RRP Regulates Autophagy through the AMPK Pathway to Alleviate the Effect of Cell Senescence on Atherosclerosis

Autophagy is closely associated with atherosclerosis and other cardiovascular diseases (CVD). Compound Danshen prescription is widely used as a clinical antiatherosclerotic drug. In our previous studies, we have shown that the combined active component, ginsenoside Rg1-notoginsenoside R1-protocatechualdehyde (RRP), can effectively alleviate endothelial dysfunction and reduce atherosclerotic plaques. However, the association between cellular senescence, caused by reduced autophagy, and atherosclerosis remains unclear. In this study, we investigated whether RRP can enhance autophagy and alleviate cell senescence through the AMPK pathway. Our results showed that RRP reduced the secretion of inflammatory factors in the serum of atherosclerotic mice, enhanced autophagy, and alleviated aortic aging in mice, thus reducing atherosclerotic plaques. In human aortic endothelial cells (HAECs), RRP effectively enhanced autophagy and inhibited senescence by activating the AMPK pathway. When AMPKα was silenced, the effect of RRP was inhibited, thus reversing its antiaging effect. Overall, our results show that RRP regulates autophagy through the AMPK pathway, thereby inhibiting cell senescence and alleviating the progression of atherosclerosis, suggesting that RRP may be a potential candidate drug for the treatment of atherosclerosis.


Introduction
Atherosclerosis can be caused by various factors, which lead to changes in the tissue microenvironment and initiate the secretion of proinflammatory factors, thereby promoting the development of atherosclerotic plaques [1,2]. Autophagy is a major intracellular degradation system of dysfunctional organelles that is essential for cell renewal and homeostasis [3]. As autophagy is a mechanism that protects and kills stressed cells [4], defects in this system have been shown to promote endothelial cell senescence and apoptosis [5,6]. Notably, atherosclerosis and cell senescence are selfpromoting, leading to the development of many cardiovascular diseases (CVD) [7]. The progression of atherosclerosis can subsequently cause cell senescence, in which cells undergo unique phenotypic changes as a result of the action of a series of stimuli, causing cell cycle blockage. During senescence, cells secrete inflammatory cytokines, chemokines, growth factors, and proteases, which induce senescence-related secretory phenotypes (SASP) [8]. SASP can stimulate the immune system to clear senescent cells, but this process diminishes with aging, leading to the hypothesis that SASP affects nonsenescent neighboring cells through paracrine and autocrine mediators, thereby promoting atherosclerotic plaque progression and instability [7,9,10]. Previous studies have found that increased senescence of vascular endothelial cells leads to endothelial cell dysfunction, and senescent endothelial cells accumulate at sites of atherosclerosis. Consequently, the protection of endothelial cells from stress-induced premature senescence has become an area of focus in atherosclerosis therapy research.
AMPK functions as a receptor for cellular energy, affecting the metabolic pathways of protein, glycogen, and fatty acids. Additionally, it plays a protective role in the cardiovascular system by affecting the cell cycle and inhibiting cell proliferation in response to cardiovascular stimulation [11]. After AMPK activation, ATP supply is supplemented by autophagy and fatty acid oxidation [12]. AMPK is also a negative regulator of mTOR. AMPK phosphorylation inhibits mTOR expression, which induces autophagy in many different cell types [13]. Activating vascular wall cell autophagy can protect endothelial cells from damage and contribute to plaque stability [14], whereas inhibition of autophagy accelerates cell senescence, apoptosis, and necrosis, making plaques more susceptible to instability [15]. AMPK activation can reduce oxidative stress, promote autophagy, and play an anti-inflammatory role by delaying endothelial cell senescence and inhibiting the progression of atherosclerosis [16].
The Danshen prescription is mainly composed of ginseng, Panax notoginseng, and borneol. It can affect coronary artery contraction, inhibit platelet aggregation, and increase myocardial tolerance to hypoxia, making it an important antiatherosclerotic drug in traditional Chinese medicine [17]. In our previous study, we found that ginsenoside Rg1-notoginsenoside R1-protocatechualdehyde (RRP), the active component of Danshen, has antiatherosclerotic activity [17]. We have previously used RRP to antagonize endothelial cell injury and suggested that its protective function may be associated with the reduction of the inflammatory response caused by shear stress. Overall, the molecular mechanism through which RRP protects endothelial cells from senescence remains unclear. Based on previous studies, we speculated that the protective effect of RRP on endothelial cells may be mediated by the AMPK signaling pathway. Our results suggest that RRP can act through the AMPK signaling pathway to reduce oxidative stress, promote autophagy, and delay cell senescence. Therefore, the effects of this medicine have potential for application as a new atherosclerosis prevention and treatment strategy.

