Age-Induced Aortic Modications Are Accompanied By Alteration in the Antioxidant Defense System in Female Rats

Aging causes signicant structural and functional changes in the vasculature, disrupting homeostasis and affecting the cardiovascular system's performance. This study looked into the effects of aging on the aorta and how that affects antioxidant capability. As a result, healthy aging female SD rats (n=10) were compared to their younger counterparts. Blood pressure, serum lipid prole, aortic histology examination, and the expression of antioxidant enzymes were all assessed. When aged rats were compared to young rats, their blood pressure and serum lipid levels were dramatically different. Furthermore, histological examination of the aorta of aging rats revealed structural changes, characteristic apoptotic nuclear morphology, and the formation of fatty deposits. Different antioxidant enzyme’s protein expression was also studied. In aged rats, the expressions of SOD-1(superoxide dismutase), GSS(glutathione synthetase), NRF2(NF-E2-related factor 2), KEAP-1(Kelch-like ECH-associated protein 1), and HO-1(Heme oxygenase 1) were drastically changed. This study revealed that age-induced aortic changes are associated with an altered antioxidant system, according to this study.


Introduction
As an unavoidable aspect of life, aging is a composite natural process of becoming older. Aging introduces progressive structural and functional deterioration in almost all tissues, resulting in the gradual decline of the body's ability to maintain homeostasis, leading to natural death [1]. Vascular aging poses the highest risk for cardiovascular pathologies, and the aorta is considered a sensitive organ affected by aging [2]. Even with nonpathological aging, the aorta undergoes many structural distortions such as enlarged lumen, intimal-medial thickening (vascular remodeling), increased collagen and decreased elastin contents, and thin and fragmented elastic lamina in both human and animals [3,4]. Besides, age-associated genderbased differences in aortic structure and function have also been discovered [5] Previous studies have highlighted the association between aging and apoptosis [6]. Generally, during aging, the rate of apoptosis increases in many tissues and organ systems, including the cardiovascular system, endocrine system, immune system, and nervous system [7,8]. Earlier, aging studies have documented the link between aortic stiffness and elevated blood pressure [9]. Likewise, age-related elevation in cholesterol levels has also been reported. With aging, blood lipid contents, especially LDL level increases, accounting for the accumulation of lipids in the vascular tissues, resulting in the atherogenic burden [10].
Antioxidants are scavenger molecules that can slow or prevent the oxidation process by neutralizing the deleterious effects of ROS. Under typical physiological conditions, there is an equilibrium between oxidant production and their elimination by the antioxidative system to prevent oxidative stress and sustain homeostasis [11]. Oxidative stress is considered a signature feature of aging caused by an imbalance between the oxidant/antioxidant equilibrium. Aerobic organisms are well-equipped with the cellular defensive antioxidant system to protect cells from oxidative insult [12,13]. The antioxidant capacity comprises rst-line defense antioxidants such as SOD-1, CAT, glutathione system, phase II detoxifying enzymes such as NQO-1 and HO-1, and different cytoprotective signaling pathways such as NRF2/KEAP-1 pathway. The antioxidant defense system plays a central role in cellular protection by eliminating harmful oxidants and preventing cells from oxidative damage [12].
The superoxide SOD-1 rapidly converts superoxide anion (O 2 − ) to hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ). Subsequently, hydrogen peroxide is decomposed into water and oxygen by the CAT enzyme [11]. One of the under-explored enzymes is glutathione synthetase (GSS), a pivotal contributor in the glutathione synthesis pathway. GSS indirectly plays an antioxidant role by mediating the 2nd step in the synthesis of GSH [13]. NRF2 is a redoxsensitive transcription factor that facilitates the transcription of many antioxidants via binding with a particular antioxidant response element (ARE) in that gene's promoter. Under normal conditions, NRF2 activity is tightly regulated by its cytoplasmic inhibitor KEAP-1 [14,15]. During oxidative stress, the KEAP-1 undergoes conformational changes. Consequently, NRF2 dissociates from KEAP-1, translocates into the nucleus, and regulates the cytoprotective phase II detoxifying enzymes such as NQO-1 and HO-1 [15].
Numerous aging studies report that the decline in the antioxidant capacity results in oxidative stress-related cellular damage in different tissues [12]. Nevertheless, the mechanism of oxidative cellular damage in the aorta in response to the altered antioxidant system is not fully explored. This research was intended to explore the relationship between age-mediated aortic changes and the antioxidant defense system. In this context, blood pressure and serum lipid levels, aortic histological analysis, and the expression of different antioxidant proteins were evaluated to understand their correlation with the ageing process's complexity in the aortic wall.

