The Acute Effects of Different Spironolactone Doses on Oxidative Stress in Streptozotocin-Induced Diabetic Rats
Categoria dell'articolo: Original Scientific Article
Pubblicato online: 26 mag 2021
Pagine: 103 - 112
Ricevuto: 16 mar 2021
Accettato: 22 mar 2021
DOI: https://doi.org/10.2478/sjecr-2021-0025
Parole chiave
© 2024 Stefan Simovic et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in patients with diabetes mellitus (DM). Currently, DM type 2 affects 347 million worldwide (1), while the risk of mortality is 1.7 times higher in patients with diabetes mellitus than in the non-diabetic population (2). Inflammation, neurohumoral activation and structural remodelling are multiple mechanisms that contribute to cardiovascular disease, whereby one of the primary means contributing to CVD development is oxidative stress (3).
Increased bioavailability of reactive oxygen species (ROS) is defined as oxidative stress and is noticed in type 2 DM and reduced antioxidant enzymes expression/activity (3). One of the many mechanisms correlated with vascular damage induced by oxidative stress is decreased nitric oxide (NO) bioavailability. NO effects are impaired by increased ROS production whereby increased generation of superoxide anion (O2-), a ROS that readily reacts with vascular NO to form peroxynitrite (ONOO−), has been reported in DM2 experimental models (4).
Aldosterone, an adrenal hormone, is secreted due to renin-angiotensin-aldosterone system (RAAS) activation, representing one of the fundamental physiological reactions in CVD (5). The discovery that the concentrations of myocardial aldosterone in typical rodents are multiple times higher than plasma prompted an extraordinary comprehension of the role of mineralocorticoid receptor activation in the CVD (6).
Spironolactone, a mineralocorticoid receptor antagonist, produces positive ionotropic actions in rat heart by increasing diastolic concentration of calcium and myosin ATPase calcium sensitivity (7). This medication utilizes coronary microvascular function, proposing a beneficial role of aldosterone in preventing diabetic cardiovascular complications in patients with type 2 DM (8). Our previous finding suggests that acute application of different spironolactone doses has other effects on healthy and diabetic hearts; In contrast, the dose-dependent effect is observed in diabetic hearts. That effect is absent in healthy ones (9). Based on the above data, aldosterone affects redox status in multiple ways, whereby cardiac-specific overexpression of human MR leads to increased ROS production and expression of NADPH oxidase (10). MR blockade with spironolactone treatment in rat led to decreased ROS formation and NADPH oxidase subunit expression (10).
Regarding pro-oxidative and pro-inflammatory effects of aldosterone in the cardiovascular system (11), and considering that aldosterone concentration increases in DM (12), our study aimed to evaluate the impact of different spironolactone dosages on oxidative stress in streptozotocin-induced DM diabetic rats.
The present investigation was done on 40 grown-up male Wistar albino rats (8 weeks old and boy weight of 200 ± 30g). They were housed under controlled natural conditions: temperature 25°C with a built-up photo time of 12 h light/day. The rats had free access to food and water - ad libitum. Rats were randomly randomized into 4 groups (10 animals for each group): (1) healthy rats treated with 0.1 μM of spironolactone (n=10); (2) diabetic rats treated with 0.1 μM of spironolactone (n=10); (3) healthy rats treated with 3 μM of spironolactone (n=10); (4) diabetic rats treated with 3 μM of spironolactone (n=10). Acute administration of spironolactone was achieved through the Langedorff apparatus directly in the isolated heart.
All experimental procedures were done per current legislation (EU Directive for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes 86/609/EES) and ethics standards. Animals were cared as per the Guide for the Care and Use of Laboratory Animals (National Research Council 1996). The study was conducted under the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies (13). The investigation protocol was approved by the Ethics committee for experimental animal well being of the Faculty of Medical Sciences of the University of Kragujevac.
