Original Research Article
Effects of pomegranate seed oil on oxidant/antioxidant balance in heart and kidney homogenates and mitochondria of diabetic rats and high glucose-treated H9c2 cell line Hamid Mollazadeh1,2, Mohammad Taher Boroushaki2,3, Mohammad Soukhtanloo4, Amir Reza Afshari2, Mohammad Mahdi Vahedi2,5* 1
Department of physiology and pharmacology, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran 2 Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 3 Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran 4 Department of Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 5 Health promotion Research Center, Zahedan University of Medical Science, Zahedan, Iran
Article history: Received: Nov 19, 2016 Received in revised form: Jan 29, 2017 Accepted: Feb 19, 2017 Vol. 7, No. 4, Jul-Aug 2017, 317-333. * Corresponding Author: Tel: +98 513 8828567 Fax: +98 513 8828567 [email protected] Keywords: Pomegranate Seed Oil Diabetes mellitus Oxidant Antioxidant Oxidative stress
Abstract Objective: Oxidative stress is a major cause of diabetes complications. The present study aimed to investigate the beneficial effects of Pomegranate Seed Oil (PSO) on diabetes-induced changes in oxidant/antioxidant balance of the kidney, heart and mitochondria from rats and H9c2 cell line. Materials and Methods: In these in vivo and in vitro studies, male rats were divided into four groups (twelve each): group 1 served as control, group 2-4 received a single dose of streptozotocin (60 mg/kg, i.p), groups 3 and 4 received PSO (0.36 and 0.72 mg/kg/daily, gavage), respectively. After three weeks, six rats of each group and one week later the remaining animals were anaesthetized and the hearts and kidneys were removed and homogenized. Mitochondrial fractions were separated and enzyme activities were measured in each sample. H9c2 cells were pretreated with high levels of glucose (35 mM), and then, incubated with PSO. Finally, cell viability test, reactive oxygen species production and lipid peroxidation were evaluated. Results: Significant reduction in enzymes activity (Superoxide dismutase, Glutathione S-transferase and Paraoxonase 1), compensatory elevation in Glutathione Reductase, Glutathione Peroxidase and Catalase activity followed by reduction after one week and significant elevation in Oxidative Stress Index (OSI) were observed in diabetic group. PSO treatment resulted in a significant increase in enzymes activity and decreased OSI values compared to diabetic group in both tissue and mitochondrial fractions. PSO remarkably decreased glucose-induced toxicity, ROS level and lipid peroxidation in H9c2 cells. Conclusion: Results suggested that PSO has a protective effect against diabetes-induced alterations in oxidant/antioxidant balance in tissues, mitochondrial and H9c2 cell line.
Please cite this paper as: Mollazadeh H, Boroushaki MT, Soukhtanloo M, Afshari AR, Vahedi MM. Effects of pomegranate seed oil on oxidant/antioxidant systems in heart and kidney homogenates and Mitochondria of Induced Diabetic Rats and High Glucose-Induced Toxicity in H9c2 Cell Line. Avicenna J Phytomed, 2017; 7 (4): 317-333.
AJP, Vol. 7, No. 4, Jul-Aug 2017
317
Mollazadeh et al.
Introduction Diabetes mellitus (DM) is a chronic and life-threatening illness characterized by high amount of glucose and high levels of glycated hemoglobin (HbA1C) in serum which might be due to adverse living conditions, genetic predisposition factors and dietary habits (TA 2014). Industrialized and developing nations have higher incidence and prevalence of DM. DM as a chronic disease affects all organs and systems of the body and retinopathy, neuropathy, nephropathy and cardiovascular diseases are the main causes of morbidity and mortality in DM due to its micro and macrovascular complications. One of the most important factors responsible for cell disruption in DM is oxidative stress (OxS) caused by chronic hyperglycemia (Forbes and Cooper 2013; Mollazadeh, Sadeghnia, Hoseini, Farzadnia and Boroushaki 2016). OxS is defined as an imbalance between free radical production and antioxidant capacity in the body. In the process of oxidation-reduction, detached electrons are formed which could induce detrimental effects on human organs. OxS is increased in parallel with the development of DM and interferes with normal cellular signaling and functions (Baynes and Thorpe 1999). Glucose autooxidation, non-enzymatic glycosylation, increased production of free fatty acids, activation of sorbitol pathway, activation of pentose phosphate pathway, increased production of inflammatory cytokines and activation of hexosamine pathway alongside with reduced antioxidant defense are the main mechanisms of increasing OxS. Amelioration of OxS represents an effective way to reduce the adverse consequences of reactive oxygen species (ROS). It has been suggested that stronger antioxidant system may decrease OxS and prevent DM progression and its complications (Johansen, Harris, Rychly and Ergul 2005; Rains and Jain 2011; Valko, Leibfritz, Moncol, Cronin, Mazur and Telser 2007).
