Neuroscience Letters 558 (2014) 31–36
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Protective effect of telmisartan against oxidative damage induced by high glucose in neuronal PC12 cell Habib Eslami a,b,∗ , Ali M. Sharifi a , Hamzeh Rahimi c , Maryam Rahati d a
Department of Pharmacology, Razi Institute of Drug Research, Tehran University of Medical Sciences, Tehran, Iran Department of Pharmacology, Research Center of Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran d Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran b c
h i g h l i g h t s • We investigated the effects of telmisartan on high glucose induced oxidative damage in PC12 cells. • Telmisartan counteracts high glucose-triggered oxidative damage and cell death in neuronal PC12 cells. • The antioxidant action of telmisartan and its inhibitory effect on NADPH oxidase may, at least in part, have mediated the inhibition of cell death.
a r t i c l e
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Article history: Received 2 August 2013 Received in revised form 21 October 2013 Accepted 24 October 2013 Keywords: Telmisartan High glucose Oxidative stress PC12 cells
a b s t r a c t Telmisartan is an angiotensin II type 1 receptor blocker and partial agonist of peroxisome proliferatoractivated receptor gamma (PPAR-␥). Here, we investigated the protective capacity of telmisartan against high glucose (HG)-elicited oxidative damage in PC12 cells. The activity of lactate dehydrogenase (LDH), NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT) as well as the levels of malondialdehyde (MDA), glutathione (GSH), intracellular reactive oxygen species (ROS), cell viability and DNA fragmentation were measured in HG-treated PC12 cells with and without telmisartan co-treatment. Moreover, the direct antioxidant effect of telmisartan was determined by 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay and protein expression of Bax, Bcl-2, cleaved caspase-3 and NOX subunit p47phox by western blotting. Telmisartan exhibited antioxidant activity in the ABTS assay with the IC50 value of 37.5 M. Pretreatment of PC12 cells with telmisartan, prior to HG exposure, was associated with a marked diminution in cleaved caspase-3 expression, DNA fragmentation, Bax/Bcl-2 ratio, intracellular ROS and MDA levels. Additionally, the cell viability, GSH level, SOD and CAT activity were notably elevated by telmisartan, whereas the activity and the protein expression of NADPH oxidase subunit p47phox were attenuated. Interestingly, co-treatment with GW9662, a PPAR-␥ antagonist, partially inhibited the beneficial effects of telmisartan. These findings suggest that telmisartan has protective effects on HG-induced neurotoxicity in PC12 cells, which may be related to its antioxidant action and inhibition of NADPH oxidase. Furthermore, the results show that PPAR-␥ activation is involved in the neuroprotective effects of telmisartan. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There is a large body of evidence highlighting that chronic high glucose (HG) condition leads to neuronal cell oxidative damage via NADPH oxidase-dependent generation of ROS [12,13]. ROS can attack proteins, lipid membranes and nucleic acids, causing oxidative injury to cells [20]. As well, excessive formation of ROS by HG
∗ Corresponding author at: Department of Pharmacology, School of Medicine, Hormozgan University of Medical Sciences, P.O. Box 79196-93116, Bandar Abbas, Iran. Tel.: +98 761 666 8477; fax: +98 761 666 8478. E-mail addresses: [email protected], [email protected] (H. Eslami). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.057
increases apoptosis, a possible mechanism of glucose neurotoxicity [25,26]. High-glucose milieu in diabetes not only generates more ROS but also attenuates anti-oxidant protective mechanisms through glycation of the antioxidant enzymes [12,20]. It is now accepted that an effective approach to prevention and treatment of diabetic complications such as neuropathy must focus on both early glycemic control and reducing factors related to oxidative stress [2]. Currently, no selective and effective therapeutic agent has been identified for clinical use to prevent or treat HG-induced neuronal cell injury and dysfunction [9]. Therefore, it is crucial to explore tools by which one can reduce oxidative stress in neurons to ameliorate the impaired neuronal function produced by hyperglycemia.
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Telmisartan has been widely used as a highly selective angiotensin II type 1 (AT1 ) receptor (AT1 R) blocker (ARB) for the treatment of hypertension. Beyond its antagonistic activity at AT1 receptors, it has additional favorable metabolic effect [4,30]. Recent studies demonstrated that telmisartan provided a significant antidiabetic effect and ameliorated hyperglycemia in rats with streptozotocin-induced or spontaneous diabetes [7,11,15]. The neuroprotective effect of telmisartan has been observed against many types of damage stimuli [10]. It is also recognized to attenuate oxidative stress in human umbilical vein endothelial cell (HUVEC) and neuronal cultures [3,22]. However, whether telmisartan has a neuroprotective role in high glucose condition has not been elucidated. To the best of our knowledge, this is the first study to investigate the protective effect of telmisartan in neuronal PC12 cells. The PC12 cell line has been extensively used as a cell model to study glucose neurotoxicity [25]. Previous studies in our laboratory have demonstrated the cytotoxicity of high glucose in PC12 cells [25,26]. The present study was designed to examine the protective effect of telmisartan on PC12 cells maintained under HG concentration. We investigated whether the neuroprotective effects of telmisartan are related to its antioxidant properties using an ABTS radical cation-scavenging assay. The alterations of antioxidant defenses (GSH level, SOD and CAT activities), intracellular ROS level, NADPH oxidase activation, MDA level, cell viability, and apoptotic neuronal cell death induced by high glucose were determined. We also used the PPAR-␥ antagonist GW9662 to examine the possible involvement of PPAR-␥ activation in the protective effect of telmisartan. 2. Methods 2.1. Cell culture and drug treatment PC12 cells were cultured as described previously [25]. After cultured in serum-free medium for 24 h, the cells were pre-incubated with 0.1–50 M of telmisartan (Sigma, T8949) for 2 h and then co-treated with telmisartan and high glucose (75 mM) for 72 h. In experiments where GW9662 (Sigma, M6191) was used together with telmisartan, cells were pre-treated with GW9662 (20 M) for 1 h before the addition of telmisartan. Control cells were cultured with normal glucose (23 mM), and not treated with telmisartan. Mannitol (75 mM) was also used as negative control for osmolarity. Telmisartan and GW9662 were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in experimental conditions was 0.1%. The selected concentrations of telmisartan and GW9662 were according to the previous study [23]. 2.2. Determination of cell viability, LDH release and intracellular ROS level Cell viability was measured using MTT assay as previously described [18,25]. The LDH activity was quantified using a LDH diagnostic kit (Stanbio Laboratory, USA). In brief, at the end of the drug treatment, the medium was collected and 100 L of it was added to 1 mL of LDH reagent. The absorbance was recorded at 340 nm. The production of intracellular ROS was measured by using 2,7-dichlorofluorescein diacetate (DCFH-DA) [29]. After treatment, cells were washed and incubated with DCFH-DA for 20 min. Then the relative levels of fluorescence were quantified by a fluorescence microplate reader.