Before DM induction, 2 weeks of adjustment was given. To build up a rat model of experimentally induced type 1 DM, which imitates similar pathophysiology in the human population, over-night fasting rats were infused with a single intraperitoneal portion of streptozocin (STZ) (60 mg/kg) (Sigma-Aldrich, St. Louis, Missouri, USA) dissolved down in cold, fresh 0.01 mol/L citrate buffer of fresh or solidified 1mL aliquots at 20 °C (0.1 mol/L citric acid, 0.1 mol/L sodium citrate), pH 4.5. DM was confirmed 72h later when blood glucose levels were above 11.1 mmol/L. Animals that developed DM were held in similar conditions and followed for the next 4 weeks.
On the 29th day after inducement of type 1 DM, rats were anaesthetized with ketamine (10 mg/kg) and xylazine (5 mg/kg) and afterwards euthanized by cervical disengagement (Schedule 1 of the Animals/Scientific Procedures, Act 1986, UK). Following a fast thoracotomy and quick cardiac arrest by superfusion with super cold isotonic saline, the hearts were speedily extracted and attached to the Langendorff apparatus (Langendorff apparatus, Experimentia LTD, 1062 Budapest, Hungary) through aortic cannulation and afterwards were perfused with Krebs-Henseleit solution for retrograde perfusion under continuously expanding coronary perfusion pressure (CPP) (CPP from 40 cmH2O to 120 cmH2O). Krebs-Henseleit solution was utilized for retrograde perfusion (in mmol/L: NaCl 118, KCl 4.7, CaCl2, x 2 H2O 2.5, MgSO4 x 7 H2O 1.7, NaHCO3 25, KH2PO4 1.2, glucose 11 and pyruvate 2). The buffer was balanced with 95% O2 and 5% CO2, with a pH estimation of 7.4 and a temperature of 37 °C.
After the heart perfusion foundation, the arrangements were balanced within 30 min with a basal CPP of 70 cmH2O. Following the adjustment period, the perfusion pressure was diminished to 50 and 40 cmH2O, and afterwards, step by step expanded to 60, 80, 100, and 120 cmH2O to set up coronary autoregulation. Testing began following the control test to keep away from undesirable time-dependent consequences. The spironolactone application (0.1 and 3 _mol/L) went on until accomplishing a steady stream, yet not under 5 min for each estimation of perfusion pressure. After the sensor (transducer BS4 73-0184, Experimetria Ltd., Budapest, Hungary) placing into the left ventricle, the accompanying cardiodynamic parameters were ceaselessly enlisted: the maximum and minimum rate of pressure development in the left ventricle (dp/dtmax, dp/dtmin), systolic and diastolic left ventricle pressure (SLVP, DLVP), pulse (HR), and coronary flow (CF) on every one of foreordained estimations of perfusion pressure (40, 60, 80, 100, and 120 cmH2O). CF was estimated by flowmetry. Every heart was its control.
Markers of oxidative stress were estimated spectrophotometrically in the gathered examples of coronary venous effluent. Samples were collected after adjustment of the coronary flow and after medication administration for every perfusion pressure. We performed quantification of nitrites (the measure of NO discharged), superoxide anion radical (O2−), and hydrogen peroxide (H2O2) and indirect quantification of the index of lipid peroxidation using receptive thiobarbituric substances (TBARS), for all examples.
Estimation of hydrogen peroxide depended on the oxidation of phenol red by hydrogen peroxide in a response catalyzed by horseradish peroxidase (HRPO) (14). A sum of 200 μl of perfusate was accelerated with 800 ml of freshly arranged phenol red solution, followed by the addition of 10 μl of (1:20) HRPO (made ex-tempore). A sufficient volume of distilled water was utilized as a blank test (rather than coronary venous gushing). The level of H2O2 was measured at 610 nm.
Nitric oxide decays quickly to frame stable metabolite nitrite/nitrate products. Nitrites can, in this manner, be utilized as an index of NO generation using a spectrophotometric strategy utilizing the Griess reagent. Nitrites (NO2−) were determined as an index of NO creation with Griess reagent (forms purple diazocomplex) (15). Briefly, 0.5 ml of the perfusate was accelerated with 200 μl of 30% sulfosalicylic acid, vortexed for 30 min and centrifuged at 3000 x g. Equal volumes of the supernatant and Griess reagent containing 1 % sulphanilamide in 5 % phosphoric acid/0.1 % naphthalene ethylenediamine-dihydrochloride was added and balanced out for 10 min in the dark and estimated spectrophotometrically at 550 nm. Distilled water was utilized as a clear test.
