Cadmium (Cd) is one of the most common toxic heavy metals that have severe cardiotoxic hazards especially on excessive exposure during cigarette smoking or food and water contamination. Cd has a great ability to accumulate in our bodies for very long period [1
]. Several mechanisms mediate Cd cardiotoxicity such as induction of oxidative stress with release of free radicals, metabolic disturbance and dyslipidemia. Cardiotoxicity of Cd leads to atherosclerosis, coronary spasm, ischemic injury, myocardial infarction, hypertension, and stroke [1
]. Uncontrolled and excessive formation of free radicals is the main cause that stimulates oxidative stress and disturbs cellular function leading to membrane lipid peroxidation, DNA damage, and cell death. Furthermore, heart cells are highly sensitive to the released reactive oxygen species (ROS) and the anti-oxidative system of these cells is fully saturated by the endogenous oxidative metabolic products [1
Up-regulation of the different inflammatory pathways is considered as another important cascade in mediating Cd cardiotoxicity. Stimulation of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα), interleukin6 (IL6) and nuclear factor kappa B (NFκB) is accompanied with enhancement of Janus kinase/signal transducer and activator of transcription (JAK/STAT) which is a critical pathway during cardiac injury [3
]. STATs are considered as a family of protein that could regulate gene expression of angiogenesis, inflammation and apoptosis. This family is pre-stimulated with IL-6 and mediates different cardiac disorders as hypertrophy, myocardial infarction and heart failure [6
]. Besides that, toll-like receptors (TLRs) control innate immunity and inflammation inside the cell and increase the gene expression of different pro-inflammatory cytokines such as TNFα and interleukins that have a critical role in myocardial tissue injuries. We can conclude that modulation of the TLR2/TNFα and IL6 /STAT pathways represents essential targets in controlling cardiac damage [9
Sodium glucose co-transporter 2 (SGLT2) inhibitors including dapagliflozin (DAP) are the most recent oral anti-diabetic drug group. Its action is based on decreasing blood glucose level and inhibiting renal glucose reabsorption. Furthermore, DAP has non-glycemic effects including lowering of blood pressure, ability to control metabolic disorders and decreasing body weight [14
]. DAP could decrease inflammatory cytokines including inflammasome, IL-1β and IL-6 but increase anti-inflammatory one; IL-10, increase the M2 (anti-inflammatory)/M1(pro-inflammatory) phenotypemacrophage ratio. In addition, DAP acts as an antioxidant and mediates M2 macrophage polarization through regulating STAT3 pathway with scavenging of reactive oxygen and nitrogen species followed by decrease of cardiac inflammation. Moreover; it controls the renal renin angiotensin aldosterone system, decreases cardiac and renal complications of diabetes, improves cardiac ischemia–reperfusion injury and doxorubicin cardiotoxicity depending on its pharmacological properties but till now there are no data regarding its role in Cd cardiotoxicity [18
Increasing the critical need for finding new cardioprotective agents against Cd cardiotoxicity and the recently detected cardiopreserving properties of DAP guided us to evaluate its ability to control Cd cardiotoxicity and to investigate the different mechanisms mediating such effect including TLR2/TNFα and IL6/STAT3 pathways.
Materials and Methods
DAP and Cd were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Caspase3 (bsm-33199M) monoclonal antibody was from Bioss Antibodies Inc, USA. Moreover, ELISA kits of TLR-2 (Catalog # MBS264416), IL6 (Catalog # MBS269892), cardiac enzymes of creatine kinase-MB (CK-MB) (Catalog #MBS2515061), Lactate dehydrogenase (LDH) (Catalog # MBS043166), heme oxygenase 1(HO1) (Catalog # MBS764989), nuclear factor erythroid 2-related factor 2 (Nrf2) (Catalog # MBS1602926), troponin I (Catalog # MBS722833) were from My BioSource Co., San Diego, CA, USA. Total antioxidant capacity (TAC) (Catalog # TA2513) was obtained from Biodiagnostic, Egypt.
