Effects of atorvastatin on myocardial oxidative and

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The aim of this study was to evaluate the role of atorvastatin in improving diabetes- induced oxidative stress in the myocardium. Male Wistar rats weighing 20–25 ...
Comparative Clinical Pathology https://doi.org/10.1007/s00580-018-2652-2

ORIGINAL ARTICLE

Effects of atorvastatin on myocardial oxidative and nitrosative stress in diabetic rats Habib Yaribeygi 1 & Nastaran Faghihi 1 & Mohammad Taghi Mohammadi 1,2 & Amirhossein Sahebkar 3,4,5 Received: 13 September 2017 / Accepted: 19 January 2018 # Springer-Verlag London Ltd., part of Springer Nature 2018

Abstract Free radicals play a pivotal role in many pathophysiological states, such as myopathies. Atorvastatin is a known cholesterollowering agent with many pleiotropic actions including antioxidant properties. However, the impact of atorvastatin on myocardial oxidative stress is not well known. The aim of this study was to evaluate the role of atorvastatin in improving diabetesinduced oxidative stress in the myocardium. Male Wistar rats weighing 20–25 g were randomly divided into four groups as normal, normal-treated, diabetes, and diabetes-treated. Induction of diabetes was performed by a single dose of streptozotocin (40 mg/kg, i.v.). Treated animals received atorvastatin for 8 weeks orally (40 mg/kg/day). After 8 weeks, animals were sacrificed and myocardial tissues were removed. Then, nitrate, glutathione (GSH), and malondialdehyde (MDA) contents as well as enzymatic activities of catalase (CAT) and superoxide dismutase (SOD) in the myocardial tissues were determined. Diabetesinduced oxidative stress by increasing nitrous free radicals (nitrate) (p ≤ 0.001) and MDA content (p ≤ 0.001), but had no significant effects on SOD, CAT, and GSH activities. Atorvastatin treatment in diabetic animals decreased free radical-induced damages by decreasing nitrate and MDA content and increasing GSH and SOD activities compared to control non-diabetic animals. Uncontrolled hyperglycemia induces oxidative burden in myocardium. Treatment by atorvastatin decreases oxidative and nitrosative stress in the myocardium of diabetic animals. Keywords Oxidative stress . Myocardium . Diabetes . Atorvastatin . Malondialdehyde

Introduction Uncontrolled hyperglycemia causes a wide range of complications in many tissues (Forbes and Cooper 2013). During hyperglycemia, disturbances in normal metabolic pathways * Mohammad Taghi Mohammadi [email protected] * Amirhossein Sahebkar [email protected]; [email protected] 1

Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

2

Department of Physiology and Biophysics, School of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran

3

Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

4

Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

5

School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

lead to enhanced formation of byproducts such as reactive oxygen (ROS) and nitrogen (RNS) species (Dandona et al. 1996; Trachtenberg and Hare 2009). This increased ROS production surpasses the biological antioxidant defenses and leads to a condition known as oxidative stress which impairs normal structure and function of vital biomolecules, such as DNA, lipids, and proteins in susceptible tissues (Trachtenberg and Hare 2009; Newsholme et al. 2007). One of the vulnerable tissues to the harmful effects of oxidative stress is myocardium (Trachtenberg and Hare 2009). Several lines of evidence have indicated the detrimental effects of ROS on the cardiovascular system (Pacher and Szabó 2006; Kajstura et al. 2001; Frustaci et al. 2000; Belch et al. 1991; Mallat et al. 1998). There are several sources for the production of free radicals in the heart tissue including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, nitric oxide (NO) synthase, mitochondria, and xanthine oxidase (Trachtenberg and Hare 2009; Giacco and Brownlee 2010). Increased ROS production in myocardium can lead to toxicity, to apoptosis and oxidation of proteins, lipids, and DNA, and to subsequent malfunction and improper contraction of cardiomyocytes