Pomegranate (Punica granatum L.), from family Punicaceae, has been traditionally used as a medicinal and Ayurvedic fruit for its antioxidant, antiproliferative, antimicrobial, antiinflammatory, cardiovascular protective, anti-diabetic and anti-obesity properties. Pomegranate seed oil (PSO) which approximately comprises 12–20% of total seed weight is a rich source of conjugated octadecatrienoic fatty acids including conjugated linolenic acids (CLnAs). PSO has been reported to have beneficial therapeutic effects including body fatreducing and lipid metabolismnormalizing and antitumor properties. Also, it can reduce body weight and leptin and insulin levels while it increases glucose tolerance and peripheral insulin sensitivity. Increasing carbohydrate oxidative capacity, inhibiting progression of type-2 diabetes, and normalizing lipid profile are other therapeutic features of PSO. Beneficial health effects of PSO, especially its antioxidant and antiinflammatory activities are due to the presence of high level of CLnAs (Adams, Seeram, Aggarwal, Takada, Sand and Heber 2006; Boroushaki, Mollazadeh and Afshari 2016; Boroushaki, Mollazadeh, Rajabian, Dolati, Hoseini, Paseban and Farzadnia 2014; Goertz and Ahmad 2015; Vroegrijk, van Diepen, van den Berg, Westbroek, Keizer, Gambelli, Hontecillas, Bassaganya-Riera, Zondag and Romijn 2011). Nowadays, the role of natural products as alternative sources of medicinal compounds is highly appealing for researchers. Use of herbal antioxidant supplements has been increased due to their safety, acceptability and easy to earn; therefore, investigators try to develop therapeutic agents with antioxidant effects (Mollazadeh and Hosseinzadeh 2014). Thus, this study was designed to evaluate the effects of DM and PSO, as a rich source of unsaturated fatty acids on antioxidant-oxidant status in heart and kidney tissues as well as their
AJP, Vol. 7, No. 4, Jul-Aug 2017
318
Pomegranate seed oil against diabetes-induced oxidant/antioxidant imbalance
mitochondrial fractions, in rats with hyperglycemia induced by streptozotocin (STZ). Moreover, we decided to evaluate the effects of high glucose medium on viability of H9c2 cell line and the protective effects of PSO against glucoseinduced toxicity based on its OxS inhibition mechanism.
Materials and Methods Animals Forty-eight adult male wistar rats (8 weeks old and weighing 235–275 g) which were purchased from Animal House, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran , were used in this study. These animals were housed in a clean rodent room under a 12hr: 12-hr light-dark cycle, with ad libitum access to food and water at a temperature of 24±1°C. The rats were fed with standard diet composing of fat (2-3%), starch (55 %), protein (20-21%), crude fiber (6%), NaCl (5%), trace elements and amino acids. All animal procedures were approved by Ethics Committee of Mashhad University of Medical Sciences and were done in compliance with National Laws and with National Institutes of Health guidelines for the use and care of laboratory animals. In vivo procedures Experimental design and induction of diabetes using STZ After acclimatization, animals were randomly divided into four groups (12 each) and individually put in metabolic cages. Group 1 served as control and orally received saline (1 ml/kg/day). Groups 2, 3 and 4 were injected with a single dose of STZ (65mg/kg body weight, i. p., dissolved in 0.1M citrate buffer, pH: 4.5), three days before PSO administration. PSO (d=0.81 g/mL was diluted in DMSO at 25 0 C and it was a kind gift from Urom Narin Co, Uromeya, Iran). The induction of DM in rats was confirmed by blood glucose evaluation after 48 hr. After overnight
