The experiments described here demonstrated that daily melatonin treatment or its co-administration with HRT for 8 weeks starting by the second week of surgical menopause, upregulated the expressions of IL-10 and SIRT1, reduced the expressions of the pro-inflammatory cytokines and reduced DNA damage in the hearts and thoracic aortae of non-exercised rats, showing a significant improvement in the inflammatory status of the cardiovascular system. Similarly, co-administration of melatonin and HRT to exercised post-menopausal rats also resulted in a reduced inflammatory response in the aortic and cardiac tissues. When taken together, these results implicate that melatonin treatment by itself upregulates SIRT1 expression and reduces the intensity of menopause-associated cardiovascular inflammation more potently than either exercise or HRT alone, while addition of melatonin to HRT or exercise alleviates upregulation of the inflammatory cascades induced either by HRT, exercise or HRT plus exercise.
Since the aging process in women overlaps with the period of reduced ovarian hormone production, age-related morphological, autonomic, biochemical and functional changes in the cardiovascular system are exacerbated by menopause, increasing the incidence of CVD . Accumulating evidence has revealed that depletion of ovarian hormones results in increased sympathetic tonus, oxidative stress, and impaired endothelial function, and thereby has a dominant impact on the occurrence of atherosclerotic processes, vascular damage and cardiovascular dysfunction [9, 47–50]. Although estrogen supplementation in OVX-rats [51–55] or in younger postmenopausal women  was shown to be effective in delaying atherosclerosis and reducing the CVD risk [57, 58], randomized controlled trials were not able to confirm the CVD-preventive effect of postmenopausal hormone therapy alone . Our findings demonstrated that estradiol treatment for 8 weeks decreased TNF-α expression and elevated IL-10 expression in both the cardiac and aortic tissues, reduced IL-6 expression in the cardiac tissue and decreased oxidative DNA damage in the aortae of the non-exercised postmenopausal rats. Moreover, HRT resulted in the upregulation of SIRT-1 expression in both tissues, demonstrating the possible involvement of SIRT1 pathway in HRT-induced amelioration of menopause-associated cardiovascular inflammation. Despite these improvements reached by estradiol treatment, aortic IL-6 expression was increased, while DNA damage was elevated and GSH levels were depleted in the hearts of estradiol supplemented non-exercised postmenopausal rats, showing some augmentation in cardiovascular inflammation by HRT. In parallel to our findings, a randomized, placebo-controlled study conducted in healthy normotensive postmenopausal women has demonstrated that a short-term estradiol treatment initially increased the plasma levels of C-reactive protein (CRP), which was associated with the increased risk of cardiovascular events due to HRT . Similarly, it was demonstrated that the inflammatory markers CRP and IL-6 are predictive of incident vascular events among otherwise healthy postmenopausal women, and long-term use of HRT elevates CRP . Thus, combining estrogen replacement with other pharmacological treatment modalities would be a meaningful strategy to reduce the ongoing inflammation. Accordingly, our findings revealed that addition of melatonin to HRT, as well as melatonin per se improved all oxidative and inflammatory parameters in the non-exercised rats.
As a non-pharmacological therapeutic approach, exercise was demonstrated to have beneficial effects in attenuating cardiometabolic syndrome, improving antioxidant status, reducing proinflammatory cytokine levels and ameliorating cardiovascular dysfunction in OVX rats [60–64]. Clinical studies verified the advantageous effects of exercise training on metabolic and lipid profiles and cardiorespiratory fitness of postmenopausal women [16, 65, 66]. On the other hand, it was reported that running exercise for a 6-week period either alone or in combination with phytoestrogen treatment had no positive effect on OVX-induced deterioration of cardiovascular functions in rats . Another study demonstrated that estradiol treatment has not prevented cardiac dysfunction in OVX-rats with myocardial infarction, while adding estradiol treatment to exercise has abolished the positive effects of 8-week treadmill exercise in infarcted postmenopausal rats . Similarly, estrogen-supplemented OVX rats have presented a decrease in cardiac IL-6 levels, while this decrease in IL-6 was abolished by an 11-week treadmill program . We have previously demonstrated that 4 weeks of swimming exercise prior to myocardial injury has reduced the plasma TNF-α level in the postmenopausal rats as compared to sedentary rats, but the elevated plasma IL-6 and IL-8 levels were not reduced by exercise . Our current findings revealed that regular swimming exercise alone for 8 weeks, downregulated IL-6 and upregulated IL-10 in both the cardiac and aortic tissues of the postmenopausal rats, while aortic TNF-α expression was further increased by exercise. Despite the elevation in aortic SIRT1 expression showing the beneficial effect of exercise on the vasculature of the non-HRT-treated rats, cardiac SIRT1 was conversely downregulated. However, exercise accompanied by HRT upregulated SIRT1 expression in both tissues, but the elevations observed in cardiac 8-OHdG and aortic IL-6 expressions of only HRT-given rats were not changed by the addition of exercise. On the other hand, except for some ongoing elevations in IL-6 and TNF-α melatonin intake in the postmenopausal exercised rats resulted in elevated SIRT1 expression and reduced DNA damage in both tissues, demonstrating the ameliorative effect of melatonin on the additive cardiovascular oxidative injury triggered by exercise.