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known as cardiomyopathy (Trachtenberg and Hare 2009; Pacher and Szabó 2006). Increased RNS production has also the same results as ROS (Trachtenberg and Hare 2009; Pacher and Szabó 2006). Therefore, restoring the balance between oxidative and nitrosative species and biological antioxidant defense systems can recover myocardial contraction and improve cardiovascular function (Pacher and Szabó 2006) and suggests the use of antioxidants as potential therapeutic agents (Forbes and Cooper 2013; Dhalla et al. 2000). Statins are the cornerstone treatment for atherosclerotic cardiovascular disease owing to their efficient cholesterollowering effect (Ko et al. 2004). Additional to their cholesterol-lowering effect, statins have several effects that contribute to their protective effects on the cardiovascular system in a manner seemingly independent of cholesterol lowering; these effects include antioxidant, anti-thrombotic, antiinflammatory, and immunomodulatory activities (Chruściel et al. 2016; Moohebati et al. 2011; Sahebkar et al. 2015a; Sahebkar et al. 2016a; Sahebkar et al. 2015b; Sahebkar et al. 2016b; Sahebkar et al. 2016c; Sahebkar et al. 2015c; Sahebkar et al. 2016d; Serban et al. 2015). Statins can modify oxidative stress by lowering free radical production and enhancement of antioxidant systems (Pacher and Szabó 2006; Ferretti et al. 2015; Parizadeh et al. 2011). Statins exert their antioxidant effects via several mechanisms including inhibition of rac1 isoprenylation (Maack et al. 2003), inhibition of rho proteins (Takemoto et al. 2001), inhibition of myeloperoxidase and nitric oxide-derived oxidant production (Shishehbor et al. 2003), increasing the enzymatic activity of thioredoxin (Haendeler et al. 2004), reducing the expression of NAD(P) H oxidase subunits (Wassmann et al. 2002), and increasing catalase (CAT) gene expression and enzyme activity (Wassmann et al. 2002). In the current study, we evaluated the effects of atorvastatin treatment on myocardial oxidative stress in an animal model of diabetes.

Diabetes induction A single dose of streptozotocin (45 mg/kg i.v.; Sigma Aldrich; dissolved in cold saline) was used to induce diabetes. After 72 h from injection, blood samples were obtained from the tail vein to monitor blood glucose by a calibrated glucometer (Bionime GmbH, Berneck, Switzerland) with a precision of ±3 mg/dl and a coefficient of variation (CV) of < 5%. Rats with plasma glucose levels above 350 mg/dl were considered as diabetic and were randomly divided into two groups (diabetic treated and diabetic non-treated groups).

Treatments Atorvastatin was obtained from Sigma Aldrich (St. Louis, MO, USA) and then dissolved in distilled water daily. Two treated groups (non-diabetic and diabetic treated groups) received it for 8 weeks orally (40 mg/kg/day).

Sampling After 8 weeks, blood samples were collected for and animals were sacrificed to remove heart tissue for assessing nitrate (NOx), malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) enzymes, and reduced glutathione (GSH) content. Serum was separated by centrifugation at 3000 rpm for 15 min and the concentration of glucose was determined by using available commercial kits (Pars Azmoon, Tehran, Iran). After weighting pancreatic samples, the homogenization medium (phosphate buffer (0.1 mol, pH 7.4)) was added. After homogenizing of tissues on ice using electric homogenizer, samples were centrifuged (20 min at 4 °C and 4000 rpm) and supernatant was removed as the pancreatic cytosolic extract and stored in − 80 °C for biochemical assessments.

Materials and methods NOx assay Animals Male Wistar rats (weighing 20–25 g) were kept in standardized polyester cages (three rats per cage) in a room with normal temperature (22 ± 2 °C) and humidity (55 ± 5%) with 12-h light/dark cycle and free access to water and standard rodent feed. Experimental animals were randomly divided into four separate groups as normal, normal + atorvastatin (normal + ato.), diabetes, and diabetes + atorvastatin (diabetes + ato.) groups (N = 6 in each group). All protocols of the study were approved by the Ethics Committee of the Baqiyatallah University of Medical Sciences, which follows the NIH guidelines for care and use of animals.

The content of NOx in the cytosolic extract was determined according to a colorimetric (Griess) method. Of the cytosolic extract, 0.1 ml was deproteinized by adding 0.2 ml of zinc sulfate solution and centrifuged for 20 min at 4000 rpm and 4 °C to separate supernatant for NOx assay. 0.05 ml sulfanilamide (0.01%) and 0.05 ml N-[1-naphthyl]ethylenediamine di-hydrochloride (NED, 0.01%) were incubated for 30 min in dark place at 37 °C. Then, the absorbance of solution was read at 540 nm. A standard curve using different concentrations of sodium nitrate solution was plotted and the nitrite concentration was estimated. Finally, the nitrite-nitrate levels were expressed as μg/mg protein.

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SOD activity The SOD enzyme activity was measured using the the Winterbourn method (Winterbourn et al. 1975) which is developed based on the inhibitory effect of SOD on the reduction of nitro-blue tetrazolium by superoxide.

CAT activity CAT enzyme activity in the myocardial tissue was evaluated according to the Aebi method (Aebi 1984).

