The Comparison of Antioxidant Effect of Aspirin, Metformin, Atorvastatin and Captopril Co-administration in the Heart and Kidney Tissues of Diabetic Rats

Document Type : Research article


1 Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

2 Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.


The present study investigated the effects of co-administration of aspirin, metformin, atorvastatin and captopril on serum lipid profile and oxidative stress in the heart and kidney of streptozotocin-induced diabetic rats. In this study, rats were randomly divided into the following eleven groups: control (Cont.), and diabetic (D), as well as 9 groups that were treated with metformin (M, 300 mg/kg) or aspirin (ASA, 120 mg/kg) alone or in different combinations with captopril (C, 50 mg/kg), or atorvastatin (AT, 40 mg/kg), as follows: (D + M), (D + ASA), (D + M + ASA), (D + M + C), (D + M + AT), (D + M + C + ASA), (D + M + C + AT), (D + M + AT + ASA), and (D + M + C + AT + ASA). The rats in treatment groups daily received drugs by gavage for six weeks. Finally, serum lipid profile and levels of oxidative markers in the heart and kidney tissues were evaluated.In diabetic rats, blood levels of glucose, cholesterol, TG (triglyceride), LDL (low-density lipoprotein), MDA (malondialdehyde) and AIP (atherogenic index of plasma) significantly increased but those of HDL (high-density lipoprotein) and total thiol as well as SOD (superoxide dismutase) and CAT (catalase) activities significantly decreased. Treatment with different combinations of C, ASA, AT and M significantly ameliorated these parameters. This study showed that co-administration of ASA, M, C and AT, could improve glucose and lipid metabolism and oxidative stress markers in the kidneys and heart tissues of diabetic rats more markedly than the administration of these drugs alone.

Graphical Abstract

The Comparison of Antioxidant Effect of Aspirin, Metformin, Atorvastatin and Captopril Co-administration in the Heart and Kidney Tissues of Diabetic Rats