Animal Experiments.
Eight-week-old male ApoE -/and C57BL/6 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China; animal certificate number SCXK (Jing)2021-0011). All animal experiments were conducted in accordance with the guidelines for the Care and Use of Experimental Animals (published by the National Institutes of Health) and were approved by the Advisory Committee on Animal Care and Research of Shandong University of Traditional Chinese Medicine. During the experiment, all mice were fed water and food freely at a temperature of 22 ± 2°C in a specific pathogen-free (SPF) laboratory under a 12 h/12 h light/dark cycle. After a week of adaptation, all mice were fed a highfat diet. After 12 weeks of continuous high-fat diet, the ApoE -/mice were randomly divided into three groups: model group (receiving 0.9% sodium chloride; I.P.), Ros group (receiving 10 mg/kg -1 rosuvastatin; I.G.), and RRP group (receiving 10 mg/kg Rg1+10 mg/kg R1+14 mg/kg PCAD kg -1 ; I.P.). Fifteen C57BL/6J mice were used as the control group (receiving sodium chloride; I.P.). All animals were treated once daily for 8 weeks.
2.2. Histopathological Analysis. Aortic samples were stained with Oil Red O to evaluate the overall load and distribution of atherosclerosis. Excess tissue was peeled off and the entire aorta was opened longitudinally. Samples were fixed overnight with 4% PFA solution and then rinsed for 3 min with PBS before staining with 0.5% Oil Red O working solution for 10 min at 37°C. The entire aorta was then immersed in 70% ethanol and then rinsed with PBS. The samples were imaged using a stereo-fluorescence microscope (M205FA, LEICA). The degree of aortic atherosclerosis was evaluated based on the ratio of lesion area to aortic area.
Subsequently, the aortic sinus was fixed with a 4% PFA solution for 24 h and then treated with paraffin. The aortic sinus was cut into 5 μm thick continuous paraffin sections. The aortic lipid plaque and elastin plaque areas were measured based on hematoxylin and eosin (H&E) and Movat staining (at room temperature). A pathological section scanner (HS6, SUNNY) was used to capture images.
2.3. Immunohistochemistry. Immunohistochemistry was used to detect the levels of γ-H2AX (1 : 250; cat. no. ab81299, Abcam) in aortic tissue. Briefly, the thoracic aorta was fixed overnight with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections. Subsequently, the slices were incubated in an oven at 65°C for 2 h, dewaxed in xylene twice for 10 min, and dehydrated in 99%, 95%, and 75% ethanol for 5 min, at each concentration. The antigen was extracted using citric acid at boiling temperature for 20 min, hydrogen peroxide was then added, and the samples were incubated in a dark box at room temperature for 10 min to reduce endogenous peroxidase levels. The sections were then sealed with 5% fetal serum for 1 h, shaken, and incubated overnight with a specific primary antibody at 4°C. In the next day, the slices were warmed at room temperature for 30 min and then incubated with secondary antibodies at room temperature for 30 min before imaging using a fluorescence microscope (Vert.A1, ZEISS). The HAECs were mixed with 5 μM of MitoSOX working solution and incubated for 10 min in the dark at 37°C. The cells were then washed gently three times with complete medium before observation of red fluorescence using a fluorescent microscope. A JC-1 mitochondrial membrane potential detection kit (cat. no. C2006, Beyotime) was used to assess mitochondrial membrane potential. The cells were treated with JC-1 staining solution for 20 min in the dark and then washed twice with cold staining buffer. The stained cells were then observed under a fluorescence microscope (Vert.A1, ZEISS). Green fluorescence reflects the monomeric form of JC-1, and red fluorescence reflects the aggregated form of JC-1.