Superoxide anion radical concentrations were estimated utilizing the NTB (Nitro Blue Tetrazolium) reagent in TRIS buffer (assay mixture) with coronary venous effluent. The estimation was performed at a wavelength of 550 nm. Distilled water was utilized as a blank test (16).
The lipid peroxidation list was determined indirectly by estimating the results of the response with thiobarbituric acid (TBARS or Thiobarbituric Acid Reactive Substances). Briefly, 1% thiobarbituric acid (TBA) in 0.05 M NaOH was incubated with coronary venous effluent at 100°C for 15 min and afterwards, spectrophotometrically estimated at a wavelength of 530 nm. Distilled water was utilized as a blank test (17).
STZ and spironolactone were purchased from Sigma-Aldrich Chemie GmbH Eschenstr. 5, 82024 Taufkirchen, Germany.
Statistical investigation of experimental information incorporated the following essential descriptive statistics: the mean value (X) ± standard deviation (SD). Results and variables from healthy and diabetic rat hearts perfused with various spironolactone dosages (0.1 or 3 μM/L) were compared with every parameter at each CPP. The following factual tests were utilized to test the measurable statistical significance of the outcomes and to affirm the speculation: Independent T-test. A database analysis of the results was performed using programming software SPSS 18th (SPSS Inc., Chicago, IL, USA). P values lower than 0.05 (p<0.05) were considered to be significant, while p values lower than 0.01 (p<0.01) were viewed as highly significant.
After spironolactone application in the dose of 0.1μM, the hydrogen peroxide levels did not significantly differ in healthy and diabetic rats (Fig 1a). However, after perfusion with 3μM of spironolactone, levels of hydrogen peroxide significantly decreased in diabetic rats when compared to healthy rats at all CPPs, with higher significance at 80 – 120 cmH2O (p<0.01) (Fig 2a). When we compared levels of H2O2 between healthy rats treated with 0.1μM and 3μM of spironolactone, no significant differences were noted at any CPP (Fig 3a). Significantly lower levels of H2O2 were noticed in diabetic rats treated with 3μM at 80, 100 and 120 cm H2O compared to diabetic rats treated with 0.1μM of spironolactone (Fig 4a). The most significant increase in the level of H2O2 was achieved with 0.1μM of spironolactone at CPP of 40 cm H2O compared to control, while 3μM lowered the level of H2O2 the most in diabetic rats at CPP of 120 cm H2O (Table 1.)
Parameters of oxidative stress in healthy rats perfused with 0.1 μM of spironolactone compared with diabetic rats perfused with 0.1 μM of spironolactone. (a) Hydrogen peroxide (H2O2) (b) Nitrites (NO2-) (c) Superoxide anion radical (O2-) (d) Index of lipid peroxidation (measured as TBARS). Data is presented as mean ± SD. CPP, coronary perfusion pressure.
Parameters of oxidative stress in healthy rats perfused with 3 μM of spironolactone compared with diabetic rats perfused with 3 μM of spironolactone. (a) Hydrogen peroxide (H2O2) (b) Nitrites (NO2-) (c) Superoxide anion radical (O2-) (d) Index of lipid peroxidation (measured as TBARS). Data is presented as mean ± SD. CPP, coronary perfusion pressure.
Parameters of oxidative stress in healthy rats perfused with 0.1 μM of spironolactone compared with healthy rats perfused with 3 μM of spironolactone. (a) Hydrogen peroxide (H2O2) (b) Nitrites (NO2−) (c) Superoxide anion radical (O2−) (d) Index of lipid peroxidation (measured as TBARS). Data is presented as mean ± SD. CPP, coronary perfusion pressure.
Parameters of oxidative stress in diabetic rats perfused with 0.1 μM of spironolactone compared with diabetic rats perfused with 3 μM of spironolactone. (a) Hydrogen peroxide (H2O2) (b) Nitrites (NO2−) (c) Superoxide anion radical (O2−)(d) Index of lipid peroxidation (measured as TBARS). Data is presented as mean ± SD. CPP, coronary perfusion pressure.