Since postmenopausal hormone therapy has been linked to an increased risk of several female cancers , postmenopausal use of estrogen has been restricted and an alternative to estrogen has become crucial. Accordingly, a recent systematic review has reported that oral melatonin administration improves hemodynamic measures, glucose metabolism, bone density, sleep quality and climacteric symptoms, suggesting that melatonin could be considered as an effective treatment option for menopausal women . Clinical studies have indicated that melatonin improves lipid metabolism and protects against the atherosclerotic changes, and thereby could have benefits in the treatment or prevention of cardiovascular diseases [36, 72–74]. In support of these clinical outcomes, melatonin supplementation in OVX-rats enhances the antioxidant effects of estrogen and prevented oxidative stress more effectively than estrogen replacement [75, 76]. In agreement with the aforementioned studies, the findings of the present study demonstrated that melatonin treatment alone in postmenopausal rats with no additional treatment strategies elevated the SIRT1 expression and improved the oxidative status of the cardiovascular tissue. Furthermore, our results also showed that HRT- or exercise-induced oxidative cardiac damage and enhanced cardiovascular inflammation were ameliorated when melatonin was added to the treatment regimen.
Considering the diverse functions of melatonin throughout the body, it is expected that the reduction in endogenous melatonin is involved in the pathogenesis of several aging-associated diseases including CVD . Aging and several age-related pathologic conditions are closely linked with reduced secretion of melatonin and diminished SIRT1 activity . SIRT1, which regulates gene expression by deacetylation of histone proteins and transcription factors, plays an important role in the prevention of oxidative stress and inflammation in several tissues by mediating multiple signaling pathways [78–81]. In accordance with that, melatonin was shown to upregulate SIRT1 in cell cultures and several animal models, while the beneficial effects of melatonin were abolished by inhibition or knockdown of sirtuin, suggesting that the mechanisms contributing to anti-inflammatory effects of melatonin include its facilitatory effect on SIRT1 signaling pathway [82–85]. Both experimental and clinical studies have shown that melatonin facilitates the recovery of osteogenesis and ameliorates postmenopausal bone damage by upregulating SIRT1-mediated antioxidant enzyme capacity [86, 87]. Although the SIRT-1-mediated anti-inflammatory, antioxidant and antiapoptotic effects of melatonin on cardiovascular injury were demonstrated in several animal models [79, 82, 84, 88, 89] the involvement of SIRT-1 signaling pathway in the postmenopausal beneficial effects of melatonin was not elucidated before. A recent study in OVX mice has shown that estrogen-SIRT1 axis has a dominant modulatory role in protecting aortas against the development of atherosclerosis . In accordance with these results, our data demonstrate that administration of melatonin or melatonin plus estradiol to non-exercised postmenopausal rats improved the oxidative and inflammatory parameters via the upregulation of SIRT1, while exercise-induced downregulation of cardiac SIRT1 was reversed by melatonin intake. When taken together, our data implicate that the beneficial effects of melatonin in ameliorating postmenopausal cardiovascular injury involve the upregulation of antioxidant capacity and the stimulation of the SIRT1 activity, making it a safer alternative treatment option as compared to HRT.
Melatonin was shown to exert neuroprotective effects against sepsis-induced oxidative brain injury by decreasing the production of proinflammatory cytokines and MDA, and increasing antioxidant capacity via the SIRT1 activation . In the present study, exercise-induced ROS generation in the brain tissue of non-HRT-treated rats was not altered by melatonin, while increased lipid peroxidation due to HRT in exercised rats was abolished by melatonin. In accordance with the minor changes in the oxidative status of the brain tissue, the short- and long-term memory functions were normal in all groups and not different among of the studied menopausal treatment modalities, suggesting that the changes in the expression of inflammatory mediators in the cardiac and vascular tissues were not reflected in cognitive functional changes yet. It is well known that cognitive decline occurs due to cumulative exposure to cardiovascular risk factors and chronic atherosclerosis that are responsible for cerebral hypoperfusion and reduced brain oxygenation [91, 92]. Thus, it is possible that surgical menopause conducted in the early life of the rats has not yet impaired the memory functions.
In conclusion, the present study suggests that melatonin treatment, either alone or in combination with exercise and/or HRT, alleviates oxidative injury and inflammation in the hearts and aortas of postmenopausal rats via the activity of SIRT1 signaling. Although the mechanistic relation between melatonin and SIRT1 expression has been
reported previously in several publications, this is the first study to demonstrate that the exercise melatonin combination exerts SIRT1-mediated beneficial effects on post-menopausal complications in the cardiovascular system. Thus, our encouraging results implicate that melatonin should be considered in alleviating CVD risk in postmenopausal women, and further studies are required to elucidate the complex regulatory mechanisms involved in the melatonin-SIRT1 axis.