Enzyme-Linked
2.12. Analysis of Mitochondrial Respiration. Mitochondrial function was analyzed using an extracellular flux analyzer (Seahorse XFe96, Agilent). The HAECs were inoculated into ECM at a density of 8000 cells per well. After 12 h, the cells were treated with RRP for 2 h, supplemented with H 2 O 2 , and inoculated for another 8 h. The ECM was then replaced with the medium containing 25 mM glucose, and the cells were incubated in a carbon dioxide-free incubator at 37°C for 1 h. Oligomycin, FCCP, and antimycin A+rotenone were preloaded onto reagent ports A, B, and C, respectively, and oxygen consumption was calculated based on the slope of the change in concentration and time. Maximum respiration was calculated by subtracting the oxygen consumption rate when inhibited by rotenone+antimycin A from the oxygen consumption rate when inhibited by FCCP. ATP production was calculated by subtracting the oligomycin inhibitory oxygen consumption rate from the baseline oxygen consumption rate.
2.13. Transmission Electron Microscopy. After prefixing with 3% glutaraldehyde, the tissue was postfixed with 1% osmium tetroxide, dehydrated in a series of acetone solutions, infiltrated with Epox 812 for a longer period of time, and embedded. Ultrathin sections were cut using diamond knives and stained with uranyl acetate and lead citrate. The sections were examined using the JEM-1400-FLASH T.
2.14. Western Blot. For western blotting, the HAECs were cultured in 6-well plates. After the specified treatment, RIPA buffer, protease inhibitor, and phosphatase inhibitor were used to cleave HAECs or the mouse aorta. Protein concentrations were determined using the BCA method. Subsequently, the same amount of total protein was transferred to a PVDF membrane (90 min at 100 V) using an 8% PAGE Gel Super Fast Preparation Kit (cat. no. MA0381, Meilunbio). After transfer, the membrane was incubated at room temperature for 2 h in 5% skimmed milk dissolved at room temperature, and finally, the membrane was incubated with an antibody at 4°C overnight. The following primary antibodies were used: p16 (1 :  no. 43566, CST), and β-actin (1 : 1000, cat. no. 20536-1-AP, Proteintech). All membranes were washed with TBST three times and then incubated at room temperature for 1 h with secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG) dissolved in TBST. Finally, the membranes were washed three times with TBST, and an ECL test kit was used for detection (cat. no. WBKLS0100; Millipore). Images were analyzed using ImageJ software and normalized using an internal control.

Total RNA Isolation and Quantitative
Real-Time (qRT-PCR). Total RNA was extracted from HAECs using the Simple P Total RNA Extraction Kit (BioFlux). Total RNA was quantified using NanoDrop One (Semel Fisher Science, USA), and 1 μg of the RNA was reverse-transcribed into cDNA using the SPARKscript II RT Plus Kit (cat. no. AG0304, SparkJade). The qRT-PCR was performed using 2x SYBR Green qPCR Mix (cat. no. AH0104, SparkJade) and LightCycler 480 II (Roche). The sequences of the primers are listed in Table 1. 2.16. siRNA Transfection. HAECs were transfected with 100 nM siAMPKα according to the manufacturer's specifications. Briefly, the siRNA and transfection reagents were added to the siRNA transfection medium and incubated at room temperature for 30 min. The cells were then incubated with the siRNA transfection reagent for 6 h. The transfection mixture was subsequently removed, and cells were added to the complete medium and incubated for 48 h before the follow-up experiments. The expression of AMPKα was measured to evaluate the transfection efficiency.
2.17. Statistical Analysis. All results are expressed as mean ± standard deviation (SD). Student's t-test and one-way ANOVA were used to compare significant differences between more than two groups. Data are means of three biological repeats. All statistical analyses were carried out using GraphPad Prism 8.0 software. Differences were considered statistically significant at p < 0:05.