fasting, animals with blood glucose higher than 250 mg/dl (blood samples were obtained from the tail vein of rats and detected by a strip-operated blood glucose meter (Accu-Check Commpact Plus, Roche Diagnostics, Germany), were considered as diabetic and selected for studies. Group 3 and 4 were treated daily with oral PSO 0.4 and 0.8 ml/kg, respectively. The doses of PSO and STZ were selected on the basis of previous studies (Boroushaki, Mollazadeh, Rajabian, Dolati, Hoseini, Paseban and Farzadnia 2014; Mollazadeh, Sadeghnia, Hoseini, Farzadnia and Boroushaki 2016). All procedures were performed between 10:00 and 12:00 AM. After overnight fasting, animals were immediately anesthetized by injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). The kidneys and heart were quickly excised for enzymatic assay studies and stored at 70°C. One week later, the remaining animals in each group, underwent the previous process. Homogenization protocol One kidney and heart were scissorminced, washed with cold isotonic normal saline (0.9%, 4°C) and homogenized in ice shower containing 4ml of 0.2M phosphate buffer with pH:7.4 (Heidolph hemogenizer, Germany). Homogenates were centrifuged at 3,000 g for 15 min at 4°C to remove tissue remnants. Homogenates were restored at -70°C until further analysis. Mitochondria extraction Mitochondria Extraction was performed according to a previously reported method (Max, Garbus and Wehman 1972). In this method, the heart tissue and 1 g of kidney were homogenized in 5mL of 0.25M cold sucrose solution. Homogenates were centrifuged at 800 g for 15 minutes at 4°C and then supernatant was re-centrifuged at 20,000 g for 10 minutes. After that, supernatant was removed and the leniently inflated layer on top of the mitochondrial
AJP, Vol. 7, No. 4, Jul-Aug 2017
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Mollazadeh et al.
pellet was raised with the mild addition of sucrose solution. Pellet was later prepared for analysis by suspension with 1.2 mL of 0.02 M phosphate buffer at pH: 7.4. Activity analysis All markers analysis was performed in tissue homogenates and mitochondria extraction separately as mentioned below: TAS, TOS and OSI assay Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) levels were measured using the commercial kit manner (Rel Assay Diagnostic, Turkey) with spectrophotometer (Cecil: CE-9500) autoanalyzer. Oxidative Stress Index (OSI) was calculated according to the following formula: OSI: TOS/TAS×100. Catalase activity assay Catalase (CAT) activity was determined according to the method of Abei (Aebi 1984). Homogenates were kept at 4 ◦C in 50 mM potassium phosphate buffer (pH: 7.4) and centrifuged at 5000 rpm for 10 min. Ethanol equivalent to 0.01 ml/mL of supernatant was included and brooded for 30 min in ice. Triton 100× was added to a last centralization of 1%. Supernatant was added to a cuvette containing 1.95 ml of 50 mM phosphate buffer. At that point 1.0 ml of 30 mM hydrogen peroxide (H2O2) was included and rate of deterioration of H2O2 was measured at 240 nm. Catalase activity was calculated according to the following formula as unit/mg protein: Activity = [ΔA × 0.73] / 0.00348 Superoxide dismutase activity assay Superoxide dismutase (SOD) activity was measured by the calorimetric method (Madesh method) based on the inhibition of superoxide anion production due to auto-oxidation of pyrogallol. Reaction between 4, 5-dimethylthiazole-2-yl, 2, 5diphenyl tetrazolium (MTT) and superoxide anion was diminished by the enzyme activity (Madesh and
Balasubramanian 1998). SOD activity was expressed as U/mg protein. Glutathione S-transferase activity assay Glutathione S-transferase (GST) activity was measured spectrophotometrically at 340 nm according to the method described by Habig et al. In this method, the reaction between 2, 4-dinitrochlorobenzene (CDNB) and glutathione in the presence of GST, leads to the enhancement of absorbance. GST activity was expressed as U/mg protein (Habig, Pabst and Jakoby 1974). Glutathione reductase activity assay Glutathione Reductase (GR) activity was measured according to the method defined by Paglia (Paglia and Valentine 1967), based on the amount of oxidized glutathione (GSSG) oxidation in the presence of NADPH into reduced glutathione (GSH) and the reduction in light absorption at 340 nm. GR activity was expressed as U/mg protein. Glutathione peroxidase activity assay Glutathione Peroxidase (GPx) activity was measured spectrophotometrically at 340 nm according to the commercial kit's instructions (GPx Assay Kit, Cayman, USA). GPx activity was expressed as U/mg protein. Paraoxonase 1 activity assay Paraoxonase 1 (PON1) activity was measured spectrophotometrically at 412 nm according to the method described by Beltowsli et al (Beltowski, Wójcicka and Marciniak 2002). Para-nitrophenol production from paraoxon by PON1, leads to the reduction in light absorption. PON1 activity was expressed as U/l. In vitro studies Cell line and substances H9c2 cell line was purchased from Pasteur Institute (Tehran, Iran). MTT, Dichloro-dihydro-fluorescein diacetate
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Pomegranate seed oil against diabetes-induced oxidant/antioxidant imbalance
(DCFH-DA) and Dulbecco’s phosphatebuffered saline (PBS) were purchased from Sigma (St Louis, MO, USA). Glucose-high Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Grand Island, NY). Dimethyl sulfoxide (DMSO), 2thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were purchased from Merck. Sodium citrate and Triton X100 were purchased from Sigma (St. Louis, MO, USA). Cell culture H9c2 cells were cultured in high glucose DMEM (4.5 g/L) supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin. All cells were maintained in a 90% humidified atmosphere containing 5% CO2 at 37°C. Cell proliferation (MTT) assay H9c2 cells (5000/well) were seeded out in 96-well culture plates, and then the cells were treated with PSO (12-800 𝜇g/ml). After this procedure, non-toxic concentrations of PSO were used for treatment of cells. For this purpose, cells were pretreated with glucose solution (35 mM) for 24 hr and then incubated for 24 or 48 hr with non-toxic concentrations of PSO. MTT solution in phosphate-buffered saline (5 mg/ml) was added to each well at the final concentration of 0.05%. After 3 hr, the absorbance was measured at 570 and 620 nm (background) using a Stat FAX303 plate reader. All treatments were carried out in triplicate. Measurement of reactive oxygen species Intracellular reactive oxygen species (ROS) production was measured in both PSO-treated and control cells using DCFH-DA. Briefly 2×105 cells/well were exposed to various concentrations of PSO for different incubation times (24 and 48 hr). After incubation, cells were washed once with PBS. Treated and control cells were re-suspended in 0.5 ml PBS
containing 10 μM DCFH-DA at 37 °C for 30 min and then incubated with glucose (as the inducer of ROS production) at 37 °C for 24 hr. ROS production was exposed by an spectrophotometer. Then, the fluorescence intensity was detected at excitation/emission of 485/530 nm. All treatments were carried out in triplicate (Moongkarndi, Kosem, Kaslungka, Luanratana, Pongpan and Neungton 2004). Lipid peroxidation assay The level of lipid peroxidation was estimated by measuring MDA which is the end-product of lipid peroxidation. Cells were seeded in 96-well culture plates. After 24 hr, the cells were pretreated (for 24 hr) with PSO (12-200 μg/ml) and then incubated with glucose solution (35 mM) for 3 hr. After that, the cells were scraped and centrifuged for 30 min. Then, 400 μl of TCA (15%) and 800 μl of TBA (0.7%) were added to 500 μl of cell samples. The mixture was vortexed and then, heated for 40 min in a boiling water bath. Then, 200 μl of the sample was transferred to a 96well plate and the fluorescence intensity was read at excitation/emission of 480/530 nm. All treatments were carried out in triplicate. MDA contents were measured by following formula and showed by percent of MDA production in controls: MDA (mol-1 cm-1) = absorbance / 1.56 × 105 Statistical analysis Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test for multiple comparisons (GraphPad Prism, Version 6.01) in in vivo experiments and one-way ANOVA followed by Tukey post-hoc test was used for multiple comparisons for data analysis in in vitro experiments. Data were expressed as mean±SEM and the p-values less than 0.05 were considered to be statistically significant.
