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A comparative study on the cardioprotective potential of atorvastatin and simvastatin in hyperhomocysteinemic rat hearts
Author’s Accepted Manuscript A comparative study on the cardioprotective potential of atorvastatin and simvastatin in hyperhomocysteinemic rat hearts Ankur Rohilla, Ayaz Ahmad, M.U. Khan, Razia Khanam www.elsevier.com/locate/ejphar
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S0014-2999(15)30110-2 http://dx.doi.org/10.1016/j.ejphar.2015.06.045 EJP70080
To appear in: European Journal of Pharmacology Received date: 9 September 2014 Revised date: 19 June 2015 Accepted date: 23 June 2015 Cite this article as: Ankur Rohilla, Ayaz Ahmad, M.U. Khan and Razia Khanam, A comparative study on the cardioprotective potential of atorvastatin and simvastatin in hyperhomocysteinemic rat hearts, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.06.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
--Research Paper--
A comparative study on the cardioprotective potential of atorvastatin and simvastatin in hyperhomocysteinemic rat hearts
Ankur Rohillaa*, Ayaz Ahmada, M.U.Khanb, Razia Khanamc
a
Department of Pharmacy, NIMS University, Shobha Nagar, Jaipur - 303121, Rajasthan, India
b
Sri Sai College of Pharmacy, Badhani, Pathankot - 145001, Punjab, India c
Faculty of Pharmacy, Jamia Hamdard University, Delhi - 110062, India
*Address for Correspondence: Ankur Rohilla Research Scholar Department of Pharmacy, NIMS University, Shobha Nagar, Jaipur - 303121, Rajasthan, India (M): +91-9896561778 E-mail: [email protected] ABSTRACT The present study has been deliberated in order to compare the cardioprotective potential of 3-hydroxymethyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, Atorvastatin and Simvastatin in hyperhomocysteinemic rat hearts. L-methionine (1.7 g/kg/day orally) was administered to rats for 4 weeks to produce experimental hyperhomocysteinemia (Hhcy). Isolated Langendorff-perfused normal and hyperhomocysteinemic rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 120 min. The extent of myocardial damage was assessed in terms of myocardial infarct size using
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triphenyltetrazolium chloride (TTC) staining, and release of creatine kinase (CK) and lactate dehydrogenase (LDH) in the coronary effluent; whereas the oxidative stress in the heart was assessed by measuring lipid peroxidation, superoxide anion generation and reduced glutathione. Ischemia-reperfusion (I/R) was noted to produce myocardial injury in normal and hyperhomocysteinemic rat hearts, assessed in terms of increase in myocardial infarct size, LDH and CK in coronary effluent and oxidative stress. Treatment with Atorvatstain (50 µM) and Simvastatin (10 µM) afforded cardioprotection against I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts as assessed in terms of reductions in myocardial infarct size, LDH and CK levels in coronary effluent and oxidative stress. It may be concluded that reductions in the high degree of oxidative stress may be responsible for the observed cardioprotective potential of Atorva-and Simvastatin, and both statins can be used interchangeably to afford cardioprotection against I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts. Key Words: Hyperhomocysteinemia, Atorvastatin, Simvastatin, Oxidative stress 1. Introduction The initial ischemic damage followed by further damage induced by reperfusion is referred to as myocardial I/R injury (Qian et al., 2014). It is believed that I/R-induced myocardial injury is related to the increased reactive oxygen species generation, calcium overloading, apoptotic and necrotic myocytes death, and the loss of membrane phospholipids, especially during reperfusion (Ferdinandy et al., 2007; Balakumar et al., 2008; Rohilla et al., 2011). Hhcy, a pathological condition characterized by abnormally high levels of homocysteine in the blood, is considered as an independent risk factor for
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various cardiovascular diseases like atherosclerosis, endothelial dysfunction, hypertension, myocardial infarction, chronic heart failure and obesity Thiengburanatham, 2009; Ciaccio and Bellia, 2010; Baszczuk et al., 2014; Zhou et al., 2014). Additionally, Hhcy has been well reported to increase the generation of reactive oxygen species, downregulate endothelial nitric oxide synthase (eNOS), reduce the generation of nitric oxide (NO), and increase the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), which may affect the function of coronary endothelium (Suematsu et al., 2007; Bogdanski et al., 2008). Statins, the HMGCoA reductase inhibitors, have been extensively used for the treatment of hyperlipidemia (Law et al., 2003). Atorvastatin and Simvastatin, the potent inhibitors of HMG-CoA reductase, have been well reported to possess various beneficial effects in the treatment of various cardiovascular diseases such as myocardial infarction, stroke, unstable angina and revascularization due to their potent antioxidant properties (Law et al., 2003; DiNicolantonio et al., 2013; Owens et al., 2014). Atorvastatin treatment reduced the infarct size in isolated Langendorff-perfused heart model by activating pro-survival kinases and NO levels (Bell and Yellon, 2003; Efthymiou and Yellon, 2005). In addition, Simvastatin has been reported to minimize the myocardial contractile dysfunction and lethal ischemic injury in isolated Langendorff-perfused rat heart model (Zheng and Hu, 2007; Yin et al., 2007). Moreover, treatment with Atorvastatin reduced the thiobarbituric acid reactive oxygen substances (TBARS) levels and lipid peroxidation levels, the oxidative stress markers, which further evidenced the antioxidant potential of it in affording cardioprotection (Ozacmak et al., 2007). Also, experimental studies have shown that treatment with Simvastatin resulted in reductions of malondialdehyde (MDA)
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levels and increases in the superoxide dysmutase (SOD) and NO levels accounting for its antioxidant and cardioprotective potential (Delbosc et al., 2002). Furthermore, Atorvastatin treatment reduced the vascular and cardiac free radical formation, and normalized the NADPH oxidase expression (Bolayirli et al., 2007). Treatment with Simvastatin prevented the aortic production of reactive oxygen species and inhibited the lipid peroxidation products like TBARS, confirming its antioxidant potential in affording cardioprotection (Naiyra et al., 2010). Therefore, the present study has been undertaken in order to compare and investigate the cardioprotective effect of Statins, i.e. Atorvastatin and Simvastatin against I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts.
