A growing body of evidence demonstrates that cardiovascular disease risk increases after the onset of menopause, which may related to metabolic and hormonal changes (Posa et al., 2015a; Rosano et al., 2007). While estrogen plays a fundamental role in antioxidant and anti-inflammatory mechanisms and positively regulates lipid and glucose metabolisms (Chakrabarti et al., 2008; Mauvais-Jarvis et al., 2013), postmenopausal women more likely tend to develop obesity, inflammation and oxidative stress (Posa et al., 2015a). Body fat accumulation causes low-grade chronic inflammation by enhancing the production of proinflammatory cytokines such as tumour necrosis factor alpha (TNF-[alpha]), interleukin-6 (IL-6), and IL-1 (Monteiro and Azevedo, 2010). This obesity-related inflammatory state can be associated with disruption of oxidant/antioxidant homeostasis and enhancement of oxidative stress. Furthermore, obesity could decrease the expression and activity of key cytoprotective systems, including heme oxygenase (HO) (Ndisang, 2010). HO is a rate-limiting enzyme responsible for the catabolism of heme into carbon monoxide (CO), ferrous iron, and biliverdin, which converted to bilirubin. CO and biliverdin/bilirubin metabolites have important functions in the cardiovascular system (Wu et al., 2011). CO can confer modulatory effects on blood vessels and causes vasodilatation. In addition, its antiapoptotic and anti-inflammatory actions are also significant (De Leon et al., 2003). Bilirubin is a powerful antioxidant by scavenging oxidants and inhibiting the production of superoxide anion. These effects verify that HO and its metabolites are key targets during chronic diseases and its modulation could influence the obesity and inflammation-related conditions.
Though, estrogen deficiency in itself increases overweight and obesity in postmenopausal women, many genetic as well as environmental/ behavioural effects (e.g. lifestyle, nutrition, and smoking) can further determine the pathophysiology of body fat accumulation. Postmenopausal women spend the third of their lives in estrogen-depleted state, therefore the management of obesity and obesity-related comorbidities has important health significance in the 21th century.
Cumulative evidence of studies indicates that physical activity plays an important role in weight management, reduces the risk of developing metabolic syndrome and seems to be an important component of cardiovascular diseases (CVD) prevention. In our earlier study we proved that 12 weeks of voluntary physical exercise is a potential therapeutic strategy to improve the metabolic parameters in ovariectomized female rats fed with high-triglyceride diet (Posa et al., 2015c). Beside metabolic homeostasis, the inflammatory state and oxidant/antioxidant homeostasis plays a pivotal role in the life expectancy of postmenopausal women.
To understand the effects of detrimental environmental factors, such as high-triglyceride diet and sedentary lifestyle and the potential preventive role of physical exercise on antioxidant and inflammatory status during menopause, HO enzyme system and inflammatory parameters, such as TNF-[alpha], IL-6 and myeloperoxidase enzyme were determined in this current study.
Animals and experimental design
All experimental procedures were performed in accordance with the standards of the European Community guidelines on the care and use of laboratory animals and had been approved by the Institutional Ethics Committee (XX.4802/2015).
Ten-week-old female Wistar rats were divided into two groups and were subjected to sham-operation (SO group) or bilateral ovariectomy (OVX group) under anesthesia (Figurel). A bilateral muscle wall incision was made and both the ovaries and the fallopian tubes were visualized. In the OVX group ovaries were bilaterally removed and the uterine horns were tied, while the uterus was left intact. The abdominal wall was sutured. In the control group the abdominal wall was opened, but the ovaries were not removed and uterine horns were not tied. After a 4-week recovery period serum levels of estrogen were measured using a quantitative enzyme-linked immunosorbent assay (Quantikine rat Estrogen Elisa kit, R&D Systems Inc.) according to the manufacturer's directions to verify estrogen deficiency (Madhu, 2010).
At the end of week 4, both OVX and SO rats were randomly subdivided into two subgroups that would differ in the amount of regular daily activity and in diet for the next 12 weeks (Figure 1). The exercising (running) subgroups of rats (R) were placed in cages mounted with running wheels. Training was defined as a voluntary wheel-running model, allowing free access to wheel for 24 h per day for 12 weeks, i.e. physical activity (running) was an inherent part of the animals' daily routine (Posa et al., 2015b). Indeed, we have previously demonstrated that the average running distance for rats placed in cages mounted with running wheels highly exceeds that for the control group placed in standard holding cages during the exercising period (Posa et al., 2015b). The animals in both groups were maintained on either standard (CTRL) or high triglyceride (HT, 40% fat content) chow and had free access to water (Posa et al., 2015c). At the end of the 12-week exercising period the animals were killed. Blood (plasma) and tissue (aorta and heart) samples were frozen for further analysis. Body weight of each rat was followed during the 12-week training/feeding period.
Measurement of heme-oxygenase activity within the aorta and the heart
Heme-oxygenase activity was measured by spectrophotometric assay. Cardiac and aortic tissues of approximately 50 mg were homogenized (Ultra-turrax, T25; 13,500 rpm twice for 20 s) in a mixture of 10 mM N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), 32 mM sucrose, 1 mM DTT, 0.1 mM EDTA, 10 [micro]g/mL soybean trypsin inhibitor, 10 [micro]g/mL leupeptin and 2 ug/mL aprotinin; pH: 7.4. The supernatants were centrifuged for 20 min at 15,000g at 4[degrees]C. The reaction mixture prepared for the measurement of HO activity contained the following compounds in a final volume of 1.5 mL: 2 mM glucose 6-phosphate, 0.14 U/mL glucose-6-phosphate dehydrogenase, 15 [micro]M heme, 150 [micro]M [beta]-NADPH, 120 [micro]g/mL rat liver cytosol as a source of biliverdin reductase, 2 mM Mg[Cl.sub.2], 100 mM potassium phosphate buffer and 150 [micro]L of the supernatant. The standard incubation mixture was kept at 37[degrees]C for 60 min in dark. The reaction was terminated by placing the samples on ice. Optical density was measured at 460 and 530 nm wave length. Protein content was assayed spectrophotometrically (Bio-Rad, Protein Assay). HO activity was expressed as bilirubin formed per hour per mg protein (Posa et al., 2015a).
Measurement of aortic and cardiac HO-1, plasma IL-6 and TNF-[alpha] concentrations
Tissue levels of HO-1, and plasma TNF-[alpha] and IL-6 concentrations were detected by using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (SunRedBio rat HO-1, SunRedBio rat IL-6 and SunRedBio rat TNF-[alpha] Elisa kits). Concentrations were expressed as ng/mg protein (HO-1) pg/ml protein (IL-6) and ng/ml protein (TNF-[alpha]). Optical density was measured at 450 nm (Benchmark Microplate reader; BioRad) (Posa et al., 2015a).
Aorta and heart myeloperoxidase activity
Aortic and cardiac tissue samples were homogenized (Ultra Turrax, T25, twice for 30 s, 13 500r.p.m.) in ice-cold phosphate buffer (50 mM, pH: 6.0), and subjected to 3 freeze/thaw cycles. The homogenate was centrifuged (at 15 000g for 15 min at 4[degrees]C) and the supernatant (12 [micro]L) was mixed with 280 [micro]L of phosphate buffer (50 mM, pH: 6) containing 0.167 mg/mL of O-dianisidine dihydrochloride. Next 10 [micro]L of 0.03% hydrogen peroxide was added to the...