Percentage of reduction or increase in the each of the parameters of oxidative stress in healthy and diabetic rats treated with different spironolactone doses.
| 0.1 μM spironolactone healthy vs. control | +20.49 | +1.34 | +1.30 | −7.57 | 0.00 | |
| 3 μM spironolactone healthy vs. control | +11.12 | −3.88 | +0.79 | −12.62 | +9.40 | |
| 0.1 μM spironolactone diabetic vs. diabetic | −0.12 | −11.62 | −0.28 | +8.73 | −8.08 | |
| 3 μM spironolactone diabetic vs. diabetic | −12.22 | −33.73 | −37.84 | −20.12 | −43.83 | |
| 0.1 μM spironolactone healthy vs. control | +13.69 | −10.83 | −20.12 | −19.92 | −31.26 | |
| 3 μM spironolactone healthy vs. control | −2.03 | −13.64 | −0.36 | −4.74 | +0.90 | |
| 0.1 μM spironolactone diabetic vs. diabetic | +4.12 | −6.74 | −4.91 | +1.26 | −0.98 | |
| 3 μM spironolactone diabetic vs. diabetic | −3.46 | −11.98 | −14.09 | −9.54 | −11.25 | |
| 0.1 μM spironolactone healthy vs. control | +5.55 | −53.58 | −14.75 | +41.34 | −16.96 | |
| 3 μM spironolactone healthy vs. control | +16.83 | −6.04 | −16.97 | −20.30 | −11.92 | |
| 0.1 μM spironolactone diabetic vs. diabetic | −21.85 | −3.80 | +32.66 | +19.34 | +67.14 | |
| 3 μM spironolactone diabetic vs. diabetic | +6.31 | −32.84 | −25.24 | −15.17 | −46.94 | |
| 0.1 μM spironolactone healthy vs. control | +7.85 | +1.32 | −6.91 | −10.89 | −9.93 | |
| 3 μM spironolactone healthy vs. control | +7.64 | +5.60 | +27.74 | +28.50 | +31.63 | |
| 0.1 μM spironolactone diabetic vs. diabetic | +5.25 | −6.02 | −7.06 | +0.60 | −3.87 | |
| 3 μM spironolactone diabetic vs. diabetic | −13.61 | −48.17 | −49.53 | −46.99 | −47.15 | |
Percentage of reduction or increase in the each of the parameters of oxidative stress in healthy rats, healthy rats treated with 0.1 μM and 3 μM of spironolactone and diabetic rats treated with 0.1 μM and 3 μM of spironolactone. Sign (−) stands for reduction and sign (+) stands for increase in the level of the parameters of oxidative stress. Data is presented as percentage.
NO activity was measured as nitrite. Its level was significantly higher in diabetic rats perfused with 0.1 μM of spironolactone than healthy rats perfused with the same dose of spironolactone at CPPs at 100 and 120 cm H2O (p<0.05). Simultaneously, the level of nitrites did not significantly differ at lower CPPs (Fig 1b). When diabetic and healthy hearts were perfused with 3μM of spironolactone, levels of nitrites were markedly higher in healthy hearts only at CPP at 120 cm H2O (p<0.05), while at lower CPPs, no significant differences were observed, but there is a noticeable trend in the rise of the level of nitrites in healthy hearts (Fig 2b). The same evident trend was also observed when we compared levels of nitrites between healthy rats treated with 0.1μM and 3μM of spironolactone, with significantly higher levels of nitrites in healthy rats treated with 3μM of spironolactone when compared to healthy rats treated with 0.1μM of spironolactone (Fig 3b). In contrast, when we compared the level of nitrites in diabetic rats treated with 0.1μM and 3μM of spironolactone, there were no significant differences in nitrites' levels (Fig 4b). The dose of 0.1μM of spironolactone increased the level of NO2− the most in healthy rats compared to healthy control at CPP 40 cm H2O, while the most significant decrease in the level of NO2− was observed between the same rats at CPP 120 cm H2O (Table 1.).