2. Materials and Methods 2.1 Experimental Animals The experimental protocol used in the present study was approved by the Institutional Animal Ethical Committee. Wistar albino rats of either sex weighing 175-225 gm were used. They were housed in Institutional animal housing and were maintained on rat feed (Kisan Feeds Ltd., Chandigarh, India) and tap water ad libitum. 2.2 Isolated Rat Heart Preparation
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Rats were heparinized (500 IU i.p.) and killed by stunning. The heart was rapidly excised and immediately mounted on a Langendorff apparatus (Langendorff, 1895). The heart was enclosed in a double walled jacket, the temperature of which was maintained at 37°C by circulating hot water. The preparation was perfused with Krebs-Henseleit (K-H) solution pH 7.4, maintained at 37 °C and bubbled with 95% O2 and 5% CO2. The coronary flow rate was maintained at around 7 ml/min, and the perfusion pressure was kept at 80 mmHg. Global ischemia was produced for 30 min by blocking the inflow of physiological solution and it was followed by perfusion for 120 min. 2.3 Laboratory Assays Myocardial infarct size was measured macroscopically using TTC staining employing volume method (Parikh and Singh, 1999). The myocardial injury was assessed by measuring the release of CK-MB and LDH in the coronary effluent using the commercially available enzymatic kits (Vital Diagnostics, Thane, Maharashtra, India). The level of TBARS, an index of lipid peroxidation in the heart was estimated according to the method of Ohkawa et al. (Ohkawa et al., 1979). The superoxide anion generation was assessed by estimating the reduced nitro blue tetrazolium (NBT) using the method of Wang et al. (Wang et al., 1998). Moreover, the reduced glutathione (GSH) content in each heart was estimated using the method of Beutler et al. (Beutler et al., 1963). 2.4 Experimental Protocol In the present study, twelve groups were employed each comprising of 8-10 animals (Fig 1). In all groups, each isolated perfused heart was allowed to stabilize for 10 min by perfusing with K-H solution.
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Group I (Normal Control): Isolated normal rat heart was perfused for 150 min using K-H solution after 10 min of stabilization. Group II (I/R-Control): Isolated normal rat heart after 10 min of stabilization was subjected to 30 min of global ischemia followed by 120 min of reperfusion Group III (Ator per se Normal Control): After 10-min stabilization, the isolated normal rat heart was infused with Atorvastatin (50 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution Group IV (Sim per se Normal Control): After 10-min stabilization, the isolated normal rat heart was infused with Simvastatin (10 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution Group V (Ator Treated I/R-Control): After 10 min of stabilization, isolated normal rat heart was infused with Atorvastatin (50 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion. Group VI (Sim Treated I/R-Control): After 10 min of stabilization, isolated normal rat heart was infused with Simvastatin (10 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion. Group VII (Hhcy control): The isolated Hhcy rat heart was perfused for 150 min using K-H solution after 10-min stabilization. Group VIII (Hhcy-I/R control): Isolated Hhcy rat heart after 10 min of stabilization was subjected to 30 min of global ischemia followed by 120 min of reperfusion Group IX (Ator per se Hhcy-control): After 10-min stabilization, the isolated Hhcy rat heart was infused with Atorvastatin (50 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution
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Group X (Sim per se Hhcy-control): After 10-min stabilization, the isolated Hhcy rat heart was infused with Simvastatin (10 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution Group XI (Ator Treated Hhcy-I/R Control): After 10 min of stabilization, isolated Hhcy rat heart was infused with Atorvastatin (50 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion Group XII (Sim Treated Hhcy-I/R Control): After 10 min of stabilization, isolated Hhcy rat heart was infused with Simvastatin (10 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion 2.5 Statistical Analysis The results were expressed as mean ± S.D. The data obtained from various groups were statistically analyzed using two-way ANOVA followed by Tukey’s multiple-comparison test. A P value < 0.05 was considered to be statistically significant. 2.6 Drugs and Chemicals The LDH and CK enzymatic estimation kits were purchased from Vital Diagnostics, Thane, Maharastra, India. DTNB and NBT were obtained from Loba Chem, Mumbai, India. Atorvastatin, Simvastatin, 1,1,3,3-tetramethoxy propane and reduced glutathione were procured from Sigma-Aldrich, USA. All other reagents used in this study were of analytical grade.
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3. Results L-methionine (1.7 g/kg/day, p.o.) was given to rats for 4 weeks via oral gavage which produced hyperhomocysteinemia (21.25±1.93 µM/L) when compared with normal rats (4.51±0.61 µM/L). In addition, L-methionine administration did not produce mortality in rats. 3.1 Effect of Atorva-and Simvastatin in I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts
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Global ischemia followed by reperfusion significantly increased the infarct size in normal and hyperhomocysteinemic rat hearts. In addition, I/R was noted to increase the CK and LDH release in the coronary effluent in normal and hyperhomocysteinemic rat hearts (Fig 2). The maximum release of CK was noted at 5 min of reperfusion, whereas the maximum release of LDH was noted immediately after reperfusion (Fig 3-4). The hyperhomocysteinemic rat hearts showed enhanced myocardial injury when compared with normal rat hearts subjected to I/R. Treatment with Atorvastatin (50 μM) and Simvastatin (10 μM) significantly attenuated I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts to a similar extent, as assessed in terms of reduction in myocardial infarct size and decreased release of CK and LDH in the coronary effluent (Fig 2-4). 3.2 Effect of Atorva-and Simvastatin in I/R-induced oxidative stress in normal and hyperhomocysteinemic rat hearts Lipid peroxidation, measured in terms of TBARS, and superoxide anion generation, assessed in terms of reduced NBT, were significantly increased and levels of reduced GSH were found to be decreased in normal and hyperhomocysteinemic rat hearts subjected to I/R (Fig 5-7). In addition, hyperhomocysteinemic rat hearts showed high oxidative stress when compared with normal rat hearts. Atorvastatin (50 μM) and Simvastatin treatment (10 μM) attenuated the I/R-induced oxidative stress to a similar extent in normal and hyperhomocysteinemic rat hearts, as assessed in terms of reduction in TBARS and superoxide anion generation, and the consequent increase in GSH (Fig 5-7).