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Results TAS, TOS and OSI TAS, TOS and OSI levels in heart and kidney homogenates are shown in Tables 1 and 2, respectively. Induction of diabetes resulted in a non-significant elevation in TOS in both tissue homogenates. PSOtreated group resulted in a non-significant reduction in TOS value compared with diabetic group and significant reduction
compared to control group (p0.05). PSO-treated group resulted in a significant elevation in TAS in both tissue homogenates and this elevation was significant in heart after 4 weeks (p
Effects of pomegranate seed oil on oxidant/antioxidant balance in heart and kidney homogenates and mitochondria of diabetic rats and high glucose-treated H9c2 cell line Hamid Mollazadeh1,2, Mohammad Taher Boroushaki2,3, Mohammad Soukhtanloo4, Amir Reza Afshari2, Mohammad Mahdi Vahedi2,5* 1
Department of physiology and pharmacology, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran 2 Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 3 Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran 4 Department of Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 5 Health promotion Research Center, Zahedan University of Medical Science, Zahedan, Iran
Article history: Received: Nov 19, 2016 Received in revised form: Jan 29, 2017 Accepted: Feb 19, 2017 Vol. 7, No. 4, Jul-Aug 2017, 317-333. * Corresponding Author: Tel: +98 513 8828567 Fax: +98 513 8828567 [email protected] Keywords: Pomegranate Seed Oil Diabetes mellitus Oxidant Antioxidant Oxidative stress
Abstract Objective: Oxidative stress is a major cause of diabetes complications. The present study aimed to investigate the beneficial effects of Pomegranate Seed Oil (PSO) on diabetes-induced changes in oxidant/antioxidant balance of the kidney, heart and mitochondria from rats and H9c2 cell line. Materials and Methods: In these in vivo and in vitro studies, male rats were divided into four groups (twelve each): group 1 served as control, group 2-4 received a single dose of streptozotocin (60 mg/kg, i.p), groups 3 and 4 received PSO (0.36 and 0.72 mg/kg/daily, gavage), respectively. After three weeks, six rats of each group and one week later the remaining animals were anaesthetized and the hearts and kidneys were removed and homogenized. Mitochondrial fractions were separated and enzyme activities were measured in each sample. H9c2 cells were pretreated with high levels of glucose (35 mM), and then, incubated with PSO. Finally, cell viability test, reactive oxygen species production and lipid peroxidation were evaluated. Results: Significant reduction in enzymes activity (Superoxide dismutase, Glutathione S-transferase and Paraoxonase 1), compensatory elevation in Glutathione Reductase, Glutathione Peroxidase and Catalase activity followed by reduction after one week and significant elevation in Oxidative Stress Index (OSI) were observed in diabetic group. PSO treatment resulted in a significant increase in enzymes activity and decreased OSI values compared to diabetic group in both tissue and mitochondrial fractions. PSO remarkably decreased glucose-induced toxicity, ROS level and lipid peroxidation in H9c2 cells. Conclusion: Results suggested that PSO has a protective effect against diabetes-induced alterations in oxidant/antioxidant balance in tissues, mitochondrial and H9c2 cell line.
Please cite this paper as: Mollazadeh H, Boroushaki MT, Soukhtanloo M, Afshari AR, Vahedi MM. Effects of pomegranate seed oil on oxidant/antioxidant systems in heart and kidney homogenates and Mitochondria of Induced Diabetic Rats and High Glucose-Induced Toxicity in H9c2 Cell Line. Avicenna J Phytomed, 2017; 7 (4): 317-333.
AJP, Vol. 7, No. 4, Jul-Aug 2017
317
Mollazadeh et al.
Introduction Diabetes mellitus (DM) is a chronic and life-threatening illness characterized by high amount of glucose and high levels of glycated hemoglobin (HbA1C) in serum which might be due to adverse living conditions, genetic predisposition factors and dietary habits (TA 2014). Industrialized and developing nations have higher incidence and prevalence of DM. DM as a chronic disease affects all organs and systems of the body and retinopathy, neuropathy, nephropathy and cardiovascular diseases are the main causes of morbidity and mortality in DM due to its micro and macrovascular complications. One of the most important factors responsible for cell disruption in DM is oxidative stress (OxS) caused by chronic hyperglycemia (Forbes and Cooper 2013; Mollazadeh, Sadeghnia, Hoseini, Farzadnia and Boroushaki 2016). OxS is defined as an imbalance between free radical production and antioxidant capacity in the body. In the process of oxidation-reduction, detached electrons are formed which could induce detrimental effects on human organs. OxS is increased in parallel with the development of DM and interferes with normal cellular signaling and functions (Baynes and Thorpe 1999). Glucose autooxidation, non-enzymatic glycosylation, increased production of free fatty acids, activation of sorbitol pathway, activation of pentose phosphate pathway, increased production of inflammatory cytokines and activation of hexosamine pathway alongside with reduced antioxidant defense are the main mechanisms of increasing OxS. Amelioration of OxS represents an effective way to reduce the adverse consequences of reactive oxygen species (ROS). It has been suggested that stronger antioxidant system may decrease OxS and prevent DM progression and its complications (Johansen, Harris, Rychly and Ergul 2005; Rains and Jain 2011; Valko, Leibfritz, Moncol, Cronin, Mazur and Telser 2007).
