and MyD88 activation, suggesting a local immune response in the isolated heart [8]. However, the connection between the cardioprotective effects of metformin and AMPK activation was not clearly known. As demonstrated in our study, metformin provoked phosporylation of α subunit of AMPK on Thr172 and hence increased phosphorylated Ser79 on ACC, which is the prototypical AMPK target site on ACC. Since metformin had very little effect on phosphorylation of ACC in the presence of compound C as an AMPK antagonist, we can
Fig. 7. TNF-α (A) and IL-6 (B) level in cardiac effluent and tissue of isolated heart of different groups at the end of 180 min perfusion period. LPS: Lipopolysaccharide (0.2 μM); Met: metformin (10 mM); CC: compound C (10 μM). Results are presented as mean ± SEM (n = 6). ⁎⁎⁎ p b 0.001 from control group; ## p b 0.01 from LPS group, + p b 0.05, ++ p b 0.01 from LPS + Met group using one way ANOVA with TUKEY post-hoc test.
conclude that phosphorylation of ACC is primarily mediated by activated AMPK. In this study, we used the isolated rat heart model to eliminate the effects of confounding, pro-inflammatory factors such as circulatory monocytes on TRL4 signaling pathway in the heart. The principal ligand for TLR4 is LPS. The interaction of LPS with TLR4 can induce an immune response either through MyD88-dependent or MyD88-independent pathway. The MyD88 adaptor connects to IRAK and then triggers a critical signaling pathway which culminates in the translocation of NF-κB to the nucleus and transcription of many genes involved in the immune response, including the genes of proinflammatory cytokines such as TNF-α and IL-6 [34]. In this study, we revealed that metformin exerts its anti-inflammatory properties by interfering in all critical steps of LPS-induced innate immunity response, including TLR4 ligation, MyD88 and NF-κB protein expression, and cytokine synthesis. As demonstrated, AMPK activation is indispensable for preserving the anti-inflammatory effects of metformin. Therefore, we can conclude that AMPK serves a critical role in the regulation of myocardial immunity in inflammatory conditions such as sepsis and myocardial infarction. The negative inotropic effect of LPS was evident within ≈90 min of perfusion and it progressed steadily until the end of the experiment, while CPP remained unaffected. The most sensible explanation for this outcome, which is also demonstrated in other studies, is that LPS provokes both the release of thromboxane and prostacyclin; the vasodilatory effects of prostacyclin might antagonize the vasoconstrictor effects of TxA2 [35]. We know that both endothelial and smooth muscle cells of the coronary vasculature and cardiomyocytes are capable of synthesizing cytokines [36]; moreover, LPS depresses contractility of isolated rat hearts by inducing TNF-a synthesis and subsequent activation of the sphingomyelinase pathway [37]. As a consequence, we can assume that LPS exerts its negative inotropic effects by local cardiac synthesis of cytokines; and metformin in part contributes to its cardioprotective effects through impeding cytokine synthesis. Over the inflammatory conditions such as LPS perfusion, while the contractile activity of the heart declines, inhibition of protein synthesis by AMPK preserves ATP for vital cellular functions such as ion transportation. As demonstrated in our study, metformin augmented phosphorylation of ACC which in turn increases mitochondrial beta-oxidation and thereby increases ATP production, contributing to metabolic effects of metformin. The immune system is a complex network of cells and tissues that protect the body against invasive pathogens; it is typically divided into innate and acquired immunity. Cardiac injury activates the innate immunity which in turn triggers an organized inflammatory reaction that results in the healing process and ventricular remodeling. Although cardiac remodeling may initially have some short-term benefits, if left unchecked, it can become maladaptive and lead to heart failure. Recently there has been a great interest in developing therapeutic strategies which aim at reducing the inflammation after myocardial injury. Anti-inflammatory therapies need to be delicately balanced, since either broad suppression of inflammatory response - for example by glucocorticoids- or unbridled inflammation may impair the reparative process. Therefore, we need to develop therapies which regulate