supernatant was measured using the Bradford method [1]. The CAT activity was quantified according to the method of Shen et al. [27]. Briefly, 1.0 mL of hydrogen peroxide (19 mM) and 50 L of the supernatant were mixed with 1.95 mL of phosphate buffer (0.05 M, pH 7.0). The CAT activity was then estimated from the decrease in absorbency at 240 nm for 2 min. The SOD activity was measured using a SOD activity assay kit (BioVision, Mountain View, CA). Briefly, 20 L of the supernatant was added to 200 L tetrazolium and 20 L of enzyme solution. After incubating at 37 ◦ C for 20 min, the absorbance was read at 450 nm. The GSH level was measured according to the method of Gulati et al. [8]. Briefly, 100 L of the supernatant was mixed with 200 L of trichloroacetic acid and centrifuged. The resulting supernatant was mixed with phosphate buffer and its absorbance measured at 412 nm. MDA content was measured as previously described [21]. Briefly, 100 L of the supernatant was mixed with 1.5 mL of acetic acid (20%), 1.5 mL of thiobarbituric acid (0.8%), and 200 L of sodium dodecyl sulphate (8%). Each reaction mixture was heated for 60 min at 95 ◦ C and cooled to room temperature. Then, 5 mL of n-butanol was added. After mixing and centrifugation at 3000 × g for 10 min, the organic layer was collected and the absorbance measured at 532 nm. 2.4. ABTS radical cation-scavenging assay The antioxidant activity of telmisartan was determined by the method of Ghanta et al. [6] and expressed as IC50 , which was defined as the concentration of test material required to cause a 50% decrease in initial ABTS radical cation concentration. Briefly, 10 L of telmisartan (0.1–70 M) was mixed with 990 L of ABTS solution. The absorbance at 734 nm was recorded. Trolox was used as the standard. 2.5. Quantification of DNA fragmentation To examine DNA fragmentation, we used Cell Death Detection ELISAPlus kit (Roche Applied Sciences, Germany) and performed assay according to the manufacturer’s instructions, except that cells were cultured in 96-well plates at a density of 2 × 104 cells/well for the ELISA analysis. 2.6. Western blot analysis Western blot analysis was performed using whole-cell lysate from PC12cells as previously described [25]. Briefly, proteins samples were separated by SDS-PAGE and transferred onto a PVDF membrane which was incubated with primary antibodies including NADPH oxidase p47phox subunit (Santa Cruz, sc-14015), Bcl-2 (BioVision, 3033-100), Bax (BioVision, 3032-100), caspase-3 (BioVision, 3015-100) and secondary antibody (BioVision, 6401-05). Bands were visualized using ECL detection reagents. The resultant bands were quantified by densitometric analysis using the Image master program and normalized against -actin protein. 2.7. Estimation of NADPH oxidase activity in PC12 cells The activity of NADPH oxidase was quantified using lucigeninenhanced chemiluminiscence with a commercial kit (Genmed Scientifics Inc.) according to manufacturer’s protocols. 2.8. Statistical analysis
2.3. MDA, GSH, SOD, and CAT assay After drug treatment, the cells were washed and homogenized. The homogenate was centrifuged and the protein content of
Data were presented as mean ± S.E.M, and were analyzed by ANOVA followed by post hoc Dunnett’s t-test. The criterion for significance was a P value < 0.05.
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Fig. 1. Effects of telmisartan (TL) on cell viability (A), LDH release (B), ROS formation (C) and DNA fragmentation (D) in PC12 cells under HG condition. Cells were incubated with HG (75 mM) for 72 h. Telmisartan (0.1–50 M) was added to the culture 2 h prior to HG and 20 M of GW9662 (GW) 1 h before the addition of telmisartan. Data are mean ± SEM, n = 6. ## P < 0.01 compared with control. *P < 0.05, **P < 0.01 compared with the HG group, † P < 0.05 compared to telmisartan (10 M) treatment.
3. Result 3.1. Effect of telmisartan on HG-induced cytotoxicity as measured by cell viability and LDH release HG significantly reduced cell viability and increased LDH release compared to control cells (P < 0.01). However, incubation of cells with telmisartan (0.1–50 M) concentration-dependently reversed HG-induced cell death (Fig. 1A) and remarkably blocked LDH leakage (Fig. 1B). Mannitol (75 mM), which was used to create a high osmotic pressure mimicking HG condition, did not show significant change in cell viability. This result indicated that the effect of high glucose on proliferation was not secondary to osmotic load. Co-treatment with GW9662 (20 M) partially prevented the protective effects of telmisartan in MTT assay. Under the condition of normal glucose (23 mM d-glucose), DMSO (0.1%), GW9662 (20 M) or telmisartan (50 M) itself had no significant effect on the PC12 cells viability and also on none of the other following measurements in 3–8 experiments (data not shown). 3.2. Effects of telmisartan on MDA, GSH, SOD, CAT and ROS The GSH level, SOD activity, and CAT activity significantly decreased after exposure of PC12 cells to HG (# P < 0.05 vs. control, Table 1). However, intracellular MDA (Table 1) and ROS (Fig. 1C) were significantly raised. Following drug treatment, the MDA and ROS level significantly decreased (*P < 0.05), whereas CAT, SOD activity and the GSH level significantly increased (*P < 0.05) compared with HG-treated PC12 cells.
Fig. 2. Percentage inhibition of ABTS radical cations by telmisartan. Data are mean ± SEM, n = 3.
3.3. ABTS radical cation-scavenging capacity of telmisartan As shown in Fig. 2, telmisartan at different concentrations (0.1–70 M) was found to effectively scavenge ABTS radical cations (2.3–100% inhibition), with an average IC50 value of 37.5 M. IC50 value for Trolox, used as the reference compound, was 21.3 M. 3.4. Effect of telmisartan on the DNA fragmentation When PC12 cells were exposed to HG, there was a significant increase in DNA fragmentation vs. untreated controls (Fig. 1D),
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Table 1 Effects of telmisartan (TL) on antioxidant enzyme activities and level of MDA and GSH in rat PC12 cells under high glucose (HG) condition.a % of Control MDA Control HG (100 mM) HG + TL (0.1 M) HG + TL (1 M) HG + TL (10 M) HG + TL (50 M)
100 159.3 152.7 138.4 126.5 119.4
GSH ± ± ± ± ± ±
7.4 9.1# 8.9 6.5* 6.9* 5.7*
100 47.5 53.6 63.5 74.2 79.6
SOD ± ± ± ± ± ±
8.1 3.5# 3.4 4.3* 4.2* 6.1*
100 43.6 47.9 58.7 68.3 76.1
CAT ± ± ± ± ± ±
6.8 4.4# 3.8 3.5* 3.6* 5.1*
100 55.2 59.9 64.5 71.1 75.4
± ± ± ± ± ±
4.6 3.7# 4.4 4.7* 4.9* 6.8*
a Cultured PC12 cells were exposed to 75 mM glucose (HG). Telmisartan (TL) was added 2 h prior to glucose addition and the levels of antioxidant enzymes, MDA and GSH were measured 72 h later. Data are means ± SEM, n = 3–8. # P < 0.05 vs. control. * P < 0.05 vs. HG.