Animals and Experimental Design
40 male albino rats of Wistar species weighing about 200–250 g were included in current study. These animals were from Animal Research Centre, Giza, Egypt. Rats were kept in suitable conditions (3 rats/cage) and the duration of acclimatization was about 2 weeks with free access to chow and tap water. Our study was performed in accordance with the ethical standards of faculty of medicine, Minia University, Egypt in agreement with EU Directive 2010/63/EU for animal experiments.
Rats were randomly divided into 5 groups (n = 8 each).
DAP was dissolved in 1% carboxymethylcellulose and Cd was dissolved in drinking water, and then administered by oral gavage.
Group Ӏ received the vehicle orally for 7 days.
was given Cd (5 mg/kg/day) orally [23
] for 7 days.
was administered DAP (2.5 mg/kg/day) orally and Cd (5 mg/kg/day) orally [23
] for 7 days.
was given DAP (5 mg/kg/day) [24
] and Cd (5 mg/kg/day) orally [23
] for 7 days.
received DAP (10 mg/kg/day) [25
] and Cd (5 mg/kg/day) orally [23
] for 7 days.
Preparation of Samples and Storage
Rats were euthanized on 7th day and each rat was anesthetized by i.p. injection of 20% Urethane hydrochloride (1 mg/kg). Arterial blood of each rat was collected from abdominal aorta then centrifuged at 5000 rpm for 15 min to obtain the sera (JanetzkiT30 centrifuge, Germany) for measuring cardiac enzymes and TAC. Each heart was excised, washed with saline and weighed. One part of each ventricle was fixed in 10% formalin and embedded in paraffin for further histopathological and immunohistochemical examination and another part was kept at − 80 °C. For preparing cardiac tissue homogenates specimens were homogenized in PBS 20%w/v using GLAS-Col homogenizer, USA then centrifuged at 3000 rpm for 20 min and the supernatant was separated and kept at − 80 °C till used.
Assessment of Cardiac Enzymes (Troponin I, CK-MB and LDH) in Serum
Serum cardiac enzymes were measured using the available commercial ELISA kits according to the manufacturers’ instruction based on sandwich ELISA immunoassay technique. The microtiter plate was pre-coated with a monoclonal antibody specific for each parameter then standards or samples were added to the microtiter plate wells and the detected enzyme bound to the antibody pre-coated wells. The enzyme–substrate reaction was terminated by addition of a sulphuric acid solution and the color change was measured spectrophotometrically. The color intensity was proportional to the concentration of the detected enzyme.
Evaluation of Oxidative Stress Parameters
We used colorimetric kit for measuring TAC and results were expressed as mmol/ml. MDA was the major product of lipoperoxidation that was detected colormetrically with the formation of MDA-thiobarbituric acid pink colored Schiff base adduct at 535 nm using 1, 1, 3, 3-tetramethoxypropane as standard and results were expressed as nmol/g tissue [26
]. Measurement of GSH was based on binding of sulfhydryl group with Ellman’s reagent followed by formation of a yellow color that was detected colormetrically at 405 nm by Beckman DU-64 UV/VIS spectrophotometer, USA as µmol/g tissue [27
Measurement of TLR2, HO1, Nrf2 and IL6
Detection of these markers was performed according to manufacturer’s instructions of the available ELISA kit depending on the double-sandwich ELISA technique. The pre-coated antibody of the measured parameters and the detecting antibody were labeled with biotin. Samples and biotin labeling antibody were added into ELISA plate wells and washed out then Avidin-peroxidase conjugates were added to ELISA wells. Finally, yellow color formed and the intensity of the color was positively correlated with the testing factors in samples.