RRP Attenuated the Vascular Senescence Induced by
Atherosclerosis. Atherosclerosis is an aging-related disease, and studies have shown that alleviating the effects of aging can effectively delay the progression of atherosclerosis [7,18]. In this study, we evaluated the inhibitory effect of RRP on the progression of atherosclerosis in mice. We found that the Oil Red O-positive area in the model group was significantly larger than that in the control group, while RRP treatment significantly reduced the size of atherosclerotic plaques in the aorta (Figure 1(a)). Next we analyzed atherosclerotic plaques in the aortic root tissue sections and evaluated the severity of atherosclerosis based on H&E and Movat staining of the paraffin-embedded sections of the aorta. The aorta of ApoE -/mice showed severe atherosclerotic lesions with thicker plaques in the inner wall, outer wall, and intima, and a large number of cholesterol crystals were revealed by Movat staining (Figure 1(b)); however, these changes were greatly inhibited in ApoE -/mice administered with RRP.
Compared to untreated ApoE -/mice, the number of blue-stained areas in aortic SA β-gal staining was significantly reduced in the RRP group ( Figure 1(c)). Immunohistochemical detection of γ-H2AX in the mouse aorta revealed that RRP decreased the levels of H2AX in ApoE -/mice ( Figure 1(d)). We then detected senescence-related secretory phenotypes in the serum of these mice. Compared with the control group, the serum levels of TNF-α, IL-1 β, IL-6, IL-1 α, MMP-3, TGF-β1, and MCP-1 were significantly elevated in the model group (Figure 1(e)), whereas the overall level of SASP in the mice treated with RRP decreased, suggesting that RRP reduced the secretion of inflammatory factors and delay aging. We also found that RRP can reduce the levels of NF-κB, which is an upstream activator of SASP, further demonstrating that RRP can reduce SASP expression (Figure 1(f)). Next we measured the expression levels of the aging markers p53, p21, p16, and γ-H2AX in the mouse aorta (Figures 1(f) and 1(g)). Compared with the model group, the expression of aging markers in the RRP group was lower than that in the model group. Taken together, these results show that RRP can delay vascular senescence in ApoE -/mice.

RRP Can Regulate the AMPK Pathway to Promote
Autophagy in Atherosclerotic Mice. Cell senescence is closely related to autophagy, which can reduce oxidative stress injury, eliminate damaged organelles, and protect cells [19,20]. Next, we examined whether RRP could improve vascular aging in mice by regulating autophagy. Western blotting and qRT-PCR analysis revealed higher levels of AMPKα in mice treated with RRP than that in the model group and decreased levels of m-TOR, suggesting that RRP might inhibit m-TOR through the AMPK pathway, thereby promoting autophagy (Figures 2(a) and 2(b)). We also found that RRP increased the levels of beclin-1 and LC3B-II/I and decreased the levels of p62 protein, suggesting autophagy-associated damage (Figure 2(c)). These results demonstrated that RRP can mitigate aortic senescence in mice by enhancing autophagy through the regulation of the AMPK pathway. 4 Oxidative Medicine and Cellular Longevity