Determination of glutathione level Glutathione (GLT) content was evaluated according to the Tietz method (Tietze 1969). Cellular protein content was precipitated using 5% sulfosalicylic acid addition and then removed by centrifugation (2000 g in 20 min). GLT content of the supernatant was assayed as follows: 100 μl of protein-free supernatant of the cell lysate, 800 μl of 0.3 mM Na2HPO4, and 100 μl of 0.04% 5–5′-dithiobis[2-nitrobenzoic acid] in 0.1% sodium citrate were mixed. The 5–5′-dithiobis[2nitrobenzoic acid] absorbance was recorded at 412 nm for 5 min. A standard curve was plotted for GLT assay and sensitivity of the assay was found to range between 1 and 100 μM. The level GLT was expressed as nmol/ml.

Lipid peroxidation assay Lipid peroxidation was assessed by determining MDA content based on the Satoh method (Satoh et al. 2005).

Statistical analyses The results are expressed as the mean ± SEM. Statistical assessments were done using two-way analysis of variance (ANOVA) and Tukey tests as post hoc. A difference between values giving a probability of P < 0.05 was considered to be statistically significant.

Fig. 1 Representative changes of nitrate content (μg/mg protein) in the myocardium of normal, normal + atorvastatin, diabetic, and diabetic + atorvastatin groups. All values are presented as mean ± SEM. ***(P < 0.001) significant differences with control group (N). ###(P < 0.001) significant differences with diabetic group

significantly lower in the atorvastatin-treated (4.34 ± 0.08 μg/mg protein) compared with diabetic untreated animals (P < 0.001).

Effects of atorvastatin and diabetes on CAT enzyme activity Figure 2 shows representative changes of CAT enzyme activity in all animals. The mean value of CAT enzyme activity in normal animals was 22.93 ± 0.92 U/mg protein which was significantly higher than that in the normal atorvastatintreated group (13.71 ± 0.61 U/mg protein; P < 0.001). The CAT enzyme activity was not found to be different between normal control and diabetic control groups (P > 0.05). Likewise, there was no significant difference in CAT enzyme activity between atorvastatin-treated and untreated diabetic groups (P > 0.05).

Effects of atorvastatin and diabetes on SOD enzyme activity Figure 3 represents the mean value of SOD enzyme activity in different groups. In normal animals, the mean value of SOD

Results Effects of atorvastatin and diabetes on nitrate level Figure 1 shows representative changes of nitrate content in all groups. The mean value of nitrate content in normal group was 3.69 ± 0.08 μg/mg protein which was significantly higher than that in the non-diabetic atorvastatin-treated group (2.41 ± 0.07 μg/mg protein) (P < 0.001). Mean nitrate level in the diabetic control group (5.73 ± 0.09 μg/mg protein) was significantly higher than that in the normal untreated animals (P < 0.001). In diabetic animals, myocardial nitrate levels were

Fig. 2 Representative changes of CAT activity (unit/mg protein) in the myocardium of normal, normal + atorvastatin, diabetic, and diabetic + atorvastatin groups. All values are presented as mean ± SEM. ***(P < 0.001) significant differences with control group (N)

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Effects of atorvastatin and diabetes on MDA content

Fig. 3 Representative changes of SOD activity (unit/mg protein) in the myocardium of normal, normal + atorvastatin, diabetic, and diabetic + atorvastatin groups. All values are presented as mean ± SEM. ***(P < 0.001) significant differences with control group (N). ###(P < 0.01) significant differences with diabetic group

Figure 5 shows representative changes of MDA content in all experimental animals. The mean MDA content in normal animals was 4.04 ± 0.27 μg/mg protein which was not significantly different from the normal atorvastatin-treated animals (P > 0.05). MDA levels in the diabetic group (7.7 ± 0.92 μg/ mg protein) was significantly higher than those in the normal untreated group (P < 0.001). MDA content in the diabetic atorvastatin-treated group (3.5 ± 0.21 μg/mg protein) was significantly lower compared with the diabetic untreated group (P < 0.001).

Discussion enzyme activity was 2.01 ± 0.19 U/mg protein, which was significantly lower than the value in the normal atorvastatintreated animals (P < 0.001). SOD enzyme activity was not found to be different between normal control and diabetic control groups (P > 0.05). However, in diabetic animals, SOD enzyme activity was higher in the atorvastatin-treated group (2.39 ± 0.22 U/mg protein) compared with the untreated group (P < 0.01).

Figure 4 shows representative changes of GSH content in all groups. The mean value of GSH in normal animals was 197 ± 2 μg/ml which was significantly lower than the value in the normal atorvastatin-treated group (256 ± 4 μg/ml; P < 0.01). GSH content in myocardial cells was not found to be different between normal control and diabetic control groups (P > 0.05). However, GSH content was found to b higher in diabetic atorvastatin-treated animals (229 ± 3 μg/ml) compared with diabetic untreated animals (P < 0.01).