Western Blotting of TNFα, NFκB, GPx and STAT3
Heart tissue homogenates about 50 μg of total proteins were boiled for 5 min with a buffer containing 2‑mercaptoethanol then loaded on 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) with running for 2 h at 100 V. After electrophoresis, proteins were blotted to polyvinylideneflouride (PVDF) membranes. Blocking step was for 1 h in a trisbuffered saline (TBS-T) and a blocking solution which contained 5% (w/v) non-fat milk and 0.05% Tween-20. Overnight incubation was at 4 °C with primary antibodies (1:1000) for rabbit anti-TNF-α (Catalog# 11948), rabbit anti-STAT3 (Catalog# 12640), rabbit anti-pY705 STAT3 (Catalog# 9145S), (Cell Signaling Technology, Danvers, MA, USA), anti-NFκB p65 antibody (Catalog# ab32536, Abcam, UK), anti-Glutathione Peroxidase 3/GPx-3 antibody (Catalog# ab256470, Abcam, UK) and anti-GAPDH antibody (Catalog# ab8245, Abcam, UK). Goat anti-rabbit polyclonal immunoglobulin conjugated with horseradish peroxidase (Cell Signaling Technology Inc., MA, USA) was used as a secondary antibody (1:5000) in blocking buffer. Bands were visualized by chemiluminescence, using an enhanced chemiluminescence kit (ECL, GE Healthcare, Chicago, IL, USA. Protein bands were evaluated densitometrically relative to GAPDH using Image J Software [28
After sacrifice; part of each ventricle was prepared for histopathological examination, dissected, fixed in formaldehyde for 24 h and embedded in paraffin wax. Five µm sections were cut for hematoxylin and eosin stain and immunohistochemistry. The pathologist examined these slides in a blinded fashion using light microscopy (Olympus microscope, Japan). Scoring of the histopathological changes in different groups was assessed according to the degree of vascular congestion; hemorrhage; hemosiderosis; muscle striation; apoptosis and necrosis. The grades were as follows: score − normal, score + mild, score ++ moderate and score +++ severe [3
Slides of the cardiac tissue specimens were de-paraffinized with xylene, hydrated in a descending graded ethyl alcohol then treated by 3% hydrogen peroxide for 30 min to block endogenous peroxides. Slides were washed in PBS solution and boiled for 20 min in citrate buffer (pH 6.0) by microwave for antigen retrieval [1
After rinse in PBS, caspase3 antibody was applied and incubated overnight then rinsed in PBS before applying the biotinylated secondary antibody for 30 min. The streptavidin–biotin complex reagent was added for 30 min after washing in PBS. For detection of brown color; 3, 3-diaminobenzidinetetra hydrochloride (DAB) was used. Slides were washed in distilled water, counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene and covered slipped. The mean area percentage of active caspase 3 reaction was measured [30
Data of current model were analyzed using one-way ANOVA then Tukey’s multiple comparison test. The values were expressed as Mean ± SEM using GraphPad Prism software (version 5) for statistical analysis. The differences were considered as significant results when the P value < 0.05.
Cd is a well-known environmental contaminant, cardiotoxic, and carcinogenic heavy metal [2
]. Our model is the first step in exploring the cardioprotective properties of DAP in Cd induced cardiac injury with studying of the different mechanisms that mediate such effect. Cd induced cardiac injury was detected in our results in form of significant increase of cardiac enzymes, heart weights, MDA, TLR2, IL6, caspase3, TNFα, NFκB but significant decrease in P-STAT3, GPx, GSH, TAC with cardiotoxic features in the histopathological examination in form of hemorrhage, inflammation, necrosis and fibrosis.
Indiscriminate exposure to Cd causes generation of free radicals and oxidative dysfunction that leads to lipid peroxidation. This associates with alteration in the antioxidant defense mechanism followed by oxidative injury of proteins, DNA, and lipids that is found in our model as significant increase in MDA levels; the most reliable marker for evaluating oxidative stress and lipid peroxidation [32
]. In addition, other oxidative stress markers including lipid hydroperoxides and protein carbonyls increased in Cd induced toxicity and they have a critical role in mediating such injury.
