The present study evaluated the association between vascular aging and the antioxidant defense system by investigating age-induced alteration in blood pressure, serum lipid levels, histological analysis, and antioxidant capacity in the aorta of young and aging rats.
Various studies have reported the rise in blood pressure that is considered an unavoidable part of healthy aging and predominantly accompanied by structural alterations in the arteries [20]. Our results showing significant elevation in the SBP, DBP, and PP in our aging model agrees with previous data. Additionally, previous data reported that high blood pressure is associated with loss of balance between oxidants and antioxidants [21]. Notably, the correlation between the age-associated decline in circulating sex hormones and elevated blood pressure has also been reported [22]. A gradual loss of gonadal hormones such as estrogen with aging has also been reported to play a role in blood pressure elevation in aging female rats. These findings and our data suggest that there is considerable correspondence between an age-related rise in blood pressure, which could result from aortic structural changes, and variation in antioxidant status.
It is well-known that aging affects normal cholesterol metabolism, resulting in an alteration in serum lipid profile [23]. This study detected significantly high serum LDL levels and significantly low serum HDL levels in aging rats than young rats. However, no significant change in the serum TG and TC levels was observed with aging. Consistent with our findings, age-related higher LDL and lower HDL levels have also been mentioned in previous reports [24, 25]. Likewise, no significant change in TG and TC levels was observed between the 24-month-old and 10-month-old rats [26]. It is well acknowledged that high LDL/low HDL levels in blood account for the accumulation of fat in the aorta and increase atherosclerosis incidence [10]. We further examined whether these age-related alterations in serum cholesterol levels are associated with the accumulation of fat/lipid in the aorta. As expected, high lipid accumulation in the aged aorta was observed. Notably, no apparent atheromatous plaque was observed in the aorta from aging rats. These findings suggest that increased fat/lipid components in the aorta might be due to altered blood cholesterol levels and can be considered a normal part of aging, independent of atherosclerosis.
Previous studies have reported that aging brings about profound structural and histological alterations in the aortic wall [1, 3]. By examining the histological changes in the aorta of young and aging rats using H&E staining, the aortic wall's basic architecture was found to be altered in aging rats. By comparison, aorta obtained from young rats exhibited normal aortic morphology. These observations in the aorta of aging rats agree with previous reports using different aging models [27, 28]. Consistent with our results, similar structural alterations in the aorta were observed in aged male albino rats [4]. In humans, age-associated modifications in the vascular structure have also been reported [29, 30]. We further investigated the effect of aging on nuclear morphology and apoptosis. Hoechst staining results revealed that aorta from aging rats exhibited typical apoptotic nuclear morphology such as blebbing, nuclear fragmentation, chromatin condensation, and cell shrinkage. The percentage of apoptotic nuclei was significantly higher in the aged aorta. These results agree with previously reported findings [6]. Importantly, increased apoptosis during aging is considered in one instance as a protective mechanism of the body against an accumulation of injurious defective cells and in another related to an age-related decline in the structure and functional integrity of various tissues [6].
Data from various studies have described that the accumulation of oxidative damage resulting from oxidant/antioxidant imbalance might play an essential role in structural alterations and increase apoptosis in various tissues with aging [6, 8]. These findings lead us to hypothesize that physiological aging accompanied by degenerative structural changes and increases apoptosis in the aorta could involve the oxidative mechanism. However, this underlying mechanism in the aged aorta is poorly understood.
Antioxidant enzymes protect the body against the deleterious effect of oxidative stress, and a decline in the antioxidant capacity results in the production of oxidative stress-related cellular damage in the tissues [12]. To evaluate the role of aging on the aorta's antioxidant capacity, we analyzed and compared some antioxidative and cytoprotective proteins' expression levels, i.e., SOD-1, GSS, CAT, NRF2, KEAP-1, HO-1, and NQO-1 in the aorta from young and aging rats. Western blot results and immunohistochemical analyses revealed that SOD-1 and GSS significantly decreased, and NRF2, KEAP-1, and HO-1 significantly increased in the aorta of aging rats in comparison with young rats. Additionally, the protein expression levels of CAT and NQO-1 remained unaltered. These results suggest that the age-related decline in the expression of SOD-1 and GSS could account for the oxidative stress-induced cellular damage to the aortic cells leading to the morphological and physiological alteration in the aorta with senescence. Similar to our findings, a study was performed in male Wister rats with ages ranging from 1 month to 24 months. The findings demonstrated an age-related decline in SOD-1 while CAT expression was unchanged in the aorta [31]. We could not find any research related to GSS alteration with aging in the aorta. However, few studies have reported changes in GSS protein expression in different organs with aging [32]. Surprisingly, the observed noticeable increase in the basal expressions of NRF2, KEAP-1, and HO-1 proteins in the aorta of aging rats is supported by others' findings on different organs other than the aorta [33]. L. Zhou et al. found that the basal expression of NRF2 regulated genes increased with aging in human bronchial epithelial cells, while inducible expression was declined in these cells [34]. Contrary to our results, impaired NRF2/KEAP-1 signaling and down-regulation of both NQO-1 and HO-1 with aging have been reported in various studies [35]. Z. Ungvari et al. reported that vascular oxidative stress is accompanied by a decreased expression of NRF2, NQO-1, and HO-1 in aged Fischer 344 _ Brown Norway rats [36]. A possible reason for this inconsistency might be using the different cells, tissues, and animals, and further investigations are needed to elucidate these controversial results.
Many studies have highlighted that, upon exposure of cells to oxidative stress, eukaryotic cells induce complex redox-sensitive NRF2/KEAP-1 signaling pathway to protect cells against oxidative injury and maintain cellular homeostasis [37]. NRF2/KEAP-1 signaling pathway up-regulates various antioxidants and phase II detoxifying enzymes such as HO-1 and NQO-1. HO-1 and NQO-1 are essential components of the cellular stress response and play a critical role in cellular protection against oxidative insult [35]. Importantly, NRF2 was mainly located in the nucleus of aortic cells, indicating the possible basal induction of NRF2/KEAP-1 signaling in the aorta in response to the occurrence of oxidative stress during aging. These findings suggest no cellular injury or oxidative damage in the aorta from young rats accounting for the basal levels of NRF2/KEAP-1 signaling pathway and its subsequent downstream regulation of NQO-1 and HO-1 proteins. However, in the aging rats, increased levels of NRF2 and KEAP-1 protein in the aortic tissues explained the age-associated overactivation of NRF2/KEAP-1 signaling pathway. Furthermore, the increased expression of HO-1 in the aorta of aging rats further confirmed that the induction of this enzyme is governed by the activation of NRF2/KEAP-1 pathway in response to oxidative stress in the aortic tissues. Interestingly, we found that the expression of NQO-1 protein was not significant in the young and aging rats. It confers the notion that in the aging aorta, the transcriptional antioxidant response of NRF2/KEAP-1 pathway is involved in the up-regulation of HO-1 compared to NQO-1 in female SD rats. Further investigations are however required to fully elucidate the basic mechanism involved in the activation of the NRF2/KEAP-1 pathway and its regulation in the aged aorta using knockdown or overexpression studies.
In summary, it is believed that there is a substantial correlation between degenerative histological changes and decreased antioxidant capacity in the aorta during physiological aging. It is possible that the degenerative structural changes associated with aging are the result of oxidative stress caused by decreased tissue production of main antioxidants such as SOD-1 and GSS. In response, eukaryotic cells may activate NRF2/KEAP-1 signalling and increase HO-1 enzyme production as a compensatory strategy to preserve normal aortic function in the aged aorta, hence promoting healthy aging in the absence of antecedent pathological illness.