The present study demonstrated that uncontrolled hyperglycemia may increase free radical production and suppress intrinsic anti-oxidative defense system elements in the myocardial tissue, resulting in oxidative stress. We observed that atorvastatin treatment may not only reduce free radicals but also enhance GSH content and SOD enzyme activity and inhibit lipid peroxidation as reflected by decreased MDA levels in the myocardial tissue during uncontrolled diabetes. It has already been shown that the increase of free radical production during uncontrolled hyperglycemia may overcome the intrinsic buffering capacity, thereby leading to oxidative stress (Siti et al. 2015). Oxidative stress is a pivotal underlying mechanism in many pathophysiologic conditions and is strongly associated with the onset and progression of various forms of cardiovascular disorders (Siti et al. 2015; Dhalla et al. 2000). It has been recognized that free radicals enhance Ca2+ entry into the cardiomyocytes and this can lead to altered gene expression and cellular dysfunction (Dhalla et al. 2000). Free radicals can induce the activity of transcription factors, such as Nf-κB which in turn upregulates inflammatory cytokines that

Fig. 4 Representative changes of glutathione content (μg/mg protein) in the myocardium of normal, normal + atorvastatin, diabetic, and diabetic + atorvastatin groups. All values are presented as mean ± SEM. ***(P < 0.01) significant differences with control group (N). ###(P < 0.01) significant differences with diabetic group

Fig. 5 Representative changes of glutathione content (μg/mg protein) in the myocardium of normal, normal + atorvastatin, diabetic, and diabetic + atorvastatin groups. All values are presented as mean ± SEM. ***(P < 0.001) significant differences with control group (N). ###(P < 0.001) significant differences with diabetic group

Effects of atorvastatin and diabetes on GSH content

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are involved in the pathogenesis of cardiomyopathy (Dhalla et al. 2000). Moreover, free radicals can directly attack biological structures, such as membrane lipid bilayer (resulting in lipid peroxidation and MDA formation), DNA (resulting in strand breaks and mutation), and bioactive carbohydrates (resulting in deactivation of these molecules) (Dhalla et al. 2000). Thus, minimizing the production of free radicals can protect myocardial cells against detrimental effects of oxidative stress. Atorvastatin is a widely used cholesterol-lowering agent that acts through its inhibitory effect on HMG-COA reductase and cholesterol biosynthesis (Takemoto et al. 2001). However, compelling evidence during the recent past decades has shown that statins possess lipid-independent effects that are crucial for their protective effects against cardiovascular diseases. One of the pleiotropic actions of statins is antioxidant activity that has been shown in different tissues, such as vascular smooth muscle cells (Wassmann et al. 2002), hepatocytes (Foster et al. 2011), and in plasma against oxidation of lipids (Oranje et al. 2001). It seems that atorvastatin exerts antioxidative impact via different pathways, such as inhibiting LDL-induced hydroxyl and peroxyl radical production, lowering LDL oxidation, metal-chelating capacity, radical scavenging, and upregulation of anti-oxidant enzymes expression (Sezer et al. 2011). In the current study, atorvastatin treatment was associated with SOD enzyme activity and GSH content and reduced levels of MDA and nitrate in myocardial cells of diabetic animals. This suggests that atorvastatin mitigates features of oxidative stress in diabetic rats. Ying et al. demonstrated that atorvastatin can improve oxidative stress independent of its lipid-lowering effects via increasing SOD and glutathione peroxidase gene expression and lowering MDA production in plasma of T2DM patients (Li et al. 2010). Also, Vipul and colleagues reported that atorvastatin directly enhanced Zn/ Cu SOD and glutathione, thereby improving oxidative status in diabetic patients (Save et al. 2006). Here, we showed enhancement of SOD and GSH activities plus reduction of MDA levels in atorvastatin-treated animals compared with untreated animals which strongly suggests that atorvastatin is a potent agent for restoring oxidative balance in the myocardial tissue in diabetic state.

Conclusion Findings of the present study suggested that treatment with atorvastatin is associated with amelioration of myocardial oxidative stress induced by experimental hyperglycemia. Since diabetes-induced oxidative stress has a major role in pathophysiology of cardiac disorders and myocardial malfunction, direct antioxidant effects of statin therapy in the myocardial tissue can serve as an important

protective mechanism against cardiovascular complications of diabetes and further supports the use of statins in diabetic subjects who are at a high absolute risk of cardiovascular disease. Source of funding This study was financially supported by the Baqiyatallah University of Medical Sciences, Tehran, Iran.

Compliance with ethical standards This manuscript complies with the ethical standards of Comparative Clinical Pathology. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval and informed consent All protocols of the study were approved by the Ethics Committee of the Baqiyatallah University of Medical Sciences. Informed consent was not applicable for this animal study.

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