In diabetic rats perfused with 0.1 μM of spironolactone compared with healthy rats perfused with the same dose of spironolactone level of O2− was significantly increased at 80 – 120 cm H2O (p<0.01) (Fig 1c), but when both groups were perfused with 3μM of spironolactone, levels of O2− were significantly higher in healthy rats at 40, 60 and 120 cm H2O (p<0.05) (Fig 2c). Level of O2− did not significantly differ between healthy rats treated with 0.1μM and 3μM of spironolactone (Fig 3c). However, a significantly higher level of O2− was observed in diabetic rats treated with 0.1μM of spironolactone compared to diabetic rats treated with 3μM od spironolactone at 60, 80, 100 and 120 cm H2O (Fig 4c). Spironolactone in the dose of 0.1μM increased the level of O2− for 67.14% in diabetic rats when compared with diabetic control at CPP 120 cm H2O, while the same quantity of spironolactone decreased the level of O2− for 53.58% in healthy rats when compared to healthy control at CPP 60 cm H2O (Table 1.).
After spironolactone application in the dose of 0.1μM, the levels of TBARS did not significantly differ in healthy and diabetic rats (Fig 1d). After perfusion with 3μM of spironolactone in diabetic rats, significantly lower levels of TBARS was observed at 80 – 120 cm H2O (Fig 2d). When we compared the level of TBARS between healthy rats treated with 0.1μM and 3μM of spironolactone, in the group treated with 3μM of spironolactone, we noticed significant higher values compared to the group treated with 0.1μM of spironolactone at 80 – 120 cm H2O (Fig 3d). Significantly lower levels of TBARS were observed in diabetic rats treated with 3μM of spironolactone when we compared them to diabetic rats treated with 0.1μM of spironolactone (Fig 4d). Levels of TBARS increased the most when 3μM of spironolactone was applied to healthy rats at CPP of 120 cm H2O compared to healthy control. In contrast, spironolactone in the same dose lowered TBARS the most in diabetic rats at CPP 80 cm H2O (Table 1.).
The present study aimed to assess the acute effect of spironolactone administration on oxidative stress parameters in STZ-induced DM isolated heart rats. Besides, we have also examined the acute effect of different spironolactone doses on oxidative stress parameters in the above mentioned isolated heart rats.
Besides hypertension, DM is one of the most common factors contributing to CVD, putting CVD at the top of the scale of mortality worldwide. Acceleration of atherosclerosis increases the arteries' stiffness; thus, earlier onset of CVD, such as coronary artery disease, heart failure, arrhythmias, is one of the primary mechanisms by which DM contributes to the development of CVD.
Development of diabetic cardiomyopathy, characterized by interstitial and perivascular fibrosis, thickening of basal membranes of capillaries and hypertrophy of cardiomyocytes are one of the most critical consequences of DM (18, 19, 20, 21). State of hyperglycemia through activation of different signal pathways leads to inflammation, increased ROS synthesis, and remodelling of the extracellular matrix of heart muscle (22). Those mechanisms lead to myocardial fibrosis, respectively increased rigidity, reduced ability for myocardial relaxation and increased stiffness of the endocardium. Abnormalities in calcium homeostasis, increased synthesis of ROS, and mitochondrial dysfunction are also some of the mechanisms involved in the development of diabetic cardiomyopathy. Simultaneously, activation of RAAS, one of the main ones, plays a crucial role in manifesting the consequences of chronic hyperglycemia (23, 24). One of RAAS activation effects is increased ROS synthesis and thus changes in activation genes leading to increased fibrosis of the heart, apoptosis, and necrosis (25). Beside increased ROS synthesis, consequential activation of angiotensin II receptors type 1 and MR can also lead to myocardial fibrosis (26). When activation of angiotensin II receptors type 1 and MR are compared, AT1 receptor blockade protects the heart from diabetic complications short-termly, while MR blockade provides long-term protection (27).