Pomegranate (Punica granatum L.), from family Punicaceae, has been traditionally used as a medicinal and Ayurvedic fruit for its antioxidant, antiproliferative, antimicrobial, antiinflammatory, cardiovascular protective, anti-diabetic and anti-obesity properties. Pomegranate seed oil (PSO) which approximately comprises 12–20% of total seed weight is a rich source of conjugated octadecatrienoic fatty acids including conjugated linolenic acids (CLnAs). PSO has been reported to have beneficial therapeutic effects including body fatreducing and lipid metabolismnormalizing and antitumor properties. Also, it can reduce body weight and leptin and insulin levels while it increases glucose tolerance and peripheral insulin sensitivity. Increasing carbohydrate oxidative capacity, inhibiting progression of type-2 diabetes, and normalizing lipid profile are other therapeutic features of PSO. Beneficial health effects of PSO, especially its antioxidant and antiinflammatory activities are due to the presence of high level of CLnAs (Adams, Seeram, Aggarwal, Takada, Sand and Heber 2006; Boroushaki, Mollazadeh and Afshari 2016; Boroushaki, Mollazadeh, Rajabian, Dolati, Hoseini, Paseban and Farzadnia 2014; Goertz and Ahmad 2015; Vroegrijk, van Diepen, van den Berg, Westbroek, Keizer, Gambelli, Hontecillas, Bassaganya-Riera, Zondag and Romijn 2011). Nowadays, the role of natural products as alternative sources of medicinal compounds is highly appealing for researchers. Use of herbal antioxidant supplements has been increased due to their safety, acceptability and easy to earn; therefore, investigators try to develop therapeutic agents with antioxidant effects (Mollazadeh and Hosseinzadeh 2014). Thus, this study was designed to evaluate the effects of DM and PSO, as a rich source of unsaturated fatty acids on antioxidant-oxidant status in heart and kidney tissues as well as their
AJP, Vol. 7, No. 4, Jul-Aug 2017
318
Pomegranate seed oil against diabetes-induced oxidant/antioxidant imbalance
mitochondrial fractions, in rats with hyperglycemia induced by streptozotocin (STZ). Moreover, we decided to evaluate the effects of high glucose medium on viability of H9c2 cell line and the protective effects of PSO against glucoseinduced toxicity based on its OxS inhibition mechanism.
Materials and Methods Animals Forty-eight adult male wistar rats (8 weeks old and weighing 235–275 g) which were purchased from Animal House, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran , were used in this study. These animals were housed in a clean rodent room under a 12hr: 12-hr light-dark cycle, with ad libitum access to food and water at a temperature of 24±1°C. The rats were fed with standard diet composing of fat (2-3%), starch (55 %), protein (20-21%), crude fiber (6%), NaCl (5%), trace elements and amino acids. All animal procedures were approved by Ethics Committee of Mashhad University of Medical Sciences and were done in compliance with National Laws and with National Institutes of Health guidelines for the use and care of laboratory animals. In vivo procedures Experimental design and induction of diabetes using STZ After acclimatization, animals were randomly divided into four groups (12 each) and individually put in metabolic cages. Group 1 served as control and orally received saline (1 ml/kg/day). Groups 2, 3 and 4 were injected with a single dose of STZ (65mg/kg body weight, i. p., dissolved in 0.1M citrate buffer, pH: 4.5), three days before PSO administration. PSO (d=0.81 g/mL was diluted in DMSO at 25 0 C and it was a kind gift from Urom Narin Co, Uromeya, Iran). The induction of DM in rats was confirmed by blood glucose evaluation after 48 hr. After overnight