Fig. 3. Effects of telmisartan (TL) on the ratio of protein expression of Bax/Bcl-2 (A), upregulation of NADPH oxidase p47phox subunit protein expression (B), NADPH oxidase activity (C) and the expression of caspase-3 (D) in PC12 cells exposed to HG in the presence or absence of telmisartan (10 M) or 20 M GW9662 (GW) for 72 h. Data are mean ± SEM, n = 3. **P < 0.01 as compared to HG group, † P < 0.05 compared to telmisartan (10 M) treatment.
which was efficiently prevented by telmisartan (0.1–50 M). However, this effect of telmisartan was antagonized by GW9662.
as well as NADPH oxidase activity in PC12 cells. Pre-incubation of PC12 cells with telmisartan attenuated these changes.
3.5. Expression of NADPH oxidase p47phox subunit, Bcl-2, Bax and caspase-3 proteins
4. Discussion
Treating PC12 cells with HG led to a significant increase of the Bax/Bcl-2 ratio (Fig. 3A) and cleaved caspase-3 expression (Fig. 3D) as compared with the control group. Pre- and co-treatment with 10 M telmisartan decreased significantly the Bax/Bcl-2 ratio and caspase-3 expression, as compared with HG group. GW9662 reduced this effect of telmisartan (Fig. 3D). As shown in Fig. 3B and C, high glucose significantly up-regulated p47phox expression
This study found that telmisartan can exert a protective effect against ROS-mediated oxidative damage and apoptosis induced by high glucose in neuronal PC12 cells. Glucose induction of ROS is critical to the pathogenesis of diabetic neuropathy [20]. In addition, Hydroxyl radical is one of the highly ROS that causes the oxidative damage associated with diabetes [16]. Our results revealed that glucose enhanced ROS concentration in PC12 cells while simultaneously treating with telmisartan and glucose effectively
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attenuated ROS generation. This finding corroborates with previous studies which have shown that telmisartan acted as efficient scavenger of ROS particularly hydroxyl radicals [3]. To evaluate the antioxidant capacity of telmisartan, the ABTS cation radical-scavenging assay, was employed [6]. The results clearly showed that telmisartan displays antioxidant properties, which is consistent with previous studies that have demonstrated excellent antioxidant activity for telmisartan [3]. Moreover, it has been suggested that telmisartan, an AT1 receptor antagonist, inhibits intracellular oxidative stress, at least in part, in a receptor-independent manner, possibly owing to its lipophilic and antioxidant structure [24]. The chemical structure of telmisartan contains the benzimidazolic and benzoic groups that probably confer selective scavenging properties for hydroxyl radicals [3]. On the other hand, some studies have previously shown that PC12 cells derived from rat pheochromocytoma predominantly express AT2 receptor [14,19]. Based on these observations, it is very likely that in PC12 cells, the neuroprotective effects of telmisartan are independent of AT1 receptor inhibition. In the present study, we also found that NADPH-oxidase activity and protein expression of p47phox was significantly higher in GH-treated than control PC12 cells. p47phox is a cytosolic subunit of NOX and several reports support its importance in diabetes. For instance, it has been shown that deletion of p47 (phox) attenuates diabetes-induced glomerular injury and beta cell dysfunction [17]. Furthermore, superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47phox subunit of NADPH oxidase [28]. Our results demonstrated that telmisartan pre- and co-treatment suppressed the upregulated p47phox, and NADPH oxidase activity. In agreement with our present findings, previous observations have also shown that ARBs decrease NADPH oxidase activation associated with oxidative stress and neuronal apoptosis [23]. ROS also is a well-known initiator of apoptosis in many cell types [20]. In this study DNA fragmentation assay, a hallmark of apoptosis, Bax/Bcl-2 index and cleaved caspase-3 expression were used for measurements of apoptotic cell death. Telmisartan prevented HG-induced increase in caspase-3 expression, DNA fragmentation in PC12 cells and decreased the ratio of Bax/Bcl-2. This ratio is supposed to dictate the relative sensitivity or resistance of cells to a wide variety of apoptotic stimuli [26]. Interestingly, telmisartan has the unique character of having both ARB and PPAR-␥ agonistic effect [10], and Fuenzalida et al. reported that PPAR␥ up-regulates Bcl-2 in neurons [5]. Also, Tamami Haraguchi et al. suggested that telmisartan reduces neuronal apoptosis via a PPAR␥-dependent caspase-3 inhibiting mechanism [10]. We used the PPAR-␥ antagonist GW9662 to examine the possible involvement of PPAR-␥ activation in the protective effect of telmisartan. Results demonstrated that the beneficial effects of telmisartan were weakened by co-treatment with GW9662. Previous reports are in line with our current data [5,10], which indicate that telmisartan diminishes HG-elicited apoptosis in neuronal PC12 cells, possibly, via downregulation of Bax/Bcl-2 and caspase-3 expression. To mitigate cumulative burden of oxidative stress, cells generally utilize antioxidant defense systems and scavenge ROS. SOD and CAT are two important antioxidant enzymes [20]. In this regard, our present study indicated that PC12 cells treated with high glucose showed a marked rise in oxidative stress as evidenced by excessive ROS and MDA production, together with depletion of “endogenous antioxidant reserve,” including GSH contents, SOD and CAT activity level. However, co-treatment with telmisartan significantly attenuated oxidative damage to PC12 under high glucose condition, as reflected by the augmentation of antioxidant defense system (CAT, SOD and GSH) with accompanying decrease in MDA and ROS levels. These results confirm the in vitro antioxidant activity of telmisartan.
