Dietary Nucleotides Retard Oxidative Stress-Induced Senescence of Human Umbilical Vein Endothelial Cells

Several lines of evidence suggest an inhibitory role of dietary nucleotides (NTs) against oxidative stress and inflammation, which promote senescence in age-associated cardiovascular diseases. We sought to test whether the dietary NTs could retard the hydrogen peroxide (H2O2)-induced senescence of human umbilical vein endothelial cells (HUVECs) and to elucidate the efficiency of different NTs as well as the potential mechanism. Senescence was induced in HUVECs by 4 h exposure to 200 µM H2O2 and was confirmed using senescence-associated-β-galactosidase staining (SA-β-gal), cell viability, and Western blot analyses of p16INK4A and p21Waf1/Cip1 after 24 h administration of growth medium. We find that NTs retards oxidative stress-induced HUVECs senescence, as shown by a lower percentage of SA-β-gal-positive cells, lower expression of p16INK4A, and p21Waf1/Cip1 as well as higher cell viability. GMP100 was the most excellent in delaying HUVECs senescence, which was followed by the NTs mixture, NMN, CMP50, and UMP50/100, while AMP retards HUVECs senescence by specifically reducing p15INK4b expression. NTs all have significant anti-inflammatory effects; AMP and CMP were more prominent in restoring mitochondrial function, GMP and CMP were more competent at eliminating ROS and MDA, while AMP and UMP were more efficient at enhancing antioxidant enzyme activity. The role of the NTs mixture in retarding HUVECs senescence is full-scaled. These results stated that the mechanisms of NTs retarding HUVECs senescence could be related to its antioxidant and anti-inflammation properties promoting cell proliferation and protecting mitochondrial function activities.


Introduction
Cardiovascular diseases (CVDs) are the leading cause of death globally [1], and the strongest independent risk factor for CVDs is age: more than 90% of CVDs occur in adults age 40 and older [2]. As a hallmark of aging, senescent cells could play a detrimental role in age-associated pathologies [3,4]. Cellular senescence is a stable cell cycle arrest [5] that is characterized by an inflammatory phenotype known as the senescence-associated secretory phenotype (SASP) [6], the accumulation of oxidative stress-induced damage [7,8], telomere shortening [9], and mitochondrial dysfunction [10]. The accumulation of senescent cells with age might trigger a chronic inflammation with detrimental effects on neighboring cells and the whole organism, therefore contributing to the initiation and progression of CVDs [11]. Indeed, there is a large body of evidence that senescent cells are present in the pathological tissues of patients with CVDs [12]. The expression of senescence markers including SA-β-gal, p16 INK4A , and p21 Waf1/Cip1 was upregulated in endothelial cells from human atherosclerotic plaques [13]. Nevertheless, a treatment that attempts to eliminate p16-positive senescent cells could be used to prevent and treat the CVDs [14][15][16].
The data from experimental studies suggest that NTs may have the potential to ameliorate cell senescence. NTs are the basic units of nucleic acid macromolecules, including cytosine, adenine, guanine, thymine, and uracil. These are a group of bioactive
The induction of HUVECs senescence by H 2 O 2 was performed as previously described; HUVECs were maintained for 4 h in a growth medium containing 200 µM H 2 O 2 (determined by preliminary tests, Figure S1) and then cultured in growth medium with or without different concentrations of NTs for 24 h. Our experiment set up 16 groups in total: a control group (growth medium), model group (growth medium), NTs mixture group (growth medium containing 100 µM NTs mixture, AMP:CMP:GMP:UMP = 22.80: 25.80:30.20:20.40), NMN group (growth medium containing 0.5 mM NMN), and low, middle, and high doses of GMP/UMP/CMP/AMP groups (growth medium containing 50/100/200 µM GMP/UMP/CMP/AMP). Following exposure to NTs, the cells were harvested for further analysis.

Morphology Observation
The cell morphological changes were observed by transmission electron microscope. In brief, cells were collected and washed twice with PBS before being fixed with 2.5% glutaraldehyde overnight at 4 • C. Then, cells were washed three times with PBS and fixed with 1% osmic acid for 1 h. Graded acetone was dehydrated (30%, 70%, 95%, 100% in PBS, each for 15 min), the resin was embedded, and ultrathin sections (Mode: OMU3, Leica Reichert, Munich, Germany) were stained with uranyl acetate and citric acid (Beyotime, Shanghai, China). Cellular morphology and mitochondria were observed by transmission electron microscopy (JEM-1400. Leica Reichert, Wetzlar, Germany).