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4. Discussion The ischemic heart disease (IHD) is a major cause of morbidity and mortality worldwide. The myocardial ischemia denotes a condition in which the heart is deprived of oxygen and other nutrients resulting in myocardial damage or dysfunction (Yu et al., 2014). The reperfusion refers to the restorations of the blood supply to the affected myocardium. Cardiac tissue damage caused when blood supply returns after a prolonged period of ischemia resulting in inflammation and oxidative damage is known as I/R
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injury. It has been extensively reported that the increase in infarct size and the release of LDH and CK are documented to be an index of I/R-induced myocardial injury (Rohilla et al., 2012 a,b). In the present study, 30 min of global ischemia followed by 120 min of reperfusion produced myocardial injury, as assessed in terms of increased infarct size in the heart and elevated release of CK and LDH in the coronary effluent. The maximal release of CK was observed after 5 min of reperfusion, whereas peak release of LDH was noted immediately after reperfusion, which are in accordance with our earlier studies (Rohilla et al., 2012a,b). Moreover, increase in lipid peroxidation and superoxide anion generation alongwith consequent decrease of reduced GSH have been reported to be the indicators of oxidative stress (Ungvari et al., 2003; Devi et al., 2006). In the present study, the lipid peroxidation measured in terms of TBARS, and superoxide anion generation assessed in terms of reduced NBT, were noted to be increased, whereas the reduced GSH level was decreased as a result of I/R. These indicators suggest the development of I/R-induced oxidative stress, which may be responsible for the noted I/Rinduced myocardial injury. L-methionine administration (1.7 g/kg/day orally) in rats for 4 weeks produced Hhcy (Balakumar et al., 2009). In the present study, a marked increase in the infarct size of heart and, release of CK and LDH in the coronary effluent were noted in the hyperhomocysteinemic rat heart when compared with the normal rat heart. Hhcy has been well reported to downregulate NO bioavailability and produce high amount of oxidative stress in the heart by activating NADPH oxidase-mediated reactive oxygen species generation (Balakumar et al., 2007). Also, the Hhcy-induced oxidative stress occurs as a result of decreased expression and activity of key antioxidant enzymes, as
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well as increased enzymatic generation of superoxide anion (Shah and Singh, 2007). Thus, it may be suggested that the development of high degree of oxidative stress may be responsible for the observed marked increase in myocardial injury in the hyperhomocysteinemic rat heart. This contention is supported by the fact that a marked increase in lipid peroxidation and superoxide anion generation and a subsequent decrease in GSH levels were noted in the hyperhomocysteinemic rat heart when compared with the normal rat heart. Statins, referred to as the HMG-CoA reductase inhibitors, have been regarded as the potent inhibitors of cholesterol biosynthesis and possess valuable effects in the prevention of coronary heart disease (Liao, 2003). The statins have been well accepted to possess various extrahepatic and cholesterol independent effects, termed as the pleiotropic effects, which include direct effects on vascular tissue, heart, kidney, bone and glucose metabolism (Liao, 2003; Laufs and Bohm, 2005). Administration of Atorvastatin has been reported to reduce the infarct size in isolated Langendorff-perfused heart model by activating pro-survival kinases and increasing NO levels (Bell and Yellon, 2003; Efthymiou and Yellon, 2005), whereas, Simvastatin administration lessened the myocardial contractile dysfunction and lethal ischemic injury in isolated Langendorff-perfused rat heart model (Zheng and Hu, 2007; Yin et al., 2007). The present study investigated the cardioprotective potential of Atorva-and Simvastatin against I/R injury in rat hearts administered at the onset of reperfusion. The data demonstrates that Atorvastatin (50 μM) and Simvastatin (10 μM) administered at the onset of reperfusion afforded cardioprotection, by significantly attenuating I/R-induced myocardial injury in rat hearts as assessed in terms of reductions in myocardial infarct
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size and decreased release of CK and LDH in the coronary effluent, which is in accordance with our recent studies (Rohilla et al., 2012 a,b). Numbers of experimental studies have reported the antioxidant potential of Atorva-and Simvastatin in order to afford cardioprotection. Treatment with Atorvastatin lowered the increased levels of serum homocysteine in rats, providing an evidence of inhibitory role of Atorvastatin in Hhcy (Bhandari et al., 2011). Atorvastatin treatment has been noted to reduce the oxidative stress markers such as TBARS and lipid peroxidation levels in a rat model of I/R (Ozacmak et al., 2007). Moreover, Atorvastatin treatments reduced the vascular and cardiac free radical formation and normalized the expression of the NADPH oxidase, evidencing its anti-oxidative properties (Bolayirli et al., 2007). Also, administration of Atorvastatin induced a significant decrease in MDA levels alongwith a significant increase of SOD activity that further accounts for its antioxidant potential in order to afford cardioprotecton (Castro et al., 2008). Furthermore, Simvastatin treatment inhibited the protein and lipid peroxidation products such as TBARS and prevented the aortic production of reactive oxygen species, confirming its antioxidant potential. Additionally, experimental studies have shown that treatment with Simvastatin resulted in reductions of MDA levelas alongwith increase of SOD and NO levels, accounting for its cardioprotective potential (Delbosc et al., 2002). Another experimental study in rats showed a significant decrease in oxidative stress in Simvastatin treated diabetic-hypercholesterolemic rat hearts that further confirmed its antioxidant potential in affording cardioprotection (Adameova et al., 2006). In the present study, Atorvastatin (50 μM) and Simvastatin (10 μM) treatment was noted to significantly reduce the oxidative stress in normal and hyperhomocysteinemic
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rat heart subjected to I/R, as assessed in terms of reduction in TBARS, and superoxide anion generation and consequent increase in the reduced form of GSH. This strongly suggests that the high degree of oxidative stress in hyperhomocysteinemic rat hearts in response to I/R injury was attenuated by Atorva-and Simvastatin in order to mimic cardioprotection. Moreover, the present study is the first of its kinds to compare the cardioprotective potential of statins, i.e. Atorva-and Simvastatin in the I/R-induced myocardial injury in normal and in hyperhomocysteinemic rat hearts. The results obtained in the present study showed that both the statins, i.e. Atorvastatin and Simvastatin can be used interchangeably in order to afford cardioprotection against I/Rinduced myocardial injury in normal and hyperhomocysteinemic rat hearts as assessed in terms of reduction in infarct size, CK and LDH in the coronary effluent alongwith reduction in oxidative stress.