Antioxidant enzymes are considered as the first line of defense against the oxidative challenges to protect cell membrane and to keep the cellular integrity and prevent the pathogenesis of different degenerative diseases. Superoxide is converted by superoxide dismutase to hydroxyl radicals and hydrogen peroxide then GPx utilizes GSH to reduce hydrogen peroxide with formation of oxidized glutathione and H2
O. During oxidative stress, the level of enzymatic antioxidant enzymes notably GPx as well as the non-enzymatic antioxidant, GSH, decreases due to interaction with the excessively released free radicals [5
]. The same was observed in our results as there is general reduction in the measured GSH, GPx and TAC in Cd treated rats and this is in accordance with previous studies [31
Nrf2/HO1 pathway is also highly essential in evaluating Cd cardiotoxicity. Nrf2 is kept inactive in the cytoplasm binding with keap under healthy conditions. However, upon stimulation of oxidative stress, it travels into the nucleus and attaches to its DNA sequence followed by stimulation of gene transcription of several antioxidants including HO1 that catalyzes the rate-limiting step in the process of heme catabolism leading to anti-inflammatory, anti-oxidant, anti-apoptotic effects; it also controls cell proliferation and differentiation [43
]. Our model reveals significant downregulation of Nrf2/HO1 pathway and the same was detected with others [43
Oxidative stress induces cell membrane lipid peroxidation and damage with release of the intracellular cardiac enzymes and increasing their serum level including troponin I, CK-MB and LDH. Troponin I is one of the contractile cardiac apparatus that activates sliding of actin over myosin. It is a specific diagnostic marker for detecting cardiac injury. Moreover, CK-MB is widely distributed in skeletal muscles all over the body and highly sensitive to the occurrence of cardiac injury [1
]. This is in accordance with our data that revealed marked increase in troponin I, CK-MB and LDH in Cd cardiotoxic group.
Initiation of inflammation is another essential signaling cascade in mediating Cd cardiotoxicity. We evaluated two essential pathways; IL6/STAT3 and TLR2/TNFα which have a great role in mediating different tissue injuries. We found significant disturbance of both pathways in Cd given group reflecting occurrence of inflammation. In addition, JAK/STAT signaling cascade is considered as a critical inflammatory pathway in cardiac injury that stimulates release of other inflammatory and apoptotic mediators including TNFα and NFκB [45
]. One of STATs family is STAT3 which is controlled by IL6 and highly expressed in many heart cells including cardiomyocytes, fibroblasts, smooth muscle cells, endothelial cells, and inflammatory cells. Disturbances in IL6/STAT3 pathway could enhance Cd induced cardiac damage. Moreover, STAT3 is a crucial regulator of various intracellular events and it can regulate autophagy by promoting its mitochondrial localization. However, downregulation of STAT3 leads to cell apoptosis and damage. Notably, recent studies have been carried out based on the cardioprotective effect of STAT3 [46
]. Besides that, there is another inflammatory signaling pathway; TLR2/TNFα that has an essential role in Cd induced toxicities. Toll-like receptors enhance further release of TNFα and other interleukins followed by activation of an important inflammatory and apoptotic molecule; NFκB which is highly involved in Cd induced cardiotoxicity [2
]. This is in accordance with our current findings that showed significant increases of TNFα, NFκB and IL6 in Cd given group compared to normal control group.