Cell Viability Assay
Cell viability was evaluated by the cell-counting kit-8 (CCK-8) assay (KeyGEN, Jiangsu, China) according to the manufacturer's protocol. In brief, 100 µL/well cells (about 1 × 10 4 ) were seeded in 96-well plates. After treatment according to the protocol, 10 µL CCK-8 was added to each well and incubated at 37 • C for 1-4 h. The absorbance of each well was measured at 450 nm with a microplate reader (BMG FLUOstar Omega, Offenburg, Germany).

Senescence-Associated Beta-Galactosidase (SA-β-gal) Activity
The SA-β-Gal activity was assayed by the SA-β-gal staining kit (Beyotime, Shanghai, China) following the manufacturer's instructions. In brief, 1 mL/well cells (about 1 × 10 5 ) were seeded in 24-well plates. After treatment according to the protocol, the cells were washed once with PBS and fixed with SA-β-gal staining stationary liquid for 15 min at room temperature. After removing the stationary liquid, the cells were washed three times with PBS. Then, cells were incubated overnight at 37 • C in darkness with the SA-β-gal staining working solution. After being rinsed with PBS, the cells were observed at the microscope for the development of the blue coloration. The percentage of SA-gal-positive cells was calculated from three random fields by ImageJ software.

Flow Cytometry
First, 2 mL/well cells (about 2 × 10 5 ) were seeded in 6-well plates and treated according to the protocol. For apoptosis analysis, cells were harvested and washed once with PBS and then resuspended in PI/Annexin-V solution (KeyGEN, Jiangsu, China) and analyzed using a Flow Cytometer (Beckman Coulter, Brea, CA, USA). For intracellular ROS analysis, cells were harvested and washed once with PBS before being incubated for 20 min at 37 • C with the 10 µM 2,7-dichlorofluorescein diacetate (Beyotime, Shanghai, China). After being washed with PBS three times, the cells were analyzed using a Flow Cytometer (Beckman Coulter, Brea, CA, USA). For mitochondrial membrane potential (∆Ψm) analysis, cells were harvested and stained with 500 µL 1× JC-1 dye solution (Beyotime, Shanghai, China) at 37 • C for 20 min in the dark. Then, the cells were washed twice and resuspended by 1× JC-1 staining buffer. The change of fluorescence color was analyzed using flow cytometry (Beckman Coulter, Brea, CA, USA).

Western Blot Analysis
First, 2 mL/well cells (about 2 × 10 5 ) were seeded in 6-well plates and treated according to the protocol. Cells were collected and washed twice with PBS; then, they were resuspended in RIPA Lysis Buffer (Biosharp, HeFei, China) and supplemented with 1 mM of phenylmethanesulfonyl fluoride. Protein was extracted by centrifugation at 14,000× g for 15 min at 4 • C, and the concentration of protein was measured with a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (80-150 µg) were separated by 10-20% SDS-PAGE gel and transferred to PVDF membranes at different electric currents according to the size of protein molecules. The membranes were blocked for 2 h in 5% non-fat milk dissolved with Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature. Protein expression was detected using a primary antibody p16 INK4A

Statistical Analysis
Statistical analyses were performed using the SPSS software version 24 (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) test; the difference of parametric samples among groups multiple comparisons of least significant difference (equal variances assumed) or Dunnett's T3 test (equal variances not assumed) was used. p < 0.05 indicated a statistically significant difference. The calculating formula of the relative rate is as follows: decreased rate = (model group − treated group)/model group, increased rate = (treated group − model group)/model group.

Effect of NTs on Senescent HUVECs Morphological Changes
Senescent HUVECs displayed an enlarged nucleus, chromatin pyknosis, invaginated nuclear membranes, and a reduced amount of mitochondria. When exposed to 100 µM NTs mixture, the normal nucleus size, homogeneous chromatin, flat nuclear membranes, and relatively major amount of mitochondria was generally maintained in HUVECs ( Figure 1A).

Effect of NTs on Cell Apoptosis in H 2 O 2 -Induced Senescent HUVECs
Significant differences in apoptotic rate were witnessed between the control and model groups. Significantly decreased apoptotic rate was seen in the NTs mixture as well as the CMP200, GMP50, and UMP50 groups ( Figure 2A). The decreased rate relative to the model group was 51.16% (NTs mixture) > 45.42% (UMP50) > 42.65% (CMP200) > 40.75% (GMP50) respectively, while no effect on apoptosis rate occurred with the administration of NMN and AMP.