5. Conclusion Hhcy transforms the myocardium susceptible to I/R-induced oxidative stress, and this high degree of oxidative stress produced by hyperhomocysteinemic rat heart in response to I/R, may be responsible for the prevention of cardioprotection. Treatment with Atorvastatin and Simvastatin afforded cardioprotection in normal and hyperhomocysteinemic rat hearts by abolishing the high degree of oxidative stress in Hhcy. Further studies are underway in our laboratory in order to explore the cardioprotective mechanisms of Statins in hyperhomocysteinemic rat hearts.
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6. Acknowledgments We wish to express our gratitude to Dr. Balvir Singh Tomar, Honorable Chairman; Dr. (Mrs) Shobha Tomar, Honorable Managing Director; Dr. K.C. Singhal, Honorable Vice Chancellor; Dr. K.P. Singh, Honorable Registrar and Prof. Govind Mohan, Director Pharmaceutical Sciences, NIMS University, Shobha Nagar, Jaipur-303121, Rajasthan for their praiseworthy suggestions and constant support for excellant research area.
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Figure 1.
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Figure 2.
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5 min Ator per se Normal Control Sim Treated I/R-Control Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
Figure 4.
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I/R-Control Ator Treated I/R-Control Hhcy-I/R control Ator Treated Hhcy-I/R Control
24
Ator per se Normal Control Sim Treated I/R-Control Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
Figure 5.
120
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100
TBARS (nM/gm)
* 80
¤ װ
60
¤
װ
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0 Normal Control Sim per se Normal Control
I/R-Control Ator Treated I/R-Control
Ator per se Normal Control Sim Treated I/R-Control
Hhcy control Sim per se Hhcy-control
Hhcy-I/R control Ator Treated Hhcy-I/R Control
Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
25
Figure 6.
120
○,≈
Reduced NBT (pM/min/mg)
100
80
* ¤ ¤
60
װ
40
װ
20
0 Normal Control Sim per se Normal Control
I/R-Control Ator Treated I/R-Control
Ator per se Normal Control Sim Treated I/R-Control
Hhcy control Sim per se Hhcy-control
Hhcy-I/R control Ator Treated Hhcy-I/R Control
Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
26
Figure 7.
GSH (uM/mg wet tissue weight)
1.2
װ
1
װ
0.8
¤
¤
* 0.6
○,≈ 0.4
0.2
0 Normal Control Sim per se Normal Control Hhcy control Sim per se Hhcy-control
I/R-Control Ator Treated I/R-Control Hhcy-I/R control Ator Treated Hhcy-I/R Control
27
Ator per se Normal Control Sim Treated I/R-Control Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
Figure Legends
Figure 1:
Diagram showing schematic representation of experimental protocol
# represents 10 min stabilization; © represents 150 minutes of K-H perfusion, Ξ represents 30 minutes of global ischemia; ® represents 120 minutes of K-H reperfusion; Θ represents 10 minutes of Atorvastatin infusion; Ω represents 10 minutes of Simvastatin infusion
Figure 2:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in infarct size Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 3:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in CK levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 4:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in LDH levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 5:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in TBARS levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 6:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in superoxide anion generation Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 7:
Effect of Atorvastatin and Simvastatin in I/R-induced decrease in reduced GSH levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
28
PII: DOI: Reference:
S0014-2999(15)30110-2 http://dx.doi.org/10.1016/j.ejphar.2015.06.045 EJP70080
To appear in: European Journal of Pharmacology Received date: 9 September 2014 Revised date: 19 June 2015 Accepted date: 23 June 2015 Cite this article as: Ankur Rohilla, Ayaz Ahmad, M.U. Khan and Razia Khanam, A comparative study on the cardioprotective potential of atorvastatin and simvastatin in hyperhomocysteinemic rat hearts, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.06.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
--Research Paper--
A comparative study on the cardioprotective potential of atorvastatin and simvastatin in hyperhomocysteinemic rat hearts
Ankur Rohillaa*, Ayaz Ahmada, M.U.Khanb, Razia Khanamc
a
Department of Pharmacy, NIMS University, Shobha Nagar, Jaipur - 303121, Rajasthan, India
b
Sri Sai College of Pharmacy, Badhani, Pathankot - 145001, Punjab, India c
Faculty of Pharmacy, Jamia Hamdard University, Delhi - 110062, India