Occurrence of oxidative stress and inflammation is followed by stimulation of the apoptotic cascade that is evaluated by measuring caspases family proteins. Programmed cell death occurs due to imbalance between anti-apoptotic and pro-apoptotic factors upon stimulation with the released free radicals and other pro-inflammatory agents as TNFα and NFκB. This is followed by releasing of several caspases such as caspase3 which is the most important indicator of apoptosis. Moreover, caspase3 is essential in both extrinsic and intrinsic pathways [8
]. The extrinsic apoptotic pathway is activated upon binding of a certain ligand to the death receptor followed by activation of caspase8 then either initiates apoptosis directly by activating executioner caspase, or activates the intrinsic apoptotic pathway. This signaling cascade can be activated through various cellular stresses leading to cytochrome c release from the mitochondria with formation of APAF1, cytochrome c, ATP, and caspase9 complex (the apoptosome) which initiates apoptosis by cleaving executioner caspases [50
]. We measured caspase3 immunoexpression as an apoptotic marker in Cd given group and results revealed significant increase in its expression reflecting occurrence of apoptosis due to Cd administration and this is in accordance with previous studies [1
Sodium glucose co-transporter 2 inhibitor is a class of oral anti-diabetic drugs recently used for lowering blood glucose level in type 2 diabetic patients. This drug group acts on proximal tubule in the kidneys and decreases renal glucose reabsorption with increasing glucose excretion [53
]. Despite the great benefits of SGLT-2 inhibitors, their different mechanisms of cardioprotection are still incompletely understood. Possible actions are proposed to be systemic effects via controlling metabolic disturbance and hemodynamic properties; natriuresis and glycosuria followed by decreasing cardiac preload and afterload without changing heart rate.
SGLT is highly expressed in the heart and scientists suggested that this drug class has an indirect effect on the cardiovascular system via ameliorating volume overload, decreasing blood pressure and controlling renin angiotensin aldosterone system associated with decreasing cardiac remodeling and hypertrophy with reduction of blood pressure [54
]. Besides that, DAP could decrease calcium overload, attenuating inflammation and different pro-inflammatory and inflammatory cytokines including inflammasome/caspase1/IL1β pathway, IL6, macrophage infiltration and fibrosis. This effect is accompanied with regulation of BAX/Bcl2 apoptotic pathway and downregulation of apoptosis as a result of reducing ROS [55
]. In addition, DAP could decrease M1macrophage polymerization and superoxide free radicals with activation of STAT3 followed by reduction of inflammation, oxidative stress, apoptosis and regulation of mitochondrial function [20
]. However, Cd induces mitochondrial dysfunction, increases M1 macrophage polymerization but decreases M2 with up-regulation of oxidative stress and inflammation [37
Several studies found that this drug group could ameliorate cardiac morphologic changes including cardiac hypertrophy, fibrosis, heart failure and decrease the infarcted size. Moreover; DAP could improve systolic and diastolic LV function in cases of diabetic cardiomyopathy and control cardiac arrhythmia in I/R injury. Furthermore, they decreased cardiac preload and afterload with diminishing intracellular Na+
loading that is mostly detected in heart failure [20
This in agreement with our findings which reveal that co-administration of DAP could markedly ameliorate Cd induced biochemical and histopathological changes. Our results found significant decrease and normalization of cardiac enzymes level including serum CK-MB, LDH and Troponin I. Moreover, there is marked regulation of oxidant/antioxidant misbalance including oxidative stress parameter, MDA level but increases tissue anti-oxidants as GSH and GPx with elevation of the whole TAC causing protection of the cell membrane and preventing release of cardiac enzymes. In addition, DAP succeeded in modulating IL6/STAT3 and TLR2/TNFα pathway reflecting its ability to suppress both inflammatory and apoptotic processes as found in previous models [20
]. Caspase3; an essential apoptotic marker, decreased significantly on co-administration of DAP with Cd in comparison to Cd given group alone confirming its anti-apoptotic effect with marked improvement of the histopathological features.
This cardioprotective properties of DAP was already discussed in other models of cardiac injury including diabetic cardiomyopathy, ischemia reperfusion models, doxorubicin cardiotoxicity, myocardial infarction and heart failure [20
]. These effects were based upon anti-oxidant, anti-inflammatory and anti-apoptotic properties of DAP and its ability to regulate renin angiotensin aldosterone system and keep the myocardium. Our model may give a promise to consider DAP as cardiac protector against Cd cardiotoxicity.
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