Effect of NTs on Intracellular ROS Production
Enhanced intracellular ROS production in response to the exogenous addition of H2O2 was observed in the model group compared with the control group, whereas the treatment with NTs significantly decreased intracellular ROS production. As shown in Figure 2B, the AMP200, GMP100/200, UMP100, and the NTs mixture significantly inhibits ROS production, and the inhibition rate relative to the model group was 48.17% (NTs mixture) > 47.60% (UMP100) > 46.14% (GMP100) > 42.35% (AMP200) > 41.45% (GMP200), respectively. No effect on ROS production occurred with the administration of NMN and CMP.

Effect of NTs on Senescent HUVECs Mitochondrial Membrane Potential (JC-1)
The exogenous addition of H2O2 induced mitochondrial membrane potential depolarization in HUVECs. Meanwhile, compared with the model group, a positive effect of

Effect of NTs on Intracellular ROS Production
Enhanced intracellular ROS production in response to the exogenous addition of H 2 O 2 was observed in the model group compared with the control group, whereas the treatment with NTs significantly decreased intracellular ROS production. As shown in Figure 2B, the AMP200, GMP100/200, UMP100, and the NTs mixture significantly inhibits ROS production, and the inhibition rate relative to the model group was 48.17% (NTs mixture) > 47.60% (UMP100) > 46.14% (GMP100) > 42.35% (AMP200) > 41.45% (GMP200), respectively. No effect on ROS production occurred with the administration of NMN and CMP. The exogenous addition of H 2 O 2 induced mitochondrial membrane potential depolarization in HUVECs. Meanwhile, compared with the model group, a positive effect of NTs mixture, AMP200, CMP200, GMP200, and UMP50/100 on mitochondrial membrane potential was observed ( Figure 2C). The increased rate of red fluorescent relative to the model group was 33.23% (UMP100) > 32.43% (UMP200) > 30.91% (CMP200) > 30.38% (GMP200) > 28.90% (AMP200) > 27.34% (NTs mixture), respectively.
Although there was a tendency toward higher expression, the expression of p15 INK4b was not significantly altered in senescent HUVECs compared with the control group, and a significantly decreased level of p15 INK4b was seen in the AMP50 group compared with the model group ( Figure 4H-J). No difference in ACE-2 expression was witnessed between the control and model groups, and it was significantly increased in the CMP200 group compared with the control group ( Figure 4K-M).   Figure 4B-D), the decreased rate relative to the model group was 80.99% (CMP100) > 67.09% (GMP200) > 65.98% (GMP50) > 61.80% (UMP50) > 61.54% (NMN) > 59.80% (NTs mixture) > 57.56% (UMP200) > 47.42% (CMP50), respectively.
Although there was a tendency toward higher expression, the expression of p15 INK4b was not significantly altered in senescent HUVECs compared with the control group, and a significantly decreased level of p15 INK4b was seen in the AMP50 group compared with the model group ( Figure 4H-J). No difference in ACE-2 expression was witnessed between the control and model groups, and it was significantly increased in the CMP200 group compared with the control group ( Figure 4K-M).