*Address for Correspondence: Ankur Rohilla Research Scholar Department of Pharmacy, NIMS University, Shobha Nagar, Jaipur - 303121, Rajasthan, India (M): +91-9896561778 E-mail: [email protected] ABSTRACT The present study has been deliberated in order to compare the cardioprotective potential of 3-hydroxymethyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, Atorvastatin and Simvastatin in hyperhomocysteinemic rat hearts. L-methionine (1.7 g/kg/day orally) was administered to rats for 4 weeks to produce experimental hyperhomocysteinemia (Hhcy). Isolated Langendorff-perfused normal and hyperhomocysteinemic rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 120 min. The extent of myocardial damage was assessed in terms of myocardial infarct size using
1
triphenyltetrazolium chloride (TTC) staining, and release of creatine kinase (CK) and lactate dehydrogenase (LDH) in the coronary effluent; whereas the oxidative stress in the heart was assessed by measuring lipid peroxidation, superoxide anion generation and reduced glutathione. Ischemia-reperfusion (I/R) was noted to produce myocardial injury in normal and hyperhomocysteinemic rat hearts, assessed in terms of increase in myocardial infarct size, LDH and CK in coronary effluent and oxidative stress. Treatment with Atorvatstain (50 µM) and Simvastatin (10 µM) afforded cardioprotection against I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts as assessed in terms of reductions in myocardial infarct size, LDH and CK levels in coronary effluent and oxidative stress. It may be concluded that reductions in the high degree of oxidative stress may be responsible for the observed cardioprotective potential of Atorva-and Simvastatin, and both statins can be used interchangeably to afford cardioprotection against I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts. Key Words: Hyperhomocysteinemia, Atorvastatin, Simvastatin, Oxidative stress 1. Introduction The initial ischemic damage followed by further damage induced by reperfusion is referred to as myocardial I/R injury (Qian et al., 2014). It is believed that I/R-induced myocardial injury is related to the increased reactive oxygen species generation, calcium overloading, apoptotic and necrotic myocytes death, and the loss of membrane phospholipids, especially during reperfusion (Ferdinandy et al., 2007; Balakumar et al., 2008; Rohilla et al., 2011). Hhcy, a pathological condition characterized by abnormally high levels of homocysteine in the blood, is considered as an independent risk factor for
2
various cardiovascular diseases like atherosclerosis, endothelial dysfunction, hypertension, myocardial infarction, chronic heart failure and obesity Thiengburanatham, 2009; Ciaccio and Bellia, 2010; Baszczuk et al., 2014; Zhou et al., 2014). Additionally, Hhcy has been well reported to increase the generation of reactive oxygen species, downregulate endothelial nitric oxide synthase (eNOS), reduce the generation of nitric oxide (NO), and increase the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), which may affect the function of coronary endothelium (Suematsu et al., 2007; Bogdanski et al., 2008). Statins, the HMGCoA reductase inhibitors, have been extensively used for the treatment of hyperlipidemia (Law et al., 2003). Atorvastatin and Simvastatin, the potent inhibitors of HMG-CoA reductase, have been well reported to possess various beneficial effects in the treatment of various cardiovascular diseases such as myocardial infarction, stroke, unstable angina and revascularization due to their potent antioxidant properties (Law et al., 2003; DiNicolantonio et al., 2013; Owens et al., 2014). Atorvastatin treatment reduced the infarct size in isolated Langendorff-perfused heart model by activating pro-survival kinases and NO levels (Bell and Yellon, 2003; Efthymiou and Yellon, 2005). In addition, Simvastatin has been reported to minimize the myocardial contractile dysfunction and lethal ischemic injury in isolated Langendorff-perfused rat heart model (Zheng and Hu, 2007; Yin et al., 2007). Moreover, treatment with Atorvastatin reduced the thiobarbituric acid reactive oxygen substances (TBARS) levels and lipid peroxidation levels, the oxidative stress markers, which further evidenced the antioxidant potential of it in affording cardioprotection (Ozacmak et al., 2007). Also, experimental studies have shown that treatment with Simvastatin resulted in reductions of malondialdehyde (MDA)
3
levels and increases in the superoxide dysmutase (SOD) and NO levels accounting for its antioxidant and cardioprotective potential (Delbosc et al., 2002). Furthermore, Atorvastatin treatment reduced the vascular and cardiac free radical formation, and normalized the NADPH oxidase expression (Bolayirli et al., 2007). Treatment with Simvastatin prevented the aortic production of reactive oxygen species and inhibited the lipid peroxidation products like TBARS, confirming its antioxidant potential in affording cardioprotection (Naiyra et al., 2010). Therefore, the present study has been undertaken in order to compare and investigate the cardioprotective effect of Statins, i.e. Atorvastatin and Simvastatin against I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts.