(1) Suckling R and Gallagher H. Chronic kidney disease, diabetes mellitus and cardiovascular disease: risks and commonalities. J. Ren. Care (2012) 38:4-11.
(2) Pálsson R and Patel UD. Cardiovascular complications of diabetic kidney disease. Adv. Chronic Kidney Dis. (2014) 21:273-80.
(3) Kayama Y, Raaz U, Jagger A, Adam M, Schellinger IN, Sakamoto M, Suzuki H, Toyama K, Joshua M. Spin JM and Tsao PS. Diabetic cardiovascular disease induced by oxidative stress. Int. J. Mol. Sci. (2015) 16:25234-63.
(4) Boroushaki MT, Mollazadeh H, Rajabian A, Dolati K, Hoseini A, Paseban M and Farzadnia M. Protective effect of pomegranate seed oil against mercuric chloride-induced nephrotoxicity in rat. Renal failure (2014) 36:1581-6.
(5) Nishikawa T, Brownlee M and Araki E. Mitochondrial reactive oxygen species in the pathogenesis of early diabetic nephropathy. J. Diabetes Investig. (2015) 6:137-9.
(6) Petrie JR, Chaturvedi N, Ford I, Brouwers MC, Greenlaw N, Tillin T, Hramiak I, Hughes AD, Klein BEK,  Klein R,  Ooi TC, Rossing P,  Stehouwer DA, Sattar N and Colhoun HM. Cardiovascular and metabolic effects of metformin in patients with type 1 diabetes (REMOVAL): a double-blind, randomised, placebo-controlled trial. Lancet Diabetes Endocrinol. (2017) 5:597-609.
(7) Mousavi SM, Niazmand S, Hosseini M, Hassanzadeh Z, Sadeghnia HR, Vafaee F and Keshavarzi Z. Beneficial effects of Teucrium polium and metformin on diabetes-induced memory impairments and brain tissue oxidative damage in rats. Int. J. Alzheimers Dis. (2015) 2015:493729
(8) Rahimi Z. The role of renin angiotensin aldosterone system genes in diabetic nephropathy. Can. J. Diabetes (2016) 40:178-83.
(9) Chawla T, Sharma D and Singh A. Role of the renin angiotensin system in diabetic nephropathy. World J. Diabetes (2010) 1:141.
(10) Coughlan MT, Thallas-Bonke V, Pete J, Long DM, Gasser A, Tong DC, Arnstein M, Thorpe SR, Cooper ME and Forbes JM. Combination therapy with the advanced glycation end product cross-link breaker, alagebrium, and angiotensin converting enzyme inhibitors in diabetes: synergy or redundancy? Endocrinology (2007) 148:886-95.
(11) de Cavanagh EM, Inserra F, Toblli J, Stella I, Fraga CG and Ferder L. Enalapril attenuates oxidative stress in diabetic rats. Hypertension (2001) 38:1130-6.
(12) Wu L and Parhofer KG. Diabetic dyslipidemia. Metabolism (2014) 63:1469-79.
(13) Tang X-L, Sanganalmath SK, Sato H, Bi Q, Hunt G, Vincent RJ, Yong Peng, Shirk G, Dawn B and Bolli R. Atorvastatin therapy during the peri-infarct period attenuates left ventricular dysfunction and remodeling after myocardial infarction. PloS one. (2011) 6(9):e25320.
(14) Abdel-Hamid AA and Firgany AE-DL. Atorvastatin alleviates experimental diabetic cardiomyopathy by suppressing apoptosis and oxidative stress. J. Mol. Histol. (2015) 46:337-45.
(15) Inoguchi T, Sonta T, Tsubouchi H, Etoh T, Kakimoto M, Sonoda N, Sato N, Sekiguchi N, Kobayashi K, Sumimoto H, Utsumi H and Nawata H. Protein kinase C–dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD (P) H oxidase. J. Am. Soc. Nephrol. (2003) 14:S227-32.
(16) Briones AM, Rodríguez-Criado N, Hernanz R, García-Redondo AB, Rodrigues-Díez RR, Alonso MJ, Egido J, Ruiz-Ortega M and Salaices M. Atorvastatin prevents angiotensin II–induced vascular remodeling and oxidative stress. Hypertension. (2009) 54:142-9.
(17) Usui H, Shikata K, Matsuda M, Okada S, Ogawa D, Yamashita T, Hida K, Satoh M, Wada J and Makino H. HMG‐CoA reductase inhibitor ameliorates diabetic nephropathy by its pleiotropic effects in rats. Nephrology Dialysis Transplantation. (2003) 18(2):265-72.
(18) Calvin AD, Aggarwal NR, Murad MH, Shi Q, Elamin MB, Geske JB, Fernandez-Balsells MM, Albuquerque FN, Lampropulos JF, Erwin PJ, Smith SA and  Montori VM. Aspirin for the primary prevention of cardiovascular events: a systematic review and meta-analysis comparing patients with and without diabetes. Diabetes Care. (2009) 32:2300-6.
(19) Paseban M, Marjaneh RM, Banach M, Riahi MM, Bo S, Sahebkar A. Modulation of microRNAs by aspirin in cardiovascular disease. Trends Cardiovasc. Med. (2020) 30:249-54.
(20) American Diabetes Association. Introduction: Standards of Medical Care in Diabetes—2020. Diabetes care (2020) 43:S1-S2.