Animals
Twenty female Sprague-Dawley (SD) rats were acquired from the animal center of Harbin Medical University. Rats were divided into two groups (n=10/group): healthy young (3 months old) and aging (24 months old). Physiological aging symptoms were con rmed via vaginal smears exhibiting the disorder of the estrous cycle with low levels of senile estrogen. All rats were maintained under controlled conditions: (temperature (22 -25°C), humidity (50 ± 10 %), and a 12-h dark/light cycle), and received the standard chow diet ad libitum with free access to water. All experimental protocols used in this research were approved by the Animal Care and Use Committee (IACUC) of Harbin Medical University, China.

Blood pressure
A week before sacri cing the animals, blood pressure was measured non-invasively in conscious rats by tail and cuff detector (BP-2010E, Softron biotechnology Ltd. China). Brie y, each rat's tail was heated to 37°C for 10 min, and the cuff with a pneumatic pulse sensor was placed around the animal's tail. Systolic, diastolic, and pulse pressure measurements were recorded in each rat and then averaged.

Sample collection
All animals were anesthetized with the intraperitoneal injection of 10% chloral hydrate and euthanized. Blood samples were collected and placed at 4°C for 1 hour and then centrifuged at 3000 rpm at 4°C for 15 min. The collected serum samples were stored at -80°C for further evaluation. The thoracic aorta was excised, and all adventitial fats were removed. A part of the aorta was xed in 4% paraformaldehyde and embedded in para n, and the remaining tissues were ash-frozen in liquid nitrogen and then stored at -80˚C for further analysis.

Histological analysis by H&E staining
The para n-embedded tissues were cut cross-sectionally into 5 µm thick sections by Leica RM 2016 rotator microtome. According to the protocol described previously, the para n sections were depara nized, rehydrated, and then stained with hematoxylin and eosin (H&E) [16]. H&E stained slides were observed under a light microscope, and photographs were taken using the M8 Microscope & Scanner (Precipoint, German). Images were processed with ViewPoint BETA v1.0.0.0 software. Aortic wall thickness was calculated using Image J software.

Nuclear morphological analysis by Hoechst 33258 staining
Aortic tissues were stained with Hoechst 33258 uorescent dye (Beyotime, China) to observe apoptotic nuclear morphological changes. Brie y, the para n sections of aortic tissues were depara nized in xylene, rehydrated in gradient alcohol, washed twice with cold PBS, and then incubated with Hoechst 33258 for 10 min at room temperature in the dark. The sections were rewashed in PBS and examined under a uorescence microscope (40x), and photographs were taken for further quantitative analysis. The percentage of apoptotic nuclei was calculated by counting the nuclei with apoptotic morphology among the normal nuclei.

Oil Red O staining (ORO)
In order to examine the aortic lipid contents, the freshly frozen (liquid nitrogen) aortic tissue samples from both groups were stained with Oil red O (ORO) (Nanjing Jiancheng Bioengineering Institute, China), as previously described [17]. The ORO-stained slides were examined under a light microscope and were photographed. The ORO staining intensity was quanti ed using ImageJ software.

Western blotting
The frozen aortic tissues were lysed with RIPA buffer, and total protein extracts were obtained by spinning at 14 000rpm for 15 min. Total protein concentration was calculated using a bicinchoninic acid (BCA) protein assay kit (Thermo Scienti c, USA). Total protein (20 µg) were electrophoretically fractionated by 10% SDS-PAGE and transferred to a polyvinylidene di uoride (PVDF) membrane with a Trans-Blot SD semi-dry transfer cell (Bio-Rad Laboratories, Richmond, Calif.). The membranes were blocked with 5% non-fat milk dissolved in Tris-buffered saline with 0.1% Tween 20 for 2 hours. The blotted proteins were visualized using Pierce ECL Western Blotting Substrate (Engreen Biosystem, China). The relative density of bands was analyzed by ImageJ software.