2. Materials and Methods 2.1 Experimental Animals The experimental protocol used in the present study was approved by the Institutional Animal Ethical Committee. Wistar albino rats of either sex weighing 175-225 gm were used. They were housed in Institutional animal housing and were maintained on rat feed (Kisan Feeds Ltd., Chandigarh, India) and tap water ad libitum. 2.2 Isolated Rat Heart Preparation
4
Rats were heparinized (500 IU i.p.) and killed by stunning. The heart was rapidly excised and immediately mounted on a Langendorff apparatus (Langendorff, 1895). The heart was enclosed in a double walled jacket, the temperature of which was maintained at 37°C by circulating hot water. The preparation was perfused with Krebs-Henseleit (K-H) solution pH 7.4, maintained at 37 °C and bubbled with 95% O2 and 5% CO2. The coronary flow rate was maintained at around 7 ml/min, and the perfusion pressure was kept at 80 mmHg. Global ischemia was produced for 30 min by blocking the inflow of physiological solution and it was followed by perfusion for 120 min. 2.3 Laboratory Assays Myocardial infarct size was measured macroscopically using TTC staining employing volume method (Parikh and Singh, 1999). The myocardial injury was assessed by measuring the release of CK-MB and LDH in the coronary effluent using the commercially available enzymatic kits (Vital Diagnostics, Thane, Maharashtra, India). The level of TBARS, an index of lipid peroxidation in the heart was estimated according to the method of Ohkawa et al. (Ohkawa et al., 1979). The superoxide anion generation was assessed by estimating the reduced nitro blue tetrazolium (NBT) using the method of Wang et al. (Wang et al., 1998). Moreover, the reduced glutathione (GSH) content in each heart was estimated using the method of Beutler et al. (Beutler et al., 1963). 2.4 Experimental Protocol In the present study, twelve groups were employed each comprising of 8-10 animals (Fig 1). In all groups, each isolated perfused heart was allowed to stabilize for 10 min by perfusing with K-H solution.
5
Group I (Normal Control): Isolated normal rat heart was perfused for 150 min using K-H solution after 10 min of stabilization. Group II (I/R-Control): Isolated normal rat heart after 10 min of stabilization was subjected to 30 min of global ischemia followed by 120 min of reperfusion Group III (Ator per se Normal Control): After 10-min stabilization, the isolated normal rat heart was infused with Atorvastatin (50 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution Group IV (Sim per se Normal Control): After 10-min stabilization, the isolated normal rat heart was infused with Simvastatin (10 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution Group V (Ator Treated I/R-Control): After 10 min of stabilization, isolated normal rat heart was infused with Atorvastatin (50 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion. Group VI (Sim Treated I/R-Control): After 10 min of stabilization, isolated normal rat heart was infused with Simvastatin (10 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion. Group VII (Hhcy control): The isolated Hhcy rat heart was perfused for 150 min using K-H solution after 10-min stabilization. Group VIII (Hhcy-I/R control): Isolated Hhcy rat heart after 10 min of stabilization was subjected to 30 min of global ischemia followed by 120 min of reperfusion Group IX (Ator per se Hhcy-control): After 10-min stabilization, the isolated Hhcy rat heart was infused with Atorvastatin (50 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution
6
Group X (Sim per se Hhcy-control): After 10-min stabilization, the isolated Hhcy rat heart was infused with Simvastatin (10 μM) for 10 min. Then the heart was perfused for 150 min using K-H solution Group XI (Ator Treated Hhcy-I/R Control): After 10 min of stabilization, isolated Hhcy rat heart was infused with Atorvastatin (50 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion Group XII (Sim Treated Hhcy-I/R Control): After 10 min of stabilization, isolated Hhcy rat heart was infused with Simvastatin (10 μM) for 10 min. The heart was then subjected to 30 min of global ischemia followed by 120 min of reperfusion 2.5 Statistical Analysis The results were expressed as mean ± S.D. The data obtained from various groups were statistically analyzed using two-way ANOVA followed by Tukey’s multiple-comparison test. A P value < 0.05 was considered to be statistically significant. 2.6 Drugs and Chemicals The LDH and CK enzymatic estimation kits were purchased from Vital Diagnostics, Thane, Maharastra, India. DTNB and NBT were obtained from Loba Chem, Mumbai, India. Atorvastatin, Simvastatin, 1,1,3,3-tetramethoxy propane and reduced glutathione were procured from Sigma-Aldrich, USA. All other reagents used in this study were of analytical grade.
7
3. Results L-methionine (1.7 g/kg/day, p.o.) was given to rats for 4 weeks via oral gavage which produced hyperhomocysteinemia (21.25±1.93 µM/L) when compared with normal rats (4.51±0.61 µM/L). In addition, L-methionine administration did not produce mortality in rats. 3.1 Effect of Atorva-and Simvastatin in I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts
8
Global ischemia followed by reperfusion significantly increased the infarct size in normal and hyperhomocysteinemic rat hearts. In addition, I/R was noted to increase the CK and LDH release in the coronary effluent in normal and hyperhomocysteinemic rat hearts (Fig 2). The maximum release of CK was noted at 5 min of reperfusion, whereas the maximum release of LDH was noted immediately after reperfusion (Fig 3-4). The hyperhomocysteinemic rat hearts showed enhanced myocardial injury when compared with normal rat hearts subjected to I/R. Treatment with Atorvastatin (50 μM) and Simvastatin (10 μM) significantly attenuated I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts to a similar extent, as assessed in terms of reduction in myocardial infarct size and decreased release of CK and LDH in the coronary effluent (Fig 2-4). 3.2 Effect of Atorva-and Simvastatin in I/R-induced oxidative stress in normal and hyperhomocysteinemic rat hearts Lipid peroxidation, measured in terms of TBARS, and superoxide anion generation, assessed in terms of reduced NBT, were significantly increased and levels of reduced GSH were found to be decreased in normal and hyperhomocysteinemic rat hearts subjected to I/R (Fig 5-7). In addition, hyperhomocysteinemic rat hearts showed high oxidative stress when compared with normal rat hearts. Atorvastatin (50 μM) and Simvastatin treatment (10 μM) attenuated the I/R-induced oxidative stress to a similar extent in normal and hyperhomocysteinemic rat hearts, as assessed in terms of reduction in TBARS and superoxide anion generation, and the consequent increase in GSH (Fig 5-7).