RRP Can Reduce HAEC Senescence Induced by H 2 O 2 .
Senescent endothelial cells are considered potential pathological factors in the development of vascular diseases [21,22]. Therefore, to further study the effect of RRP on cell senescence, 800 μM H 2 O 2 was used to interfere with HAEC to induce oxidative stress damage and promote senescence (Figure 3(a)). First, we measured the level of oxidative stress damage after H 2 O 2 intervention and used a DCFH-DA fluorescence probe to detect reactive oxygen species (ROS). We found that H 2 O 2 treatment increased intracellular ROS levels ( Figure 3(b)). The expression of the antioxidant nitric oxide synthase (eNOS) protein was decreased, and this effect was alleviated in the RRP group (Figure 3(c)). Thus, RRP intervention reduced the H 2 O 2 -induced oxidative stress in HAECs. Next, we used SA-β-gal staining to detect the degree of cell senescence (Figure 3( Figure 4(b)). These results confirmed that RRP effectively alleviate the mitochondrial dysfunction in HAECs caused by H 2 O 2 . We also used a mitochondrial pressure detection kit to further investigate the protective effects of RRP on mitochondrial function and found that H 2 O 2 significantly decreased the oxygen consumption rate (OCR) of HAECs, which was allevi-ated to varying degrees in the RRP and Met groups. Compared with the untreated cells, H 2 O 2 treatment resulted in significantly reduced basal respiration and maximum respiration rates, whereas it was prevented by RRP pretreatment (Figure 4(c)). Moreover, the RRP pretreatment also restored the level of ATP, which was reduced following H 2 O 2 treatment. These results suggest that RRP treatment protected the HAECs from oxidative stress-induced mitochondrial dysfunction.  -Actin     Damage to mitochondrial function greatly affects the normal operation of autophagy, with reduced autophagic flux in senescent cells [24,25]. Restoring autophagy partially alleviated cell senescence. Therefore, we further studied the effect of RRP on autophagy in HAECs induced by H 2 O 2 . After inducing cell senescence, we found that the numbers of HEAC autophagosomes associated with RRP pretreatment increased (Figure 4(d)). When autophagosomes are formed, the conversion of the cytoplasmic protein LC3B-I into LC3B-II and LC3B-II by enzymatic hydrolysis represents the initiation of autophagy. The expression of the autophagy marker LC3B was observed following adenovirus transduction of mRFP-GFPLC3 (Figure 4(e)). In H 2 O 2treated HAECs exhibiting senescence, autophagy marker LC3B and mitochondria were rarely colocalized (yellow dot), and this effect was alleviated in the RRP group. Consistent with this result, western blotting results showed that LC3B-II increased after RRP treatment compared with the H 2 O 2 group, indicating that RRP increased the autophagy flux of HAECs. In addition, compared with the H 2 O 2 group, the levels of the cell-associated autophagy protein beclin-1 increased and the levels of the autophagy substrate P62 pro-tein decreased in the RRP group (Figure 4(f)). Together, these results suggest that RRP can mitigate mitochondrial damage and regulate autophagic flux, thereby reducing HEACs senescence.

RRP Attenuates H 2 O 2 -Stimulated HAEC Senescence by
Activating AMPK Pathway. AMPK is a late endosome/lysosomal resident protein that can be activated on the lysosomal membrane [14,26], and AMPK pathway regulates autophagy and reduces cell senescence. To further clarify the effects of RRP on autophagy and senescence, we examined the expression of the AMPK pathway in HAECs [27]. As shown in Figure 5(a), the levels of AMPKα and p-AMPKα decreased to varying degrees in response to H 2 O 2 treatment, and the downstream targets mTOR and p-mTOR were inversely activated. After treatment of senescent HAECs with RRP, the levels of AMPKα and p-AMPKα increased, and the levels of mTOR and p-mTOR decreased (Figures 5(a) and  5(b)). These results confirm that RRP affects the AMPK signaling pathway and regulates autophagy.