(21) Ou H-C, Lee W-J, Wu C-M, Chen JF-M and Sheu WH-H. Aspirin prevents resistin-induced endothelial dysfunction by modulating AMPK, ROS, and Akt/eNOS signaling. J. Vasc. Surg. (2012) 55:1104-15.
(22) Paseban M, Butler AE and Sahebkar A. Mechanisms of statin‐induced new‐onset diabetes. J. Cell Physiol. (2019) 234:12551-61.
(23) Dervisevik M, Dinevska-Kovkarovska S, Dimitrovska M, Cipanovska N and Miova B. High dose of aspirin moderates diabetes-induced changes of heart glycogen/glucose metabolism in rats. J. Physiol. Sci. (2014) 64:411-20.
(24) Fullerton MD, Ford RJ, McGregor CP, LeBlond ND, Snider SA, Stypa SA, Day EA, Lhoták S, Schertzer JD, Austin RC, Kemp BE and Steinberg GR. Salicylate improves macrophage cholesterol homeostasis via activation of Ampk. J. Lipid Res. (2015) 56:1025-33.
(25) Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE and Shulman GI. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J. Clin. Invest. (2002) 109:1321-6.
(26) Paseban M, Mohebbati R, Niazmand S, Sathyapalan T and Sahebkar A. Comparison of the Neuroprotective Effects of Aspirin, Atorvastatin, Captopril and Metformin in Diabetes Mellitus. Biomolecules (2019) 9:118.
(27) Abbasnezhad A, Niazmand S, Mahmoudabady M, Rezaee SA, Soukhtanloo M, Mosallanejad R and Hayatdavoudi P. Nigella sativa L. seed regulated eNOS, VCAM-1 and LOX-1 genes expression and improved vasoreactivity in aorta of diabetic rat. J. ethnopharmacology (2019) 228:142-7.
(28) Lee MJ, Park JT, Han SH, Kim Y-L, Kim YS, Yang CW, Kim N-H, Kang S-W, Kim HJ and Yoo T-H. The atherogenic index of plasma and the risk of mortality in incident dialysis patients: Results from a nationwide prospective cohort in Korea. PloS one (2017) 12:e0177499.
(29) Sedlak J and Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. (1968) 25:192-205.
(30) Mohebbati R, Jalili-Nik M, Paseban M, Shafei MN and Rad AK. Effects of Zataria multiflora extract and Carvacrol on doxorubicin-induced oxidative stress in rat brain. Pharm. Sci. (2018) 24:187-92.
(31) Uchiyama M and Mihara M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal. biochem. (1978) 86:271-8.
(32) Pasebasn M, Niazmand S, Soukhtanloo M, Meibodi NT, Abbasnezhad A, Mousavi SM and Niazmand MJ. The Therapeutic Effect of Nigella sativa Seed on Indomethacin-induced Gastric Ulcer in Rats. Curr. Nutr. Food Sci. (2020) 16:276-83.
(33) Madesh M and Balasubramanian K. Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide. Indian J Biochem Biophys. (1998) 35:184-8.
(34) Mohebbati R, Paseban M, Soukhtanloo M, Jalili-Nik M, Shafei MN, Yazdi AJ and Rad AK. Effects of standardized Zataria multiflora extract and its major ingredient, Carvacrol, on Adriamycin-induced hepatotoxicity in rat. Biomed. J. (2018) 41:340-7.
(35) Aebi H. Catalase in vitro.  Methods Enzymol. (1984) 105:121-6.
(36) Mohebbati R, Paseban M, Beheshti F, Soukhtanloo M, Shafei MN, Rakhshandeh H and Rad AK. The Preventive Effects of Standardized Extract of Zataria multiflora and Carvacrol on Acetaminophen-Induced Hepatotoxicity in Rat:-Zataria multiflora and Carvacrol and Hepatotoxicity. J. pharmacopuncture (2018) 21:249.
(37) Podhaisky HP, Abate A, Polte T, Oberle S and Schröder H. Aspirin protects endothelial cells from oxidative stress–possible synergism with vitamin E. FEBS Lett. (1997) 417:349-51.
(38) Sena CM, Matafome P, Louro T, Nunes E, Fernandes R and Seiça RM. Metformin restores endothelial function in aorta of diabetic rats. Br. J. Pharmacol. (2011) 163:424-37.
(39) Yaribeygi H, Faghihi N, Mohammadi MT and Sahebkar A. Effects of atorvastatin on myocardial oxidative and nitrosative stress in diabetic rats. Comp. Clin. Path. (2018) 27:691-7.
(40) Onozato ML, Tojo A, Goto A, Fujita T and Wilcox CS. Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int. (2002) 61:186-94.
(41) Zou M-H, Kirkpatrick SS, Davis BJ, Nelson JS, Wiles WG, Schlettner U, Neumann D, Brownlee M, Freeman MB and Goldman MH. Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo: role of mitochondrial reactive nitrogen species. J. Biol. Chem. (2004) 279:43940-51.
(42) Gallo A, Ceolotto G, Pinton P, Iori E, Murphy E, Rutter GA, Rizzuto R, Semplicini A and Avogaro A. Metformin prevents glucose-induced protein kinase C-β2 activation in human umbilical vein endothelial cells through an antioxidant mechanism. Diabetes (2005) 54:1123-31.