Immunohistochemistry (IHC)
The Para n-embedded sections of the aorta were depara nized and rehydrated, then treated with 0.3% H 2 O 2 to block endogenous peroxidase activity.
After blocking nonspeci c reactions with goat serum, the primary rabbit polyclonal antibodies of SOD

Statistical analysis
All results in this study were expressed as mean ± SD. For the statistical analysis, Student's t-test was applied for the group comparisons and was evaluated by GraphPad Prism version 6.0c (GraphPad, La Jolla, CA). P value less 5 (p<0.05) was considered statistically signi cant.

Aging increases blood pressure in rats
Systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure (PP) were analyzed and compared between young and aging rats. SBP, DBP, and PP were signi cantly elevated in aging rats compared to those of young rats (Fig. 1a, b & c). These results provide evidence that blood pressure increases with physiological aging.

Aging regulates serum lipid pro le
As compared to young rats, the serum HDL levels signi cantly decreased, whereas LDL levels remarkably increased in the aging rat (Fig. 2a&b). Although the total cholesterol and triglycerides levels slightly decreased in aging rats, they were not statistically signi cant (Fig. 2c & d).

Aging alters the histology of the aorta
Histological examination of the aorta from young rats expressed typical morphology. The tunica intima (TI) was very thin and composed of a continuous layer of thin attened endothelial cells with attened nuclei, and the luminal surface of the aorta was very regular and smooth. The tunica media (TM) was characterized by numerous diverse elastic laminae, which were wavy, parallel, and regularly arranged. Smooth muscle cells (SMCs) with oval to attened nuclei were regularly arranged in the narrow spaces between the concentric lamellae. The tunica adventitia (TA) was comprised of loose connective tissue (Fig. 3a).
In contrast, however, the aorta of aging rats revealed noticeable destructive morphology. TI was thick to the extent that it could easily be distinguished from the TM. The endothelial lining was featured with endothelial cell proliferation as their nuclei were close to each other with the invasion of the subendothelial connective tissues, and the luminal surface was relatively rough and irregular. TM was particularly thick with an increased number of elastic lamellae. The elastic bers of the TM were thin and remarkably spacious, with some level of fragmentation. SMCs were also proliferating in between the elastic laminae and showed binucleate and multinucleate appearance. The TA was thickened and showed brosis (Fig. 3b). Aortic wall thickness showed a signi cant increase in aging rats compared to young rats (Fig. 3c).

Aging alters nuclear morphology
Nuclear morphological changes were analyzed by Hoechst 33258 staining. As shown in Fig. 4a, the morphology of the nucleus was normal and intact and displayed fewer apoptotic nuclei in the young rats. In contrast, in aging rats, it was observed that nuclear staining exhibited typical apoptotic morphological changes such as chromatin condensation, nuclear fragmentation, and high apoptotic bodies (Fig. 4b). The percentage of apoptotic nuclei in the aging rats was signi cantly higher than that of the young rats (Fig. 4c).

There is the accumulation of lipid in the aortic wall during aging
The ORO staining was performed to visualize the quantity and intracellular localization of neutral triglycerides, lipid, and fat contents in the aorta. As illustrated in Fig. 5a, young rats displayed very weak ORO lipid staining as very few lipid droplets were seen in the aortic wall. By comparison, aging rats exhibited highly positive ORO staining in the aorta. Microscopically, the lipid droplets appeared as streaks of small red granules or grape-like clusters in all three layers. Besides, some enlarged lipid droplets were also detected within some regions in tunica media and tunica adventitia, representing the high amount of fat deposition in that particular region (Fig. 5b). The ORO positive area percentage was signi cantly increased in the aorta of aging rats (Fig.   5c).