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siAMPKα Reversed the Effect of RRP on H 2 O 2 -Induced
Senescence in HAECs. AMPK is one of the most important proteins regulating cell energy metabolism, and deletion of AMPK leads to altered autophagy [21,28]. To better understand the role of RRP in the AMPK pathway, we studied the effect of siAMPKα on RRP. For this, we transfected HAECs with siAMPKα to silence AMPKα (Figure 6(a)). After siAMPKα treatment, RRP did not effectively reduce the levels of ROS (Figure 6(b)), and the levels of eNOS protein did not change (Figure 6(c)), indicating that when AMPKα was silenced, RRP could not effectively reduce oxidative stress damage in HAECs. We further examined the effect of RRP on HAEC autophagy following AMPKα silencing. Our results showed that cotreatment with RRP and siAMPKα did not effectively change the levels of mtROS ( Figure 6(d)), MMP ( Figure 6(e)), autophagosomes ( Figure 6(f)), OCR values ( Figure 6(g)), and autophagy marker LC3B in HAECs ( Figure 6(h)). Additionally, RRP did not restore the levels of the autophagy-related proteins beclin-1 and LC3B-II, with no significant changes in the levels of m-TOR, p-m-TOR, and p62 proteins (Figures 6(c) and 6(i)). These results suggest that silencing AMPKα inhibits the effect of RRP on autophagy in HAECs. We also measured the levels of γ-H2AX and NF-κB after silencing AMPKα and found that the ameliorative effect of RRP on DNA damage was inhibited (Figures 7(a) and 7(b)). At the same time, the results of SAβ-Gal assays showed that although RRP reduced the number of blue-stained cells (Figure 7(c)), this effect was weaker than that in the nonsilent AMPKα group. The levels of senescence-related proteins P21 and P16 also did not decrease significantly (Figures 7(b) and 7(d)). Finally, AMPKα knockout inhibited cell senescence regulation by RRP. Overall, the results of this study showed that the protective effect of RRP on HAEC senescence induced by autophagy-mediated H 2 O 2 was partly dependent on the AMPK signaling pathway.

Discussion
Aging is a major risk factor for many diseases including atherosclerosis [7,29,30]. Senescent endothelial cells have been found in atherosclerotic plaques in elderly patients, and endothelial cell senescence is considered a potential pathophysiological factor in the development of cardiovascular disease [21,31,32]. This study is the first to demonstrate that RRP can attenuate H 2 O 2 -induced cellular senescence in HAECs by inducing autophagy via the activation of the AMPK signaling pathway. Autophagy is a self-protective mechanism that plays an important role in the aging process. Damaged organelles and proteins are engulfed by autophagosomes and eventually degraded, allowing cells to maintain normal functions [33,34]. Several studies have confirmed that in aged mice, the ECS expresses lower levels of autophagy proteins than in young mice [35]. Based on a recent hypothesis, the alleviation of senescence by autophagy may be associated with SASP regulation, as the accumulation of senescent endothelial cells can release SASP inflammatory cytokines, thereby promoting the development of vascular diseases [36]. Since activation of autophagy has been shown to reduce inflammation [37], SASP in senescent ECs was inhibited by the modulation of autophagy. This is consistent with our findings that RRP treatment significantly reduced the expression of inflammatory factors and alleviated aortic vascular senescence in ApoE -/mice. Although further studies are needed to determine how defective autophagy promotes ECS senescence, p62 is associated with mTORC1 or NF-κB. The  interaction between p62 and NF-κB can partially explain SASP transcription, defective autophagy, and senescent growth arrest [38]. It is known that p62 induces SASP production through TRAF6 polyubiquitination, which enables NF-κB activation [39]. Furthermore, increased p62 levels in autophagy-inhibited cells inhibited proteasomal degradation, leading to accumulation of protein aggregates. Finally, p62 is part of the mTORC1 complex and is required for           Figure 6: Continued. 16 Oxidative Medicine and Cellular Longevity