Discussion
Currently, CVDs account for approximately 39.6% of age-related diseases [29], and CVDs-induced insufficient blood supply may further accelerate the aging process. By this, we investigated the role of NTs in attenuating HUVECs senescence. The free radical theory of aging proposes that the accumulation of molecular oxidative damage increases as the organism ages and is postulated to be a major causal factor of senescence [30]. Although recent data support a role for ROS in the activation of compensatory homeostatic responses, ROS aggravate age-associated damage at certain higher doses [31]. Multiple studies have successfully built a senescence model by aggravating oxidative stress [32,33]. Based on this factual framework, we have established the model of H 2 0 2-induced senescence.
We assessed several senescence markers during the experiment, and our data showed that exposure to 200 µM H 2 O 2 for 4 h significantly upregulated p16 INK4A , p21 Waf1/Cip1 , and SA-β-gal expression in HUVECs, confirming that the senescence model was wellestablished. Given a permanent-growth arrest coupled to acute oxidative stress, reduced cell viability and increased apoptosis rate were observed in senescent HUVECs. However, our data revealed that the expression of p15 INK4b , a cyclin-dependent kinase inhibitor that mediates cell cycle arrest [34], and ACE-2, a hallmark of cardiopulmonary aging [35], were not affected in the present study.
We show that NTs retards oxidative stress-induced HUVECs senescence. The expression of p16 INK4A , p21 Waf1/Cip1 , and SA-β-gal significantly reduced in the NTs treated group. Correspondently, cell viability and apoptosis rate returned to the normal levels. Further analysis of the data supported the comprehensively positive role of the NTs mixture, CMP50, GMP100, and UMP50/100 in delaying HUVECs senescence, and GMP100 was the most excellent candidate ( Figure S2A,B). While NMN specifically downregulated the expression of p16 INK4A , it did not affect the p21 Waf1/Cip1 ( Figure S2B). AMP significantly augmented cell viability and suppressed SA-β-gal activity in HUVECs at low and middle doses, but they both demonstrated an incapacity for lowering p16 INK4A and p21 Waf1/Cip1 expression ( Figure S2C). Interestingly, AMP50 significantly downregulated p15 INK4b expression compared to the model group. Given that cellular senescence was identified as a hallmark of aging, the therapeutic strategies targeting senescent cells may attenuate ageassociated pathologies and prolong lifespan. Our data revealed the role of NTs in delaying HUVECs senescence, thus revealing its potential application for CVDs therapy. Our finding is partially consistent with that of Xu, who also found that elevated intracellular adenosine promoted proliferation in human endothelial cells [36]. Furthermore, previous studies in our lab also found that the long-term feeding of NTs prolonged the average and maximum lifespan of SD rats and reduced the death rate due to tumors [25].
According to recent studies, SASP, the hallmarks of cellular senescence [37], can drive age-related pathologies through paracrine and likely endocrine effects [38]. Along this line, the suppression of SASP is rational for retarding senescence and attenuating agerelated pathologies. Our data showed that NTs have strong anti-inflammatory properties in HUVECs, and the response intensity of each group was relatively consistent, especially to IL-6, IL-1β. The NTs mixture, NMN, CMP50/100/200, GMP50/100, and the UMP50 groups significantly decreased all of the five indexes, while NMN and GMP100 exhibited the greatest performance ( Figure S3A,B). AMP did not affect sICAM-1 secretion, but it was highly active in inhibiting IL-6, IL-1β, VCAM-1, and MMP-3 secretion, especially at a low dose ( Figure S3C). For UMP, the low dose was also more effective ( Figure S3F), while in GMP and CMP, the middle dose showed greater performance ( Figure S3D,E). Furthermore, IL-17 was pinpointed as an aging marker elevated in the aged lung, heart, and vessel [35]. Consistent with this, our data showed that IL-17 secretion was upregulated in senescent HUVECs, but NTs did not alter the upregulated state in the present study. Meanwhile, NTs are essential for maintaining the optimal immune response, and several previous studies supported their anti-inflammatory action [18].
Given that oxidative stress aggravates age-associated damage, the alleviation of oxidative damage or enhancement of antioxidant capacity by NTs treatment is a potential mechanism for retarding senescence in HUVECs. We showed that NTs exhibit superior antioxidant properties through different mechanisms ( Figure S4A). Only AMP200 significantly altered overall the four indexes, indicating the integrative competencies of AMP200 to suppress oxidative damage. GMP100 and CMP200 were more inclined to eliminate ROS and MDA ( Figure S4B), while AMP50 and UMP100 were inclined to increase GSH-Px and SOD activities ( Figure S4C). In the present study, the GHS-Px activity in the model group tended to decrease, but there was no difference. Therefore, if we ignore the effect of NTs on GSH-Px ( Figure S4D), the performance of NTs mixture was outstanding and all-sided, followed by AMP200 and GMP100 ( Figure S4E). In addition, our previous studies have demonstrated that NTs inhibited the decrease in antioxidant enzyme activity and the increase in lipid peroxidation product in aged animal models [25].