Altered expression of antioxidant proteins with aging in the aorta
To investigate the effect of aging on the aorta's antioxidant enzymes, we analyzed protein expression levels of SOD-1, CAT, GSS, NRF2, KEAP-1, HO-I, and NQO-1 by western blot. Western blot results revealed that, compared to young rats, the protein expression levels of SOD-1 and GSS decreased signi cantly, whereas the protein expression levels of NRF2, KEAP-1, and HO-1 proteins signi cantly increased in aging rats. However, the protein expression of CAT was slightly decreased, and the protein expression of NQO-1 was slightly increased in aging rats compared to young rats (Fig. 5a&b). Immunohistochemistry (IHC) was performed to visualize antioxidant proteins' expression in the aorta from young and aging rats. IHC results con rmed the western blot ndings and revealed that the protein expression levels of SOD-1 and GSS decreased signi cantly in aging rats. However, quite the opposite, the immunostaining for NRF2, KEAP-1, and HO-1 increased signi cantly in aging rats' aorta compared to the young rats. Notably, immunostaining for CAT and NQO-1 was weak in both groups, and no noticeable difference was observed between young and aging rats (Fig. 7a&b). Quantitative assessments of the staining con rmed that the mean optical intensity density (MOID) score for SOD-1 and GSS showed a signi cant decrease, whereas the score for NRF2, KEAP-1, and HO-1 showed a signi cant increase in aging rats relative to the younger group. The MOID score for CAT and NQO-1 did not differ signi cantly in both groups (Fig. 7c).

Discussion
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 signi cant elevation in the SBP, DBP, and PP in our aging model agrees with previous data. 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 ndings 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 pro le [23]. This study detected signi cantly high serum LDL levels and signi cantly low serum HDL levels in aging rats than young rats. However, no signi cant change in the serum TG and TC levels was observed with aging. Consistent with our ndings, age-related higher LDL and lower HDL levels have also been mentioned in previous reports [24,25]. Likewise, no signi cant 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 ndings 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 modi cations 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 signi cantly higher in the aged aorta. These results agree with previously reported ndings [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 ndings 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 signi cantly decreased, and NRF2, KEAP-1, and HO-1 signi cantly 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 stressinduced cellular damage to the aortic cells leading to the morphological and physiological alteration in the aorta with senescence. Similar to our ndings, a study was performed in male Wister rats with ages ranging from 1 month to 24 months. The ndings demonstrated an age-related decline in SOD-1 while CAT expression was unchanged in the aorta [31]. We could not nd 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' ndings 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]. 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 ndings 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 con rmed 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 signi cant 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.

Conclusions
Aging may result in structural and functional changes to the aorta, as well as altered antioxidant defence system expression. These age-related changes in the antioxidant system of the old aorta may represent a compensation reaction designed to preserve normal homeostasis during aging. Comparison of blood pressure analysis between young and aging rats. In aging rats, systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure (PP) signi cantly increased compared to the young rats. (a) Systolic blood pressure ** (P<0.01) (b) diastolic blood pressure *** (P<0.001),

Figure 2
Comparison of serum lipid pro le (mmol/L) between young and aging rats. Relative to young rats, (a) Serum HDL levels signi cantly decreased ** (P<0.01) whereas (b) Serum LDL levels signi cantly increased in aging rats*** (P<0.001). No signi cant differences were found in (c) TG and (d) TC levels between young and aging groups. Data represent the mean ± SD. (n= 10 rats). Abbreviations: HDL=High-density lipoprotein, LDL= Low-density lipoprotein, TG=Triglyceride, and TC=Total cholesterol.   Oil red O staining of the aorta of the young and aging rats. (a) Representative images display weak ORO staining in the aorta of young rats (b) and high ORO staining in the aorta of aging rats that represents the accumulation of lipids in the aortic wall. (c) % of ORO positive area signi cantly increased in aging rats *** (P<0.001) vs. young rats (n=3). Abbreviations: ORO = Oil red O, yellow arrow = lipid droplets appeared as red granules in the aorta. Scale bar=200 µm. Figure 6