9
4. Discussion The ischemic heart disease (IHD) is a major cause of morbidity and mortality worldwide. The myocardial ischemia denotes a condition in which the heart is deprived of oxygen and other nutrients resulting in myocardial damage or dysfunction (Yu et al., 2014). The reperfusion refers to the restorations of the blood supply to the affected myocardium. Cardiac tissue damage caused when blood supply returns after a prolonged period of ischemia resulting in inflammation and oxidative damage is known as I/R
10
injury. It has been extensively reported that the increase in infarct size and the release of LDH and CK are documented to be an index of I/R-induced myocardial injury (Rohilla et al., 2012 a,b). In the present study, 30 min of global ischemia followed by 120 min of reperfusion produced myocardial injury, as assessed in terms of increased infarct size in the heart and elevated release of CK and LDH in the coronary effluent. The maximal release of CK was observed after 5 min of reperfusion, whereas peak release of LDH was noted immediately after reperfusion, which are in accordance with our earlier studies (Rohilla et al., 2012a,b). Moreover, increase in lipid peroxidation and superoxide anion generation alongwith consequent decrease of reduced GSH have been reported to be the indicators of oxidative stress (Ungvari et al., 2003; Devi et al., 2006). In the present study, the lipid peroxidation measured in terms of TBARS, and superoxide anion generation assessed in terms of reduced NBT, were noted to be increased, whereas the reduced GSH level was decreased as a result of I/R. These indicators suggest the development of I/R-induced oxidative stress, which may be responsible for the noted I/Rinduced myocardial injury. L-methionine administration (1.7 g/kg/day orally) in rats for 4 weeks produced Hhcy (Balakumar et al., 2009). In the present study, a marked increase in the infarct size of heart and, release of CK and LDH in the coronary effluent were noted in the hyperhomocysteinemic rat heart when compared with the normal rat heart. Hhcy has been well reported to downregulate NO bioavailability and produce high amount of oxidative stress in the heart by activating NADPH oxidase-mediated reactive oxygen species generation (Balakumar et al., 2007). Also, the Hhcy-induced oxidative stress occurs as a result of decreased expression and activity of key antioxidant enzymes, as
11
well as increased enzymatic generation of superoxide anion (Shah and Singh, 2007). Thus, it may be suggested that the development of high degree of oxidative stress may be responsible for the observed marked increase in myocardial injury in the hyperhomocysteinemic rat heart. This contention is supported by the fact that a marked increase in lipid peroxidation and superoxide anion generation and a subsequent decrease in GSH levels were noted in the hyperhomocysteinemic rat heart when compared with the normal rat heart. Statins, referred to as the HMG-CoA reductase inhibitors, have been regarded as the potent inhibitors of cholesterol biosynthesis and possess valuable effects in the prevention of coronary heart disease (Liao, 2003). The statins have been well accepted to possess various extrahepatic and cholesterol independent effects, termed as the pleiotropic effects, which include direct effects on vascular tissue, heart, kidney, bone and glucose metabolism (Liao, 2003; Laufs and Bohm, 2005). Administration of Atorvastatin has been reported to reduce the infarct size in isolated Langendorff-perfused heart model by activating pro-survival kinases and increasing NO levels (Bell and Yellon, 2003; Efthymiou and Yellon, 2005), whereas, Simvastatin administration lessened the myocardial contractile dysfunction and lethal ischemic injury in isolated Langendorff-perfused rat heart model (Zheng and Hu, 2007; Yin et al., 2007). The present study investigated the cardioprotective potential of Atorva-and Simvastatin against I/R injury in rat hearts administered at the onset of reperfusion. The data demonstrates that Atorvastatin (50 μM) and Simvastatin (10 μM) administered at the onset of reperfusion afforded cardioprotection, by significantly attenuating I/R-induced myocardial injury in rat hearts as assessed in terms of reductions in myocardial infarct
12
size and decreased release of CK and LDH in the coronary effluent, which is in accordance with our recent studies (Rohilla et al., 2012 a,b). Numbers of experimental studies have reported the antioxidant potential of Atorva-and Simvastatin in order to afford cardioprotection. Treatment with Atorvastatin lowered the increased levels of serum homocysteine in rats, providing an evidence of inhibitory role of Atorvastatin in Hhcy (Bhandari et al., 2011). Atorvastatin treatment has been noted to reduce the oxidative stress markers such as TBARS and lipid peroxidation levels in a rat model of I/R (Ozacmak et al., 2007). Moreover, Atorvastatin treatments reduced the vascular and cardiac free radical formation and normalized the expression of the NADPH oxidase, evidencing its anti-oxidative properties (Bolayirli et al., 2007). Also, administration of Atorvastatin induced a significant decrease in MDA levels alongwith a significant increase of SOD activity that further accounts for its antioxidant potential in order to afford cardioprotecton (Castro et al., 2008). Furthermore, Simvastatin treatment inhibited the protein and lipid peroxidation products such as TBARS and prevented the aortic production of reactive oxygen species, confirming its antioxidant potential. Additionally, experimental studies have shown that treatment with Simvastatin resulted in reductions of MDA levelas alongwith increase of SOD and NO levels, accounting for its cardioprotective potential (Delbosc et al., 2002). Another experimental study in rats showed a significant decrease in oxidative stress in Simvastatin treated diabetic-hypercholesterolemic rat hearts that further confirmed its antioxidant potential in affording cardioprotection (Adameova et al., 2006). In the present study, Atorvastatin (50 μM) and Simvastatin (10 μM) treatment was noted to significantly reduce the oxidative stress in normal and hyperhomocysteinemic
13
rat heart subjected to I/R, as assessed in terms of reduction in TBARS, and superoxide anion generation and consequent increase in the reduced form of GSH. This strongly suggests that the high degree of oxidative stress in hyperhomocysteinemic rat hearts in response to I/R injury was attenuated by Atorva-and Simvastatin in order to mimic cardioprotection. Moreover, the present study is the first of its kinds to compare the cardioprotective potential of statins, i.e. Atorva-and Simvastatin in the I/R-induced myocardial injury in normal and in hyperhomocysteinemic rat hearts. The results obtained in the present study showed that both the statins, i.e. Atorvastatin and Simvastatin can be used interchangeably in order to afford cardioprotection against I/Rinduced myocardial injury in normal and hyperhomocysteinemic rat hearts as assessed in terms of reduction in infarct size, CK and LDH in the coronary effluent alongwith reduction in oxidative stress.