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In summary, our results indicate that telmisartan plays a protective role against HG-induced cell death in PC12 cells in a dose-dependent manner. This is possibly accomplished through diminution of NADPH oxidase activation and ROS formation in parallel with the increases in the GSH level, SOD and CAT activity. Furthermore, the results show that PPAR-␥ activation is involved in the neuroprotective effects of telmisartan. Acknowledgment The authors would like to thank research council of the Hormozganan University of Medical Sciences for financial supporting. References [1] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [2] N.C. Chilelli, S. Burlina, A. Lapolla, AGEs, rather than hyperglycemia, are responsible for microvascular complications in diabetes: a “glycoxidation-centric” point of view, Nutr. Metab. Cardiovasc. Dis. 23 (2013) 913–919. [3] S. Cianchetti, A. Del Fiorentino, R. Colognato, R. Di Stefano, F. Franzoni, R. Pedrinelli, Anti-inflammatory and anti-oxidant properties of telmisartan in cultured human umbilical vein endothelial cells, Atherosclerosis 198 (2008) 22–28. [4] M. Destro, F. Cagnoni, G.P. Dognini, V. Galimberti, C. Taietti, C. Cavalleri, E. Galli, Telmisartan: just an antihypertensive agent? A literature review, Expert Opin. Pharmacother. 12 (2011) 2719–2735. [5] K. Fuenzalida, R. Quintanilla, P. Ramos, D. Piderit, R.A. Fuentealba, G. Martinez, N.C. Inestrosa, M. Bronfman, Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis, J. Biol. Chem. 282 (2007) 37006–37015. [6] S. Ghanta, A. Banerjee, A. Poddar, S. Chattopadhyay, Oxidative DNA damage preventive activity and antioxidant potential of Stevia rebaudiana (Bertoni) Bertoni, a natural sweetener, J. Agr. Food Chem. 55 (2007) 10962–10967. [7] B.R. Goyal, K. Parmar, R.K. Goyal, A.A. Mehta, Beneficial role of telmisartan on cardiovascular complications associated with STZ-induced type 2 diabetes in rats, Pharmacol. Rep. 63 (2011) 956–966. [8] K. Gulati, A. Chakraborti, A. Ray, Modulation of stress-induced neurobehavioral changes and brain oxidative injury by nitric oxide (NO) mimetics in rats, Behav. Brain Res. 183 (2007) 226–230. [9] A.A. Habib, T.H. Brannagan 3rd, Therapeutic strategies for diabetic neuropathy, Curr. Neurol. Neurosci. Rep. 10 (2010) 92–100. [10] T. Haraguchi, K. Iwasaki, K. Takasaki, K. Uchida, T. Naito, A. Nogami, K. Kubota, T. Shindo, N. Uchida, S. Katsurabayashi, K. Mishima, R. Nishimura, M. Fujiwara, Telmisartan, a partial agonist of peroxisome proliferator-activated receptor gamma, improves impairment of spatial memory and hippocampal apoptosis in rats treated with repeated cerebral ischemia, Brain Res. 1353 (2010) 125–132. [11] G. Hasegawa, M. Fukui, H. Hosoda, M. Asano, I. Harusato, M. Tanaka, E. Shiraishi, T. Senmaru, K. Sakabe, M. Yamasaki, J. Kitawaki, A. Fujinami, M. Ohta, H. Obayashi, N. Nakamura, Telmisartan, an angiotensin II type 1 receptor blocker, prevents the development of diabetes in male Spontaneously Diabetic Torii rats, Eur. J. Pharmacol. 605 (2009) 164–169. [12] A. Hosseini, M. Abdollahi, Diabetic neuropathy and oxidative stress: therapeutic perspectives, Oxid. Med. Cell. Longev. 2013 (2013) 168039. [13] F. Jiang, Y. Zhang, G.J. Dusting, NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair, Pharmacol. Rev. 63 (2011) 218–242. [14] K. Kijima, H. Matsubara, S. Murasawa, K. Maruyama, N. Ohkubo, Y. Mori, M. Inada, Regulation of angiotensin II type 2 receptor gene by the protein kinase C-calcium pathway, Hypertension 27 (1996) 529–534. [15] S. Kushwaha, G.B. Jena, Telmisartan ameliorates germ cell toxicity in the STZinduced diabetic rat: studies on possible molecular mechanisms, Mutat. Res. 755 (2013) 11–23. [16] B. Lipinski, Hydroxyl radical and its scavengers in health and disease, Oxid. Med. Cell Longev. 2011 (2011) 809696. [17] G.C. Liu, F. Fang, J. Zhou, K. Koulajian, S. Yang, L. Lam, H.N. Reich, R. John, A.M. Herzenberg, A. Giacca, G.Y. Oudit, J.W. Scholey, Deletion of p47phox attenuates the progression of diabetic nephropathy and reduces the severity of diabetes in the Akita mouse, Diabetologia 55 (2012) 2522–2532. [18] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [19] S. Murasawa, H. Matsubara, M. Urakami, M. Inada, Regulatory elements that mediate expression of the gene for the angiotensin II type 1a receptor for the rat, J. Biol. Chem. 268 (1993) 26996–27003. [20] G. Negi, A. Kumar, R.P. Joshi, S.S. Sharma, Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle, Biochem. Biophys. Res. Commun. 408 (2011) 1–5.
36
H. Eslami et al. / Neuroscience Letters 558 (2014) 31–36
[21] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [22] M.S. Ola, M.M. Ahmed, H.M. Abuohashish, S.S. Al-Rejaie, A.S. Alhomida, Telmisartan ameliorates neurotrophic support and oxidative stress in the retina of streptozotocin-induced diabetic rats, Neurochem. Res. 38 (2013) 1572– 1579. [23] T. Pang, J. Wang, J. Benicky, E. Sanchez-Lemus, J.M. Saavedra, Telmisartan directly ameliorates the neuronal inflammatory response to IL-1beta partly through the JNK/c-Jun and NADPH oxidase pathways, J. Neuroinflammation 9 (2012) 102. [24] J. Shao, M. Nangaku, R. Inagi, H. Kato, T. Miyata, T. Matsusaka, E. Noiri, T. Fujita, Receptor-independent intracellular radical scavenging activity of an angiotensin II receptor blocker, J. Hypertens. 25 (2007) 1643–1649. [25] A.M. Sharifi, H. Eslami, B. Larijani, J. Davoodi, Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells, Neurosci. Lett. 459 (2009) 47–51.
[26] A.M. Sharifi, S.H. Mousavi, M. Farhadi, B. Larijani, Study of high glucose-induced apoptosis in PC12 cells: role of bax protein, J. Pharmacol. Sci. 104 (2007) 258–262. [27] X. Shen, Y. Tang, R. Yang, L. Yu, T. Fang, J.A. Duan, The protective effect of Zizyphus jujube fruit on carbon tetrachloride-induced hepatic injury in mice by antioxidative activities, J. Ethnopharmacol. 122 (2009) 555–560. [28] S.W. Suh, E.T. Gum, A.M. Hamby, P.H. Chan, R.A. Swanson, Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase, J. Clin. Invest. 117 (2007) 910–918. [29] F.X. Sureda, C. Gabriel, J. Comas, M. Pallas, E. Escubedo, J. Camarasa, A. Camins, Evaluation of free radical production, mitochondrial membrane potential and cytoplasmic calcium in mammalian neurons by flow cytometry, Brain Res. Brain Res. Protoc. 4 (1999) 280–287. [30] H. Takagi, M. Niwa, Y. Mizuno, S.N. Goto, T. Umemoto, Telmisartan as a metabolic sartan: the first meta-analysis of randomized controlled trials in metabolic syndrome, J. Am. Soc. Hypertens. 7 (2013) 229–235.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Protective effect of telmisartan against oxidative damage induced by high glucose in neuronal PC12 cell Habib Eslami a,b,∗ , Ali M. Sharifi a , Hamzeh Rahimi c , Maryam Rahati d a
Department of Pharmacology, Razi Institute of Drug Research, Tehran University of Medical Sciences, Tehran, Iran Department of Pharmacology, Research Center of Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran d Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran b c
h i g h l i g h t s • We investigated the effects of telmisartan on high glucose induced oxidative damage in PC12 cells. • Telmisartan counteracts high glucose-triggered oxidative damage and cell death in neuronal PC12 cells. • The antioxidant action of telmisartan and its inhibitory effect on NADPH oxidase may, at least in part, have mediated the inhibition of cell death.