Mitochondrial dysfunction occurs with cells and as organisms age, thus resulting in the loss of mitochondrial membrane potential and reduced ATP production. In turn, this opens up the possibility of retarding HUVECS senescence by improving mitochondrial function. In the present study, mitochondrial function was estimated by mitochondrial membrane potential and the ability of ATP production. From the data in Figure S5, it is apparent that the NTs mixture and a high dose of NTs exerted a protective effect on the mitochondrial membrane, while for ATP production, the effects of AMP and CMP were generally superior to those of GMP and UMP. The NTs mixture and AMP200 significantly increased both mitochondrial membrane potential and ATP production. There was a complex link between NAD + metabolism and mitochondrial function, and decreased cellular NAD + concentrations occur during aging [39]. Thus, we involved the NAD + level in our test, and the effects of the NTs mixture, AMP, and CMP were also highlighted. We supposed that the senescence ameliorative effect of NTs on HUVECs may depend on NAD + sensing by Sirt1. Increased levels of NAD + enhance Sirt1 activity and lead to the activating deacetylation of PGC-1α and increased mitochondrial biogenesis and function [40]. Sirt1 is also linked to aging by regulating inflammation through NF-κB signaling [39]. Meanwhile, this study broadly supports the work of other studies in animal models, which showed that exogenous NTs could increase oxidative phosphorylation [20], enhance the repair of damaged mitochondrial DNA [41], and promote the early recovery of mitochondrial function changes after stress [28]. However, this study has been unable to demonstrate that NTs enhance the turnover between oxidized and reduced forms of NAD + , as described previously [20].
NMN is a key NAD + intermediate; extensive studies have shown that supplementing NMN ameliorates age-associated pathophysiologies and disease conditions, including CVDs [42]. NMN is also a derivative of NTs and has a similar chemical structure, so we used the NMN administration group as a positive control for the present study and evaluated its effect on senescent HUVECs. We showed that NMN retards H 2 O 2 -induced HUVECs senescence through downregulating the p16 INK4A and SASP, promoting cell viability and ATP production. However, NMN has a weak effect on NAD + in this investigation compared to those of other studies [43,44]. The antioxidant and protecting mitochondrial function of NTs is superior to NMN in the present study.
Overall, the present study has supported the free radical theory of aging, confirming that aggravated oxidative stress triggers HUVECs senescence. These findings, while preliminary, provide the first evidence for the role of NTs in retarding HUVECs senescence, and the mechanism may relate to its anti-inflammatory, anti-oxidant activities and the effect of improving mitochondrial functions. The findings of this investigation complement those of earlier studies; we comprehensively assessed the anti-inflammatory, anti-oxidant, and protecting mitochondria activities of four kinds of NTs and their mixtures. The comprehensive analysis of NTs undertaken here has extended our knowledge of the functional characteristics, the effective dose of different purine and pyrimidine NTs, as well as its optimum proportion. This new understanding should help expand the research fields of NTs widely.
There were several limitations in our study. First, to satisfy the overwhelming cell demand of the multiple test groups, we chose the cell line in our research. Disappointingly, the H 2 O 2 -induced premature senescent model cannot fully simulate the natural cellular senescence process. The study should be repeated using replicative senescent cells or primary cultured senescent endothelial cells. Second, because of the heavy workload and long literary piece, the present study only focuses on functional assessment; the fundamental mechanisms responsible for the retarding HUVECs senescence effect of NTs have not yet been fully elucidated. Further research should be undertaken to explore how NTs are absorbed by HUVECs and their molecular mechanisms of delaying cellular senescence. Considerably more work will need to be done to determine the role of NTs in CVDs treatment, including in vivo experiments and large randomized controlled trials. Notwithstanding these limitations, the study suggests that NTs can restore the functional changes during the HUVECs senescence process and provide a theoretical foundation for future research.

Conclusions
In conclusion, we have shown that NTs retard the oxidative stress-induced senescence of HUVECs, decrease the expression of senescence markers, promote cell viability, augment antioxidant activity, ameliorate SASP and mitochondrial dysfunction, and increase NAD + production during the HUVECs senescence process. GMP100 was most excellent in delaying HUVECs senescence, while the effect of the NTs mixture was full-scaled. These results stated that the mechanisms of the amelioration of HUVECs senescence actions of NTs could be related to their antioxidant and anti-inflammation properties as well as their ability to promote cell proliferation and protect mitochondrial function activities.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu13093279/s1, Figure S1: H 2 O 2 -induced senescence in HUVECs, Figure S2: Comprehensive analysis of NTs on retarding HUVECs senescence, Figure S3: Comprehensive analysis of NTs on inhibiting SASP in HUVECs, Figure S4: Comprehensive analysis of NTs on alleviating oxidative damage in HUVECs, Figure S5. Comprehensive analysis of NTs on mitochondrial function in HUVECs.
Author Contributions: Y.L., M.X. and N.Z. conceived and designed the experiments; N.Z. and X.L. performed the experiments; N.Z. analyzed the data; N.Z. wrote and revised the paper. All authors have read and agreed to the published version of the manuscript.