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Oxidative Medicine and Cellular Longevity mediating amino acid sensing, which activates the mTOR pathway [40]. Therefore, p62 accumulation may lead to overactivation of these pathways and promote cellular senescence. In this study, we used H 2 O 2 to induce oxidative stress in HAECs, thereby damaging the cells by increasing intracellular ROS production, which subsequently led to impaired autophagy and accelerated cellular senescence. We evaluated the effect of RRP treatment on the levels of ROS, mtROS, MMP, and OCR and found that RRP restored impaired mitochondrial function and thus helped to preserve autophagy. Consistent with this result, the autophagy-related markers LC3B-II and beclin-1 were significantly upregulated, the autophagic substrate p62 was downregulated, and there was an increase in autophagic vesicles compared to the model group, together suggesting that RRP enhanced cellular autophagy, thereby delaying senescence. AMPK plays an important role in cellular and organismal energy metabolism as an energy sensor and regulator

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Oxidative Medicine and Cellular Longevity [15,16]. AMPK is sensitive to changes in ATP depletion, with growing evidence showing that AMPK regulates lipid metabolism, inflammation, and angiogenesis in various animal models and cell types [41]. It cooperates with downstream molecules, including mTOR, a major regulatory pathway regulating autophagy in mammalian cells. Another study showed that decorin (DCN), a small leucine-rich proteoglycan, activates autophagy by activating AMPK and simultaneously inhibiting mTOR [42]. Pharmacological inhibition of mTOR by AMPK activators, such as rapamycin, significantly prolonged the lifespan of nematodes, Drosophila, and mice and showed AMPK-dependent inhibition in nematodes and mice [43][44][45], with significant results for SASP inhibition [46]. It is now generally accepted that the activation of AMPK attenuates cellular oxidative stress and inhibits m-TOR, thereby protecting cellular autophagy levels and exerting anti-inflammatory effects to delay cellular senescence [14,44].
The regulation of the AMPK pathway by RRP has not been previously reported. We hypothesized that RRP would be able to activate the AMPK pathway and observed that early intervention of HAECs by RRP increased the levels of AMPK and p-AMPK and decreased the levels of downstream mTOR and p-m-TOR. Subsequently, we examined the levels of senescence markers SA-β-gal, p53, p21, p16, and γ-H2AX and found that RRP attenuated senescence in HAECs. Activation of autophagy by RRP was inhibited when AMPKα was silenced, whereas the antiaging effect of RRP was reversed. RRP attenuated ApoE -/mouse aortic SA-β-gal staining, reduced the secretion of senescencerelated markers and SASP, and restored the levels of autophagy-related proteins. We further demonstrated that RRP regulates autophagy by activating the AMPK pathway ( Figure 8).
In conclusion, our study using in vitro and in vivo models of atherosclerosis-induced cellular senescence demonstrated that RRP treatment produces a significant antiaging effect by inducing autophagy through the AMPK signaling pathway. This study provides new insights into the potential of RRP treatment and suggests that RRP may be a promising drug candidate for the prevention of endothelial cell senescence and atherosclerosis. However, this study had some limitations. First, aging is related to functions such as tissue repair, and therefore, careful evaluation of the dynamics of aging is required [7,47]. Additionally, the association between autophagy and cellular senescence can vary owing to differences in the drugs, response time, cellular activity, and other therapeutic factors [7,48].

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Oxidative Medicine and Cellular Longevity Consequently, further studies are required to explore the detailed mechanisms underlying RRP-regulated autophagy.
In conclusion, the results of this study indicate that the protective effect of RRP may be associated with autophagy, suggesting that RRP may have potential for application in the treatment of atherosclerosis.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
All authors disclosed no relevant relationships.

Authors' Contributions
DL designed and performed the experiments, analyzed the data, and wrote the manuscript. SY performed ERG, analyzed the data, and wrote the manuscript. ST analyzed the data and reviewed the manuscript. LL, LZ, QY, XN, and JY performed the experiments and conceived and wrote the manuscript. DZ and WY designed the research project, arranged the data, and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. Dekun Liu and Yueyue Song contributed to the work equally and should be regarded as co-first authors.