a r t i c l e
i n f o
Article history: Received 2 August 2013 Received in revised form 21 October 2013 Accepted 24 October 2013 Keywords: Telmisartan High glucose Oxidative stress PC12 cells
a b s t r a c t Telmisartan is an angiotensin II type 1 receptor blocker and partial agonist of peroxisome proliferatoractivated receptor gamma (PPAR-␥). Here, we investigated the protective capacity of telmisartan against high glucose (HG)-elicited oxidative damage in PC12 cells. The activity of lactate dehydrogenase (LDH), NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT) as well as the levels of malondialdehyde (MDA), glutathione (GSH), intracellular reactive oxygen species (ROS), cell viability and DNA fragmentation were measured in HG-treated PC12 cells with and without telmisartan co-treatment. Moreover, the direct antioxidant effect of telmisartan was determined by 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay and protein expression of Bax, Bcl-2, cleaved caspase-3 and NOX subunit p47phox by western blotting. Telmisartan exhibited antioxidant activity in the ABTS assay with the IC50 value of 37.5 M. Pretreatment of PC12 cells with telmisartan, prior to HG exposure, was associated with a marked diminution in cleaved caspase-3 expression, DNA fragmentation, Bax/Bcl-2 ratio, intracellular ROS and MDA levels. Additionally, the cell viability, GSH level, SOD and CAT activity were notably elevated by telmisartan, whereas the activity and the protein expression of NADPH oxidase subunit p47phox were attenuated. Interestingly, co-treatment with GW9662, a PPAR-␥ antagonist, partially inhibited the beneficial effects of telmisartan. These findings suggest that telmisartan has protective effects on HG-induced neurotoxicity in PC12 cells, which may be related to its antioxidant action and inhibition of NADPH oxidase. Furthermore, the results show that PPAR-␥ activation is involved in the neuroprotective effects of telmisartan. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There is a large body of evidence highlighting that chronic high glucose (HG) condition leads to neuronal cell oxidative damage via NADPH oxidase-dependent generation of ROS [12,13]. ROS can attack proteins, lipid membranes and nucleic acids, causing oxidative injury to cells [20]. As well, excessive formation of ROS by HG
∗ Corresponding author at: Department of Pharmacology, School of Medicine, Hormozgan University of Medical Sciences, P.O. Box 79196-93116, Bandar Abbas, Iran. Tel.: +98 761 666 8477; fax: +98 761 666 8478. E-mail addresses: [email protected], [email protected] (H. Eslami). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.057
increases apoptosis, a possible mechanism of glucose neurotoxicity [25,26]. High-glucose milieu in diabetes not only generates more ROS but also attenuates anti-oxidant protective mechanisms through glycation of the antioxidant enzymes [12,20]. It is now accepted that an effective approach to prevention and treatment of diabetic complications such as neuropathy must focus on both early glycemic control and reducing factors related to oxidative stress [2]. Currently, no selective and effective therapeutic agent has been identified for clinical use to prevent or treat HG-induced neuronal cell injury and dysfunction [9]. Therefore, it is crucial to explore tools by which one can reduce oxidative stress in neurons to ameliorate the impaired neuronal function produced by hyperglycemia.
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Telmisartan has been widely used as a highly selective angiotensin II type 1 (AT1 ) receptor (AT1 R) blocker (ARB) for the treatment of hypertension. Beyond its antagonistic activity at AT1 receptors, it has additional favorable metabolic effect [4,30]. Recent studies demonstrated that telmisartan provided a significant antidiabetic effect and ameliorated hyperglycemia in rats with streptozotocin-induced or spontaneous diabetes [7,11,15]. The neuroprotective effect of telmisartan has been observed against many types of damage stimuli [10]. It is also recognized to attenuate oxidative stress in human umbilical vein endothelial cell (HUVEC) and neuronal cultures [3,22]. However, whether telmisartan has a neuroprotective role in high glucose condition has not been elucidated. To the best of our knowledge, this is the first study to investigate the protective effect of telmisartan in neuronal PC12 cells. The PC12 cell line has been extensively used as a cell model to study glucose neurotoxicity [25]. Previous studies in our laboratory have demonstrated the cytotoxicity of high glucose in PC12 cells [25,26]. The present study was designed to examine the protective effect of telmisartan on PC12 cells maintained under HG concentration. We investigated whether the neuroprotective effects of telmisartan are related to its antioxidant properties using an ABTS radical cation-scavenging assay. The alterations of antioxidant defenses (GSH level, SOD and CAT activities), intracellular ROS level, NADPH oxidase activation, MDA level, cell viability, and apoptotic neuronal cell death induced by high glucose were determined. We also used the PPAR-␥ antagonist GW9662 to examine the possible involvement of PPAR-␥ activation in the protective effect of telmisartan. 2. Methods 2.1. Cell culture and drug treatment PC12 cells were cultured as described previously [25]. After cultured in serum-free medium for 24 h, the cells were pre-incubated with 0.1–50 M of telmisartan (Sigma, T8949) for 2 h and then co-treated with telmisartan and high glucose (75 mM) for 72 h. In experiments where GW9662 (Sigma, M6191) was used together with telmisartan, cells were pre-treated with GW9662 (20 M) for 1 h before the addition of telmisartan. Control cells were cultured with normal glucose (23 mM), and not treated with telmisartan. Mannitol (75 mM) was also used as negative control for osmolarity. Telmisartan and GW9662 were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in experimental conditions was 0.1%. The selected concentrations of telmisartan and GW9662 were according to the previous study [23]. 2.2. Determination of cell viability, LDH release and intracellular ROS level Cell viability was measured using MTT assay as previously described [18,25]. The LDH activity was quantified using a LDH diagnostic kit (Stanbio Laboratory, USA). In brief, at the end of the drug treatment, the medium was collected and 100 L of it was added to 1 mL of LDH reagent. The absorbance was recorded at 340 nm. The production of intracellular ROS was measured by using 2,7-dichlorofluorescein diacetate (DCFH-DA) [29]. After treatment, cells were washed and incubated with DCFH-DA for 20 min. Then the relative levels of fluorescence were quantified by a fluorescence microplate reader.