fasting, animals with blood glucose higher than 250 mg/dl (blood samples were obtained from the tail vein of rats and detected by a strip-operated blood glucose meter (Accu-Check Commpact Plus, Roche Diagnostics, Germany), were considered as diabetic and selected for studies. Group 3 and 4 were treated daily with oral PSO 0.4 and 0.8 ml/kg, respectively. The doses of PSO and STZ were selected on the basis of previous studies (Boroushaki, Mollazadeh, Rajabian, Dolati, Hoseini, Paseban and Farzadnia 2014; Mollazadeh, Sadeghnia, Hoseini, Farzadnia and Boroushaki 2016). All procedures were performed between 10:00 and 12:00 AM. After overnight fasting, animals were immediately anesthetized by injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). The kidneys and heart were quickly excised for enzymatic assay studies and stored at 70°C. One week later, the remaining animals in each group, underwent the previous process. Homogenization protocol One kidney and heart were scissorminced, washed with cold isotonic normal saline (0.9%, 4°C) and homogenized in ice shower containing 4ml of 0.2M phosphate buffer with pH:7.4 (Heidolph hemogenizer, Germany). Homogenates were centrifuged at 3,000 g for 15 min at 4°C to remove tissue remnants. Homogenates were restored at -70°C until further analysis. Mitochondria extraction Mitochondria Extraction was performed according to a previously reported method (Max, Garbus and Wehman 1972). In this method, the heart tissue and 1 g of kidney were homogenized in 5mL of 0.25M cold sucrose solution. Homogenates were centrifuged at 800 g for 15 minutes at 4°C and then supernatant was re-centrifuged at 20,000 g for 10 minutes. After that, supernatant was removed and the leniently inflated layer on top of the mitochondrial
AJP, Vol. 7, No. 4, Jul-Aug 2017
319
Mollazadeh et al.
pellet was raised with the mild addition of sucrose solution. Pellet was later prepared for analysis by suspension with 1.2 mL of 0.02 M phosphate buffer at pH: 7.4. Activity analysis All markers analysis was performed in tissue homogenates and mitochondria extraction separately as mentioned below: TAS, TOS and OSI assay Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) levels were measured using the commercial kit manner (Rel Assay Diagnostic, Turkey) with spectrophotometer (Cecil: CE-9500) autoanalyzer. Oxidative Stress Index (OSI) was calculated according to the following formula: OSI: TOS/TAS×100. Catalase activity assay Catalase (CAT) activity was determined according to the method of Abei (Aebi 1984). Homogenates were kept at 4 ◦C in 50 mM potassium phosphate buffer (pH: 7.4) and centrifuged at 5000 rpm for 10 min. Ethanol equivalent to 0.01 ml/mL of supernatant was included and brooded for 30 min in ice. Triton 100× was added to a last centralization of 1%. Supernatant was added to a cuvette containing 1.95 ml of 50 mM phosphate buffer. At that point 1.0 ml of 30 mM hydrogen peroxide (H2O2) was included and rate of deterioration of H2O2 was measured at 240 nm. Catalase activity was calculated according to the following formula as unit/mg protein: Activity = [ΔA × 0.73] / 0.00348 Superoxide dismutase activity assay Superoxide dismutase (SOD) activity was measured by the calorimetric method (Madesh method) based on the inhibition of superoxide anion production due to auto-oxidation of pyrogallol. Reaction between 4, 5-dimethylthiazole-2-yl, 2, 5diphenyl tetrazolium (MTT) and superoxide anion was diminished by the enzyme activity (Madesh and
Balasubramanian 1998). SOD activity was expressed as U/mg protein. Glutathione S-transferase activity assay Glutathione S-transferase (GST) activity was measured spectrophotometrically at 340 nm according to the method described by Habig et al. In this method, the reaction between 2, 4-dinitrochlorobenzene (CDNB) and glutathione in the presence of GST, leads to the enhancement of absorbance. GST activity was expressed as U/mg protein (Habig, Pabst and Jakoby 1974). Glutathione reductase activity assay Glutathione Reductase (GR) activity was measured according to the method defined by Paglia (Paglia and Valentine 1967), based on the amount of oxidized glutathione (GSSG) oxidation in the presence of NADPH into reduced glutathione (GSH) and the reduction in light absorption at 340 nm. GR activity was expressed as U/mg protein. Glutathione peroxidase activity assay Glutathione Peroxidase (GPx) activity was measured spectrophotometrically at 340 nm according to the commercial kit's instructions (GPx Assay Kit, Cayman, USA). GPx activity was expressed as U/mg protein. Paraoxonase 1 activity assay Paraoxonase 1 (PON1) activity was measured spectrophotometrically at 412 nm according to the method described by Beltowsli et al (Beltowski, Wójcicka and Marciniak 2002). Para-nitrophenol production from paraoxon by PON1, leads to the reduction in light absorption. PON1 activity was expressed as U/l. In vitro studies Cell line and substances H9c2 cell line was purchased from Pasteur Institute (Tehran, Iran). MTT, Dichloro-dihydro-fluorescein diacetate