5. Conclusion Hhcy transforms the myocardium susceptible to I/R-induced oxidative stress, and this high degree of oxidative stress produced by hyperhomocysteinemic rat heart in response to I/R, may be responsible for the prevention of cardioprotection. Treatment with Atorvastatin and Simvastatin afforded cardioprotection in normal and hyperhomocysteinemic rat hearts by abolishing the high degree of oxidative stress in Hhcy. Further studies are underway in our laboratory in order to explore the cardioprotective mechanisms of Statins in hyperhomocysteinemic rat hearts.
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6. Acknowledgments We wish to express our gratitude to Dr. Balvir Singh Tomar, Honorable Chairman; Dr. (Mrs) Shobha Tomar, Honorable Managing Director; Dr. K.C. Singhal, Honorable Vice Chancellor; Dr. K.P. Singh, Honorable Registrar and Prof. Govind Mohan, Director Pharmaceutical Sciences, NIMS University, Shobha Nagar, Jaipur-303121, Rajasthan for their praiseworthy suggestions and constant support for excellant research area.
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Figure 1.
Group I (Normal Control)
#
©
Group II (I/R Control)
#
Ξ
®
Group III (Ator Per se Normal Control)
#
Θ
©
Group IV (Sim Per se Normal Control)
#
Ω
©
Group V (Ator Treated I/R Control)
#
Θ
Ξ
®
Group VI (Sim Treated I/R Control)
#
Ω
Ξ
®
Group VII (Hhcy Control)
#
©
Group VIII (Hhcy-I/R Control)
#
Ξ
®
Group IX (Ator Per se Hhcy-Control)
#
Θ
©
Group X (Sim Per se Hhcy-Control)
#
Ω
©
Group XI (Ator Treated Hhcy-I/R Control)
#
Θ
Ξ
®
Group XII (Sim Treated Hhcy-I/R Control)
#
Ω
Ξ
®
21
Figure 2.
70
○,≈ 60
* Infarct Size (I.S.)
50
¤ ¤
40
װ
30
װ
20 10 0 Normal Control Sim per se Normal Control Hhcy control Sim per se Hhcy-control
I/R-Control Ator Treated I/R-Control Hhcy-I/R control Ator Treated Hhcy-I/R Control
22
Ator per se Normal Control Sim Treated I/R-Control Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
Figure 3.
250
○,≈ Creatine Kinase (U/L)
200
* 150
¤
¤
װ װ
100
50
0 Basal Normal Control Sim per se Normal Control Hhcy control Sim per se Hhcy-control
I/R-Control Ator Treated I/R-Control Hhcy-I/R control Ator Treated Hhcy-I/R Control
23
5 min Ator per se Normal Control Sim Treated I/R-Control Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
Figure 4.
400
○,≈ Lactate Dehydrogenase (U/L)
350
* 300
¤ ¤
250
װ װ
200 150 100 50 0 Basal
Normal Control Sim per se Normal Control Hhcy control Sim per se Hhcy-control
Imm Reperfusion
I/R-Control Ator Treated I/R-Control Hhcy-I/R control Ator Treated Hhcy-I/R Control
24
Ator per se Normal Control Sim Treated I/R-Control Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
Figure 5.
120
○,≈
100
TBARS (nM/gm)
* 80
¤ װ
60
¤
װ
40
20
0 Normal Control Sim per se Normal Control
I/R-Control Ator Treated I/R-Control
Ator per se Normal Control Sim Treated I/R-Control
Hhcy control Sim per se Hhcy-control
Hhcy-I/R control Ator Treated Hhcy-I/R Control
Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
25
Figure 6.
120
○,≈
Reduced NBT (pM/min/mg)
100
80
* ¤ ¤
60
װ
40
װ
20
0 Normal Control Sim per se Normal Control
I/R-Control Ator Treated I/R-Control
Ator per se Normal Control Sim Treated I/R-Control
Hhcy control Sim per se Hhcy-control
Hhcy-I/R control Ator Treated Hhcy-I/R Control
Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
26
Figure 7.
GSH (uM/mg wet tissue weight)
1.2
װ
1
װ
0.8
¤
¤
* 0.6
○,≈ 0.4
0.2
0 Normal Control Sim per se Normal Control Hhcy control Sim per se Hhcy-control
I/R-Control Ator Treated I/R-Control Hhcy-I/R control Ator Treated Hhcy-I/R Control
27
Ator per se Normal Control Sim Treated I/R-Control Ator per se Hhcy-control Sim Treated Hhcy-I/R Control
Figure Legends
Figure 1:
Diagram showing schematic representation of experimental protocol
# represents 10 min stabilization; © represents 150 minutes of K-H perfusion, Ξ represents 30 minutes of global ischemia; ® represents 120 minutes of K-H reperfusion; Θ represents 10 minutes of Atorvastatin infusion; Ω represents 10 minutes of Simvastatin infusion
Figure 2:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in infarct size Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 3:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in CK levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 4:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in LDH levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 5:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in TBARS levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 6:
Effect of Atorvastatin and Simvastatin in I/R-induced increase in superoxide anion generation Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
Figure 7:
Effect of Atorvastatin and Simvastatin in I/R-induced decrease in reduced GSH levels Values are expressed as mean ± S.D. * = P<0.05 vs normal control; = װP<0.05 vs I/R Control; ○ = P<0.05 vs Hhcy-Control; ≈ = P<0.05 vs I/R control; ¤ = P<0.05 vs Hhcy-IR Control.
28