supernatant was measured using the Bradford method [1]. The CAT activity was quantified according to the method of Shen et al. [27]. Briefly, 1.0 mL of hydrogen peroxide (19 mM) and 50 L of the supernatant were mixed with 1.95 mL of phosphate buffer (0.05 M, pH 7.0). The CAT activity was then estimated from the decrease in absorbency at 240 nm for 2 min. The SOD activity was measured using a SOD activity assay kit (BioVision, Mountain View, CA). Briefly, 20 L of the supernatant was added to 200 L tetrazolium and 20 L of enzyme solution. After incubating at 37 ◦ C for 20 min, the absorbance was read at 450 nm. The GSH level was measured according to the method of Gulati et al. [8]. Briefly, 100 L of the supernatant was mixed with 200 L of trichloroacetic acid and centrifuged. The resulting supernatant was mixed with phosphate buffer and its absorbance measured at 412 nm. MDA content was measured as previously described [21]. Briefly, 100 L of the supernatant was mixed with 1.5 mL of acetic acid (20%), 1.5 mL of thiobarbituric acid (0.8%), and 200 L of sodium dodecyl sulphate (8%). Each reaction mixture was heated for 60 min at 95 ◦ C and cooled to room temperature. Then, 5 mL of n-butanol was added. After mixing and centrifugation at 3000 × g for 10 min, the organic layer was collected and the absorbance measured at 532 nm. 2.4. ABTS radical cation-scavenging assay The antioxidant activity of telmisartan was determined by the method of Ghanta et al. [6] and expressed as IC50 , which was defined as the concentration of test material required to cause a 50% decrease in initial ABTS radical cation concentration. Briefly, 10 L of telmisartan (0.1–70 M) was mixed with 990 L of ABTS solution. The absorbance at 734 nm was recorded. Trolox was used as the standard. 2.5. Quantification of DNA fragmentation To examine DNA fragmentation, we used Cell Death Detection ELISAPlus kit (Roche Applied Sciences, Germany) and performed assay according to the manufacturer’s instructions, except that cells were cultured in 96-well plates at a density of 2 × 104 cells/well for the ELISA analysis. 2.6. Western blot analysis Western blot analysis was performed using whole-cell lysate from PC12cells as previously described [25]. Briefly, proteins samples were separated by SDS-PAGE and transferred onto a PVDF membrane which was incubated with primary antibodies including NADPH oxidase p47phox subunit (Santa Cruz, sc-14015), Bcl-2 (BioVision, 3033-100), Bax (BioVision, 3032-100), caspase-3 (BioVision, 3015-100) and secondary antibody (BioVision, 6401-05). Bands were visualized using ECL detection reagents. The resultant bands were quantified by densitometric analysis using the Image master program and normalized against -actin protein. 2.7. Estimation of NADPH oxidase activity in PC12 cells The activity of NADPH oxidase was quantified using lucigeninenhanced chemiluminiscence with a commercial kit (Genmed Scientifics Inc.) according to manufacturer’s protocols. 2.8. Statistical analysis
2.3. MDA, GSH, SOD, and CAT assay After drug treatment, the cells were washed and homogenized. The homogenate was centrifuged and the protein content of
Data were presented as mean ± S.E.M, and were analyzed by ANOVA followed by post hoc Dunnett’s t-test. The criterion for significance was a P value < 0.05.
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Fig. 1. Effects of telmisartan (TL) on cell viability (A), LDH release (B), ROS formation (C) and DNA fragmentation (D) in PC12 cells under HG condition. Cells were incubated with HG (75 mM) for 72 h. Telmisartan (0.1–50 M) was added to the culture 2 h prior to HG and 20 M of GW9662 (GW) 1 h before the addition of telmisartan. Data are mean ± SEM, n = 6. ## P < 0.01 compared with control. *P < 0.05, **P < 0.01 compared with the HG group, † P < 0.05 compared to telmisartan (10 M) treatment.
3. Result 3.1. Effect of telmisartan on HG-induced cytotoxicity as measured by cell viability and LDH release HG significantly reduced cell viability and increased LDH release compared to control cells (P < 0.01). However, incubation of cells with telmisartan (0.1–50 M) concentration-dependently reversed HG-induced cell death (Fig. 1A) and remarkably blocked LDH leakage (Fig. 1B). Mannitol (75 mM), which was used to create a high osmotic pressure mimicking HG condition, did not show significant change in cell viability. This result indicated that the effect of high glucose on proliferation was not secondary to osmotic load. Co-treatment with GW9662 (20 M) partially prevented the protective effects of telmisartan in MTT assay. Under the condition of normal glucose (23 mM d-glucose), DMSO (0.1%), GW9662 (20 M) or telmisartan (50 M) itself had no significant effect on the PC12 cells viability and also on none of the other following measurements in 3–8 experiments (data not shown). 3.2. Effects of telmisartan on MDA, GSH, SOD, CAT and ROS The GSH level, SOD activity, and CAT activity significantly decreased after exposure of PC12 cells to HG (# P < 0.05 vs. control, Table 1). However, intracellular MDA (Table 1) and ROS (Fig. 1C) were significantly raised. Following drug treatment, the MDA and ROS level significantly decreased (*P < 0.05), whereas CAT, SOD activity and the GSH level significantly increased (*P < 0.05) compared with HG-treated PC12 cells.
Fig. 2. Percentage inhibition of ABTS radical cations by telmisartan. Data are mean ± SEM, n = 3.
3.3. ABTS radical cation-scavenging capacity of telmisartan As shown in Fig. 2, telmisartan at different concentrations (0.1–70 M) was found to effectively scavenge ABTS radical cations (2.3–100% inhibition), with an average IC50 value of 37.5 M. IC50 value for Trolox, used as the reference compound, was 21.3 M. 3.4. Effect of telmisartan on the DNA fragmentation When PC12 cells were exposed to HG, there was a significant increase in DNA fragmentation vs. untreated controls (Fig. 1D),
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Table 1 Effects of telmisartan (TL) on antioxidant enzyme activities and level of MDA and GSH in rat PC12 cells under high glucose (HG) condition.a % of Control MDA Control HG (100 mM) HG + TL (0.1 M) HG + TL (1 M) HG + TL (10 M) HG + TL (50 M)
100 159.3 152.7 138.4 126.5 119.4
GSH ± ± ± ± ± ±
7.4 9.1# 8.9 6.5* 6.9* 5.7*
100 47.5 53.6 63.5 74.2 79.6
SOD ± ± ± ± ± ±
8.1 3.5# 3.4 4.3* 4.2* 6.1*
100 43.6 47.9 58.7 68.3 76.1
CAT ± ± ± ± ± ±
6.8 4.4# 3.8 3.5* 3.6* 5.1*
100 55.2 59.9 64.5 71.1 75.4
± ± ± ± ± ±
4.6 3.7# 4.4 4.7* 4.9* 6.8*
a Cultured PC12 cells were exposed to 75 mM glucose (HG). Telmisartan (TL) was added 2 h prior to glucose addition and the levels of antioxidant enzymes, MDA and GSH were measured 72 h later. Data are means ± SEM, n = 3–8. # P < 0.05 vs. control. * P < 0.05 vs. HG.