AJP, Vol. 7, No. 4, Jul-Aug 2017
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Pomegranate seed oil against diabetes-induced oxidant/antioxidant imbalance
(DCFH-DA) and Dulbecco’s phosphatebuffered saline (PBS) were purchased from Sigma (St Louis, MO, USA). Glucose-high Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Grand Island, NY). Dimethyl sulfoxide (DMSO), 2thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were purchased from Merck. Sodium citrate and Triton X100 were purchased from Sigma (St. Louis, MO, USA). Cell culture H9c2 cells were cultured in high glucose DMEM (4.5 g/L) supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin. All cells were maintained in a 90% humidified atmosphere containing 5% CO2 at 37°C. Cell proliferation (MTT) assay H9c2 cells (5000/well) were seeded out in 96-well culture plates, and then the cells were treated with PSO (12-800 𝜇g/ml). After this procedure, non-toxic concentrations of PSO were used for treatment of cells. For this purpose, cells were pretreated with glucose solution (35 mM) for 24 hr and then incubated for 24 or 48 hr with non-toxic concentrations of PSO. MTT solution in phosphate-buffered saline (5 mg/ml) was added to each well at the final concentration of 0.05%. After 3 hr, the absorbance was measured at 570 and 620 nm (background) using a Stat FAX303 plate reader. All treatments were carried out in triplicate. Measurement of reactive oxygen species Intracellular reactive oxygen species (ROS) production was measured in both PSO-treated and control cells using DCFH-DA. Briefly 2×105 cells/well were exposed to various concentrations of PSO for different incubation times (24 and 48 hr). After incubation, cells were washed once with PBS. Treated and control cells were re-suspended in 0.5 ml PBS
containing 10 μM DCFH-DA at 37 °C for 30 min and then incubated with glucose (as the inducer of ROS production) at 37 °C for 24 hr. ROS production was exposed by an spectrophotometer. Then, the fluorescence intensity was detected at excitation/emission of 485/530 nm. All treatments were carried out in triplicate (Moongkarndi, Kosem, Kaslungka, Luanratana, Pongpan and Neungton 2004). Lipid peroxidation assay The level of lipid peroxidation was estimated by measuring MDA which is the end-product of lipid peroxidation. Cells were seeded in 96-well culture plates. After 24 hr, the cells were pretreated (for 24 hr) with PSO (12-200 μg/ml) and then incubated with glucose solution (35 mM) for 3 hr. After that, the cells were scraped and centrifuged for 30 min. Then, 400 μl of TCA (15%) and 800 μl of TBA (0.7%) were added to 500 μl of cell samples. The mixture was vortexed and then, heated for 40 min in a boiling water bath. Then, 200 μl of the sample was transferred to a 96well plate and the fluorescence intensity was read at excitation/emission of 480/530 nm. All treatments were carried out in triplicate. MDA contents were measured by following formula and showed by percent of MDA production in controls: MDA (mol-1 cm-1) = absorbance / 1.56 × 105 Statistical analysis Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test for multiple comparisons (GraphPad Prism, Version 6.01) in in vivo experiments and one-way ANOVA followed by Tukey post-hoc test was used for multiple comparisons for data analysis in in vitro experiments. Data were expressed as mean±SEM and the p-values less than 0.05 were considered to be statistically significant.
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Results TAS, TOS and OSI TAS, TOS and OSI levels in heart and kidney homogenates are shown in Tables 1 and 2, respectively. Induction of diabetes resulted in a non-significant elevation in TOS in both tissue homogenates. PSOtreated group resulted in a non-significant reduction in TOS value compared with diabetic group and significant reduction
compared to control group (p0.05). PSO-treated group resulted in a significant elevation in TAS in both tissue homogenates and this elevation was significant in heart after 4 weeks (p