For chronic administration of spironolactone, MR's nonselective antagonist has been previously reported to lower the myocardial fibrosis, however, without effect on cardiodynamic parameters (28). Our previous research has documented a different impact of acute spironolactone administration on healthy and diabetic rats with better effects of higher spironolactone doses in diabetic rats, while in a healthy one, the same effect was observed with lower spironolactone doses (9). And our current findings are consistent with the previous one, showing the same effect of spironolactone on oxidative stress parameters as on parameters of cardiac dynamics in healthy and diabetic hearts. Levels of H2O2 were significantly lower in the group of diabetic rats treated with 3μM then in the healthy rats treated with the same dose of spironolactone. Simultaneously, no significant differences were noted when diabetic and healthy rats treated with 0.1μM of spironolactone were compared and when healthy rats treated with 0.1μM were compared with healthy rats treated with 3μM of spironolactone. Previous research has concluded that chronic administration of spironolactone lowers levels of H2O2, while it has also been proved that spironolactone enhances the activity of catalase, an antioxidant enzyme with effects on H2O2 (29, 30). Levels of NO2− did not significantly differ between our groups, except when we compared healthy and diabetic rats treated with 0.1μM of spironolactone at higher values of CPP where better effect were observed in healthy rats; and when we compared healthy rats treated with 0.1μM of spironolactone and healthy rats treated with 3μM of spironolactone again at higher values of CPP (100 and 120 cm H2O), confirming our earlier conclusion that spironolactone has better effects in heathy rats when given in lower doses. Similar results were observed when we looked at O2− levels, as well as TBARS levels.
Some of the findings we observed in our previously published research regarding different effects of different spironolactone doses on parameters of cardiodynamics could be explained by these findings of oxidative stress. In the literature, spironolactone improved eNOS, endothelial nitric oxide synthase improving coronary flow (3). It has also been reported that spironolactone improves NO/soluble guanylyl cyclase mediated response inducing vasodilatation (30). Beside improved function of eNOS, it has been shown that vitamin E levels were significantly higher in STZ-induced DM rat hearts that were treated with spironolactone, either by its enhanced production or suppressed consumption (3). It has also been shown that MR antagonists have only partial protection in diabetic eNOS knock-out mice in comparison to fully protected diabetic wild-type mice (31). Spironolactone also leads to lower levels of TBARS, which can point protective effects of spironolactone towards biomembranes, which contain numerous polyunsaturated free fatty acids.
In summary, different spironolactone doses achieved different effects on parameters of oxidative stress. While better effects in healthy rats were achieved with lower amounts of spironolactone, in diabetic rats, better outcomes were achieved with higher doses of spironolactone. These results coincide with our previous results regarding cardiodynamic parameters. Therefore, one explanation for the same effect of different spironolactone doses on cardiodynamic parameters in diabetic and healthy rats could be that the higher the dose of spironolactone, the better its impact on oxidative stress and, therefore, cardiac function. It has been shown that the state of hyperglycemia enhances MR transcriptional activity, and the reason for better spironolactone effect on diabetic hearts in higher dose could be explained by overexpression of MR in diabetic hearts, thus blocking it with higher spironolactone concentrations better result is achieved (32). In healthy hearts, the same effect is observed with lower spironolactone concentrations because there is no MR overexpression. These discoveries likewise concur with the recently revealed level after which perfusion with higher spironolactone groupings does not have any extra impact on myocardial contractility (33). Besides the reported plateau for spironolactone, it has also been shown that different spironolactone doses did not change the concentrations of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) in human coronary artery endothelial cells treated with 5.5mM dextrose (34).
Our study's main limitations are the lack of a causal relationship between the spironolactone effect on cardiodynamics and on parameters of oxidative stress. While oxidative stress induces MR activation, oxidative stress is subsequently increased by MR activation in a feedback loop, we could not determine whether the spironolactone effect is achieved directly by influencing cardiodynamic parameters and consequently changing the levels of parameters of oxidative stress or primarily by modification of parameters of oxidative stress and after that cardiodynamic parameters, however, the causal link exists (35, 36). Therefore, further research is needed to investigate the spironolactone effect on the relationship between cardiodynamic and parameters of oxidative stress and determine the definitive mechanism of action of different spironolactone dosage on diabetic hearts.
Spironolactone, a non-selective MR antagonist, achieved different effects on oxidative stress parameters when given acutely in different doses in diabetic and healthy rats. In lower doses, spironolactone's acute administration lowered the parameters of oxidative stress in healthy rats better than higher doses of spironolactone. In contrast, in the diabetic group, acute effects of higher doses of spironolactone lowered oxidative stress parameters better than lower doses of spironolactone.