Fig. 3. Effects of telmisartan (TL) on the ratio of protein expression of Bax/Bcl-2 (A), upregulation of NADPH oxidase p47phox subunit protein expression (B), NADPH oxidase activity (C) and the expression of caspase-3 (D) in PC12 cells exposed to HG in the presence or absence of telmisartan (10 M) or 20 M GW9662 (GW) for 72 h. Data are mean ± SEM, n = 3. **P < 0.01 as compared to HG group, † P < 0.05 compared to telmisartan (10 M) treatment.
which was efficiently prevented by telmisartan (0.1–50 M). However, this effect of telmisartan was antagonized by GW9662.
as well as NADPH oxidase activity in PC12 cells. Pre-incubation of PC12 cells with telmisartan attenuated these changes.
3.5. Expression of NADPH oxidase p47phox subunit, Bcl-2, Bax and caspase-3 proteins
4. Discussion
Treating PC12 cells with HG led to a significant increase of the Bax/Bcl-2 ratio (Fig. 3A) and cleaved caspase-3 expression (Fig. 3D) as compared with the control group. Pre- and co-treatment with 10 M telmisartan decreased significantly the Bax/Bcl-2 ratio and caspase-3 expression, as compared with HG group. GW9662 reduced this effect of telmisartan (Fig. 3D). As shown in Fig. 3B and C, high glucose significantly up-regulated p47phox expression
This study found that telmisartan can exert a protective effect against ROS-mediated oxidative damage and apoptosis induced by high glucose in neuronal PC12 cells. Glucose induction of ROS is critical to the pathogenesis of diabetic neuropathy [20]. In addition, Hydroxyl radical is one of the highly ROS that causes the oxidative damage associated with diabetes [16]. Our results revealed that glucose enhanced ROS concentration in PC12 cells while simultaneously treating with telmisartan and glucose effectively
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attenuated ROS generation. This finding corroborates with previous studies which have shown that telmisartan acted as efficient scavenger of ROS particularly hydroxyl radicals [3]. To evaluate the antioxidant capacity of telmisartan, the ABTS cation radical-scavenging assay, was employed [6]. The results clearly showed that telmisartan displays antioxidant properties, which is consistent with previous studies that have demonstrated excellent antioxidant activity for telmisartan [3]. Moreover, it has been suggested that telmisartan, an AT1 receptor antagonist, inhibits intracellular oxidative stress, at least in part, in a receptor-independent manner, possibly owing to its lipophilic and antioxidant structure [24]. The chemical structure of telmisartan contains the benzimidazolic and benzoic groups that probably confer selective scavenging properties for hydroxyl radicals [3]. On the other hand, some studies have previously shown that PC12 cells derived from rat pheochromocytoma predominantly express AT2 receptor [14,19]. Based on these observations, it is very likely that in PC12 cells, the neuroprotective effects of telmisartan are independent of AT1 receptor inhibition. In the present study, we also found that NADPH-oxidase activity and protein expression of p47phox was significantly higher in GH-treated than control PC12 cells. p47phox is a cytosolic subunit of NOX and several reports support its importance in diabetes. For instance, it has been shown that deletion of p47 (phox) attenuates diabetes-induced glomerular injury and beta cell dysfunction [17]. Furthermore, superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47phox subunit of NADPH oxidase [28]. Our results demonstrated that telmisartan pre- and co-treatment suppressed the upregulated p47phox, and NADPH oxidase activity. In agreement with our present findings, previous observations have also shown that ARBs decrease NADPH oxidase activation associated with oxidative stress and neuronal apoptosis [23]. ROS also is a well-known initiator of apoptosis in many cell types [20]. In this study DNA fragmentation assay, a hallmark of apoptosis, Bax/Bcl-2 index and cleaved caspase-3 expression were used for measurements of apoptotic cell death. Telmisartan prevented HG-induced increase in caspase-3 expression, DNA fragmentation in PC12 cells and decreased the ratio of Bax/Bcl-2. This ratio is supposed to dictate the relative sensitivity or resistance of cells to a wide variety of apoptotic stimuli [26]. Interestingly, telmisartan has the unique character of having both ARB and PPAR-␥ agonistic effect [10], and Fuenzalida et al. reported that PPAR␥ up-regulates Bcl-2 in neurons [5]. Also, Tamami Haraguchi et al. suggested that telmisartan reduces neuronal apoptosis via a PPAR␥-dependent caspase-3 inhibiting mechanism [10]. We used the PPAR-␥ antagonist GW9662 to examine the possible involvement of PPAR-␥ activation in the protective effect of telmisartan. Results demonstrated that the beneficial effects of telmisartan were weakened by co-treatment with GW9662. Previous reports are in line with our current data [5,10], which indicate that telmisartan diminishes HG-elicited apoptosis in neuronal PC12 cells, possibly, via downregulation of Bax/Bcl-2 and caspase-3 expression. To mitigate cumulative burden of oxidative stress, cells generally utilize antioxidant defense systems and scavenge ROS. SOD and CAT are two important antioxidant enzymes [20]. In this regard, our present study indicated that PC12 cells treated with high glucose showed a marked rise in oxidative stress as evidenced by excessive ROS and MDA production, together with depletion of “endogenous antioxidant reserve,” including GSH contents, SOD and CAT activity level. However, co-treatment with telmisartan significantly attenuated oxidative damage to PC12 under high glucose condition, as reflected by the augmentation of antioxidant defense system (CAT, SOD and GSH) with accompanying decrease in MDA and ROS levels. These results confirm the in vitro antioxidant activity of telmisartan.