(43) Piwkowska A, Rogacka D, Jankowski M, Dominiczak MH, Stępiński JK, Angielski S. Metformin induces suppression of NAD (P) H oxidase activity in podocytes. Biochem. Biophys. Res. Commun. (2010) 393:268-73.
(44) Hou X, Song J, Li X-N, Zhang L, Wang X, Chen L and Shen YH. Metformin reduces intracellular reactive oxygen species levels by upregulating expression of the antioxidant thioredoxin via the AMPK-FOXO3 pathway. Biochem. Biophys. Res. Commun.  (2010) 396:199-205.
(45) Prasad K and Lee P. Suppression of oxidative stress as a mechanism of reduction of hypercholesterolemic atherosclerosis by aspirin. J. Cardiovasc. Pharmacol. Ther. (2003) 8:61-9.
(46) Grosser N, Abate A, Oberle S, Vreman HJ, Dennery PA, Becker JC, Pohle T, Seidman DS and Schröder H. Heme oxygenase-1 induction may explain the antioxidant profile of aspirin. Biochem. Biophys. Res. Commun. (2003) 308:956-60.
(47) Vecchione C, Gentile M, Aretini A, Marino G, Poulet R, Maffei A, Passarelli F, Landolfi A, Vasta A and G Lembo G. A novel mechanism of action for statins against diabetes-induced oxidative stress. Diabetologia (2007) 50:874-80.
(48) Muchova L, Wong RJ, Hsu M, Morioka I, Vitek L, Zelenka J, Schröder H and Stevenson DK. Statin treatment increases formation of carbon monoxide and bilirubin in mice: a novel mechanism of in vivo antioxidant protection. Can. J. Physiol. Pharmacol. (2007) 85:800-10.
(49) Wassmann S, Laufs U, Müller K, Konkol C, Ahlbory K, Bäumer AT, Linz W, Böhm M and Nickenig G. Cellular antioxidant effects of atorvastatin in vitro and in vivo. Arterioscler. Thromb. Vasc. Biol. (2002) 22:300-5.
(50) Landmesser U and Drexler H. Oxidative stress, the renin-angiotensin system, and atherosclerosis. Eur. Heart. J. suppl. (2003) 5:A3-7.
(51) Wulffelé eM, Kooy A, De Zeeuw D, Stehouwer C and Gansevoort R. The effect of metformin on blood pressure, plasma cholesterol and triglycerides in type 2 diabetes mellitus: a systematic review. J. Intern. Med. (2004) 256:1-14.
(52) Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ and Moller DE. Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. (2001) 108:1167-74.
(53) Malmqvist K, Kahan T, Isaksson H and Östergren J. Regression of left ventricular mass with captopril and metoprolol, and the effects on glucose and lipid metabolism. Blood press. (2001) 10:101-10.
(54) Kawamoto S, Kawamura T, Miyazaki Y and Hosoya T. Effects of atorvastatin on hyperlipidemia in kidney disease patients. Nihon Jinzo Gakkai Shi. (2007) 49:41-8.
(55) Ford RJ, Fullerton MD, Pinkosky SL, Day EA, Scott JW, Oakhill JS, Bujak AL, Smith BK, Crane JD, Blumer RM, Marcinko K, Kemp BE, Gerstein HC, and Steinberg GR. Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity. Biochem. J. (2015)  468:125–32.
(56) Caminhotto RO, Sertié RAL, Andreotti S, Campaãa AB, and Lima FB. Renin-angiotensin system blockers regulate the metabolism of isolated fat cells in vitro. Braz. J. Med. Biol. Res. (2016) 49:e5409.
(57) De Berardis G, Sacco M, Evangelista V, Filippi A, Giorda CB, Tognoni G, Valentini U and Nicolucci A. Aspirin and Simvastatin Combination for Cardiovascular Events Prevention Trial in Diabetes (ACCEPT-D): design of a randomized study of the efficacy of low-dose aspirin in the prevention of cardiovascular events in subjects with diabetes mellitus treated with statins. Trials. (2007) 8:21.
(58) Koh KK, Son JW, Ahn JY, Kim DS, Jin DK, Kim HS, Han SH, Seo YH, Chung WJ, Kang WC and Shin EK. Simvastatin combined with ramipril treatment in hypercholesterolemic patients. Hypertension (2004) 44:180-5.
(59) Qin J, Zhang Z, Liu J, Sun L, Hu L, Cooper ME and Cao Z. Effects of the combination of an angiotensin II antagonist with an HMG-CoA reductase inhibitor in experimental diabetes. Kidney int. (2003) 64:565-71.
(60) Tousoulis D, Koniari K, Antoniades C, Miliou A, Noutsou M, Nikolopoulou A, Papageorgiou N, Marinou K, Stefanadi E and Stefanadis C. Impact of 6 weeks of treatment with low-dose metformin and atorvastatin on glucose-induced changes of endothelial function in adults with newly diagnosed type 2 diabetes mellitus: A single-blind study. Clin. Ther. (2010) 32:1720-8.