specific inflammatory pathways. Accordingly, selectively modulating AMPK-dependent pathway in TRL4 signaling downstream which both suppresses inflammation and preserves energy seems to offer a safe and efficient approach for the treatment of myocardial injuries. Multiple studies have demonstrated that circulatory immune cells like monocytes and neutrophils play a great part in orchestration of inflammatory responses after a cardiac injury; however, the possible role of local innate immune system alone is not clearly illuminated. The results of the present study indicate that TRL4 signaling pathway of local cardiac immunity plays a prominent role in the development of inflammatory reactions after a cardiac insult. Furthermore, metforminmediated AMPK activation bestows cardioprotective effects through blocking TRL4 pathway in inflammatory conditions. Our study is novel since it is the first to demonstrate that metformin exerts its
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cardiprotective effects through AMPK-dependent mechanism in TLR4 signaling pathway in isolated rat heart model. In the present study our focus was mainly on the cardioprotective effects of metformin in septic conditions; however, its pharmacologic use can extend beyond and into the protection of other solid organs (i.e. liver, kidney) against septic shock injuries. Conflict of interests The authors declare no conflict of interests. Acknowledgment This study was supported by a grant from the Research Vice Chancellors of Tabriz University of Medical Sciences, Tabriz, Iran. The authors are very grateful to Research Center for Pharmaceutical Nanotechnology (RCPN) at Tabriz University of Medical Sciences, for providing RT-PCR instruments in qPCR analysis. This article was written based on a data set of Haleh Vaez Ph.D. thesis registered in Tabriz University of Medical Sciences (NO. 90). References [1] D.J. Marchant, J.H. Boyd, D.C. Lin, D.J. Granville, F.S. Garmaroudi, B.M. McManus, Inflammation in myocardial diseases, Circ. Res. 110 (2012) 126–144. [2] J.H. Boyd, S. Mathur, Y. Wang, R.M. Bateman, K.R. Walley, Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response, Cardiovasc. Res. 72 (2006) 384–393. [3] A. Linde, D. Mosier, F. Blecha, T. Melgarejo, Innate immunity and inflammation–new frontiers in comparative cardiovascular pathology, Cardiovasc. Res. 73 (2007) 26–36. [4] S. Nemoto, J.G. Vallejo, P. Knuefermann, A. Misra, G. Defreitas, B.A. Carabello, et al., Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart, Am. J. Physiol. Heart Circ. Physiol. 282 (2002) H2316–H2323. [5] J.A. Thomas, S.B. Haudek, T. Koroglu, M.F. Tsen, D.D. Bryant, D.J. White, et al., IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction, Am. J. Physiol. Heart Circ. Physiol. 285 (2003) H597–H606. [6] A.J. Chong, A. Shimamoto, C.R. Hampton, H. Takayama, D.J. Spring, C.L. Rothnie, et al., Toll-like receptor 4 mediates ischemia/reperfusion injury of the heart, J. Thorac. Cardiovasc. Surg. 128 (2004) 170–179. [7] S.C. Kim, A. Ghanem, H. Stapel, K. Tiemann, P. Knuefermann, A. Hoeft, et al., Toll-like receptor 4 deficiency: smaller infarcts, but no gain in function, BMC Physiol. 7 (2007) 5. [8] M. Rameshrad, N. Maleki-Dizaji, H. Vaez, H. Soraya, A. Nakhlband, A. Garjani, Lipopolysaccharide induced activation of toll like receptor 4 in isolated rat heart suggests a local immune response in myocardium, Iran. J. Immunol. 12 (2015) 104–116. [9] M. Rameshrad, H. Soraya, N. Maleki-Dizaji, H. Vaez, A. Garjani, A769662, a direct AMPK activator, attenuates lipopolysaccharideinduced acute heart and lung inflammation in rats, Mol. Med. Rep. (2016). [10] A. Kelkar, A. Kuo, W.H. Frishman, Allopurinol as a cardiovascular drug, Cardiol. Rev. 19 (2011) 265–271. [11] S. Deftereos, G. Giannopoulos, N. Papoutsidakis, V. Panagopoulou, C. Kossyvakis, K. Raisakis, et al., Colchicine and the heart: pushing the envelope, J. Am. Coll. Cardiol. 62 (2013) 1817–1825. [12] P.M. Ridker, T.F. Luscher, Anti-inflammatory therapies for cardiovascular disease, Eur. Heart J. 35 (2014) 1782–1791. [13] P.M. Ridker, Targeting inflammatory pathways for the treatment of cardiovascular disease, Eur. Heart J. 35 (2014) 540–543. [14] D.A. Escobar, A.M. Botero-Quintero, B.C. Kautza, J. Luciano, P. Loughran, S. Darwiche, et al., Adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation, J. Surg. Res. 194 (2015) 262–272.
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