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In summary, our results indicate that telmisartan plays a protective role against HG-induced cell death in PC12 cells in a dose-dependent manner. This is possibly accomplished through diminution of NADPH oxidase activation and ROS formation in parallel with the increases in the GSH level, SOD and CAT activity. Furthermore, the results show that PPAR-␥ activation is involved in the neuroprotective effects of telmisartan. Acknowledgment The authors would like to thank research council of the Hormozganan University of Medical Sciences for financial supporting. References [1] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [2] N.C. Chilelli, S. Burlina, A. Lapolla, AGEs, rather than hyperglycemia, are responsible for microvascular complications in diabetes: a “glycoxidation-centric” point of view, Nutr. Metab. Cardiovasc. Dis. 23 (2013) 913–919. [3] S. Cianchetti, A. Del Fiorentino, R. Colognato, R. Di Stefano, F. Franzoni, R. Pedrinelli, Anti-inflammatory and anti-oxidant properties of telmisartan in cultured human umbilical vein endothelial cells, Atherosclerosis 198 (2008) 22–28. [4] M. Destro, F. Cagnoni, G.P. Dognini, V. Galimberti, C. Taietti, C. Cavalleri, E. Galli, Telmisartan: just an antihypertensive agent? A literature review, Expert Opin. Pharmacother. 12 (2011) 2719–2735. [5] K. Fuenzalida, R. Quintanilla, P. Ramos, D. Piderit, R.A. Fuentealba, G. Martinez, N.C. Inestrosa, M. Bronfman, Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis, J. Biol. Chem. 282 (2007) 37006–37015. [6] S. Ghanta, A. Banerjee, A. Poddar, S. Chattopadhyay, Oxidative DNA damage preventive activity and antioxidant potential of Stevia rebaudiana (Bertoni) Bertoni, a natural sweetener, J. Agr. Food Chem. 55 (2007) 10962–10967. [7] B.R. Goyal, K. Parmar, R.K. Goyal, A.A. Mehta, Beneficial role of telmisartan on cardiovascular complications associated with STZ-induced type 2 diabetes in rats, Pharmacol. Rep. 63 (2011) 956–966. [8] K. Gulati, A. Chakraborti, A. Ray, Modulation of stress-induced neurobehavioral changes and brain oxidative injury by nitric oxide (NO) mimetics in rats, Behav. Brain Res. 183 (2007) 226–230. [9] A.A. Habib, T.H. Brannagan 3rd, Therapeutic strategies for diabetic neuropathy, Curr. Neurol. Neurosci. Rep. 10 (2010) 92–100. [10] T. Haraguchi, K. Iwasaki, K. Takasaki, K. Uchida, T. Naito, A. Nogami, K. Kubota, T. Shindo, N. Uchida, S. Katsurabayashi, K. Mishima, R. Nishimura, M. Fujiwara, Telmisartan, a partial agonist of peroxisome proliferator-activated receptor gamma, improves impairment of spatial memory and hippocampal apoptosis in rats treated with repeated cerebral ischemia, Brain Res. 1353 (2010) 125–132. [11] G. Hasegawa, M. Fukui, H. Hosoda, M. Asano, I. Harusato, M. Tanaka, E. Shiraishi, T. Senmaru, K. Sakabe, M. Yamasaki, J. Kitawaki, A. Fujinami, M. Ohta, H. Obayashi, N. Nakamura, Telmisartan, an angiotensin II type 1 receptor blocker, prevents the development of diabetes in male Spontaneously Diabetic Torii rats, Eur. J. Pharmacol. 605 (2009) 164–169. [12] A. Hosseini, M. Abdollahi, Diabetic neuropathy and oxidative stress: therapeutic perspectives, Oxid. Med. Cell. Longev. 2013 (2013) 168039. [13] F. Jiang, Y. Zhang, G.J. Dusting, NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair, Pharmacol. Rev. 63 (2011) 218–242. [14] K. Kijima, H. Matsubara, S. Murasawa, K. Maruyama, N. Ohkubo, Y. Mori, M. Inada, Regulation of angiotensin II type 2 receptor gene by the protein kinase C-calcium pathway, Hypertension 27 (1996) 529–534. [15] S. Kushwaha, G.B. Jena, Telmisartan ameliorates germ cell toxicity in the STZinduced diabetic rat: studies on possible molecular mechanisms, Mutat. Res. 755 (2013) 11–23. [16] B. Lipinski, Hydroxyl radical and its scavengers in health and disease, Oxid. Med. Cell Longev. 2011 (2011) 809696. [17] G.C. Liu, F. Fang, J. Zhou, K. Koulajian, S. Yang, L. Lam, H.N. Reich, R. John, A.M. Herzenberg, A. Giacca, G.Y. Oudit, J.W. Scholey, Deletion of p47phox attenuates the progression of diabetic nephropathy and reduces the severity of diabetes in the Akita mouse, Diabetologia 55 (2012) 2522–2532. [18] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [19] S. Murasawa, H. Matsubara, M. Urakami, M. Inada, Regulatory elements that mediate expression of the gene for the angiotensin II type 1a receptor for the rat, J. Biol. Chem. 268 (1993) 26996–27003. [20] G. Negi, A. Kumar, R.P. Joshi, S.S. Sharma, Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle, Biochem. Biophys. Res. Commun. 408 (2011) 1–5.
36
H. Eslami et al. / Neuroscience Letters 558 (2014) 31–36
[21] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [22] M.S. Ola, M.M. Ahmed, H.M. Abuohashish, S.S. Al-Rejaie, A.S. Alhomida, Telmisartan ameliorates neurotrophic support and oxidative stress in the retina of streptozotocin-induced diabetic rats, Neurochem. Res. 38 (2013) 1572– 1579. [23] T. Pang, J. Wang, J. Benicky, E. Sanchez-Lemus, J.M. Saavedra, Telmisartan directly ameliorates the neuronal inflammatory response to IL-1beta partly through the JNK/c-Jun and NADPH oxidase pathways, J. Neuroinflammation 9 (2012) 102. [24] J. Shao, M. Nangaku, R. Inagi, H. Kato, T. Miyata, T. Matsusaka, E. Noiri, T. Fujita, Receptor-independent intracellular radical scavenging activity of an angiotensin II receptor blocker, J. Hypertens. 25 (2007) 1643–1649. [25] A.M. Sharifi, H. Eslami, B. Larijani, J. Davoodi, Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells, Neurosci. Lett. 459 (2009) 47–51.
[26] A.M. Sharifi, S.H. Mousavi, M. Farhadi, B. Larijani, Study of high glucose-induced apoptosis in PC12 cells: role of bax protein, J. Pharmacol. Sci. 104 (2007) 258–262. [27] X. Shen, Y. Tang, R. Yang, L. Yu, T. Fang, J.A. Duan, The protective effect of Zizyphus jujube fruit on carbon tetrachloride-induced hepatic injury in mice by antioxidative activities, J. Ethnopharmacol. 122 (2009) 555–560. [28] S.W. Suh, E.T. Gum, A.M. Hamby, P.H. Chan, R.A. Swanson, Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase, J. Clin. Invest. 117 (2007) 910–918. [29] F.X. Sureda, C. Gabriel, J. Comas, M. Pallas, E. Escubedo, J. Camarasa, A. Camins, Evaluation of free radical production, mitochondrial membrane potential and cytoplasmic calcium in mammalian neurons by flow cytometry, Brain Res. Brain Res. Protoc. 4 (1999) 280–287. [30] H. Takagi, M. Niwa, Y. Mizuno, S.N. Goto, T. Umemoto, Telmisartan as a metabolic sartan: the first meta-analysis of randomized controlled trials in metabolic syndrome, J. Am. Soc. Hypertens. 7 (2013) 229–235.
