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Resveratrol protects against atherosclerosis by downregulating the PI3K/AKT/mTOR signaling pathway in atherosclerosis model mice

Atherosclerosis is a cardiovascular disease, which is characterized by the interaction between carbohydrates, lipids, cells and various other molecules and genetic factors. Previous studies have demonstrated that resveratrol (RV) served protective roles in numerous types of human disease by regulating different signaling pathways. The aim of the present study was to investigate the therapeutic effects of RV and analyze the potential RV-mediated mechanism in umbilical vein endothelial cells (UVECS) in atherosclerosis model mice. Reverse transcription-quantitative PCR, western blotting and immunohistochemistry were used to analyze the therapeutic effects of RV both in vitro and in vivo. The results demonstrated that total cholesterol, triglycerides, low-density lipoprotein cholesterin and high-density lipoprotein cholesterin levels were significantly decreased in the RV group compared with the control group. RV demonstrated significant anti-atherosclerotic activity, which was determined through the atherogenic index, 3-hydroxy-3-methyl-glutaryl-Coa (HMG-CoA) reductase activity and marker enzymes, such as lactate dehydrogenase, creatine phosphokinase, aspartate transaminase, alanine transaminase and alkaline phosphatase. It was also observed that RV treatment significantly decreased the area of the arteriosclerotic lesion in the RV group compared with the control, as well as significantly decreasing the expression levels of matrix metalloproteinase-9 and CD40 ligand (CD40L) in arterial lesion tissue compared with the control group. Serum expression levels of tumor necrosis factor-α and C-reactive protein were also significantly decreased by RV treatment compared with the control group. Furthermore, RV treatment significantly decreased the expression levels of PI3K, AKT and mTOR in UVECS in vitro. In conclusion, these results suggested that the anti-atherosclerotic activity of RV may be due to its modulatory activity over the PI3K/AKT/mTOR signaling pathway. These findings suggested a potential novel treatment option for patients with atherosclerosis.


Introduction
Cardiovascular disease is the number one cause of death (~7.8%) worldwide, thus posing a significant threat to human health (1)(2)(3). Atherosclerosis is a cardiovascular disease that involves the biochemical narrowing of the diameter of blood vessels through the development of fatty streaks and plaques (4); and is characterized by the interaction between carbohydrates, lipids and genetic factors (5-7). Usai MV et al indicated that atherosclerosis is closely associated with the metabolic disorder of blood lipids (8); however, the mechanisms of atherosclerosis remain largely unknown. It is important to understand the mechanisms behind high fat diet-induced atherosclerosis to identify potential drugs that could relieve atherosclerosis.
Resveratrol (RV) is a multifunctional biological polyphenol that serves therapeutic roles in multiple types of human cardiovascular disease (9). A previous study demonstrated that RV exhibited neuroprotective effects in ischemic injury in rats through improving brain energy metabolism and alleviating oxidative stress (10). A study revealed that RV presented anti-oxidative and anti-inflammatory effects in the prevention or inhibition of age-related cardiovascular disease (11). In one study, the protective cardiovascular effects of RV were directly linked to improved heart contraction, which could be a potential target for the development of new clinical therapies for patients (12). Furthermore, a dietary and clinical perspective study observed that RV demonstrated cardioprotective benefits in the primary and secondary prevention of cardiovascular disease (13), with another study reporting that RV may be a potential agent for the prevention and treatment of cardiovascular disease (14); however, the potential RV-mediated mechanism remains largely understood.
Lipoprotein oxidation and oxidative processes in general serve important roles in the pathogenesis of atherosclerosis.
Disorders of lipid metabolism manifest as elevations in the levels of plasma lipids and lipoprotein fractions, which in turn results in the development of cardiovascular disease (15). The Atherogenic Index of Plasma is a marker of cardiovascular disease (16). Several studies have demonstrated that total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterin (LDL-c) and high-density lipoprotein cholesterin (HDL-c) levels are responses to increased thickness of vessel walls in patients with atherosclerosis (17,18). A previous study indicated that RV may reduce serum cholesterol levels through downregulation of HMG-CoAreductase (HMGR) mRNA expression levels in hamsters fed a high-fat diet (HFD) (19). Total panax notoginsenosides are the main ingredients found in the root of Panax notoginseng (Burk) F.H. Chen, which belongs to the Araliaceae family. They are useful in preventing the development of atherosclerosis in apolipoprotein E-knockout mice by downregulating CD40 and matrix metalloproteinase-9 (MMP-9) expression (20). By contrast, it was demonstrated that increased expression levels of tumor necrosis factor (TNF)-α and C-reactive protein (CRP) promoted the development of early atherosclerosis by increasing the transcytosis of LDL across endothelial cells (21)(22)(23). In addition, activation of the mitochondrial-related AMP kinase/PI3K/AKT/endothelial nitric oxide synthase signaling pathway was observed to improve endothelial function and alleviate atherosclerosis (24). mTOR inhibitors have also been demonstrated to prevent lipid storage, increase LDL-c levels and activate lipolysis, which further lead to a decreased risk of developing atherosclerosis (25); however, the association between RV and the PI3K/AKT/mTOR signaling pathway have not been well investigated in umbilical vein endothelial cells (UVECS) in atherosclerosis.
Thus, the aim of the present study was to investigate the effects of RV treatment in atherosclerosis murine model. The regulatory effects of RV treatment on the expression of inflammatory markers, the AIP, 3-hydroxy-3-methyl-glutaryl-Coa (HMG-CoA) reductase activity and marker enzymes were analyzed. This study also indicated that RV treatment may mediate the PI3K/AKT/mTOR signaling pathway in UVECS through the administration of a PI3K inhibitor prior to RV treatment.

Materials and methods
Animal studies. The present study was carried out in strict accordance with the guidelines set by the Committee of the People's Hospital of Weifang. All animal experiments were approved by the Ethics Committee of the People's Hospital of Weifang (approval no. TPH20170812S08). A total of 36 Apolipoprotein E-deficient (ApoE -/-) mice (age, 6 weeks; sex, male; body weight, 18-23 g) were purchased from the Laboratory Animal Science Center at Peking University People's Hospital (Beijing, China). The mice were housed in a 12-h light/dark cycle at 24-26˚C and 60±10% humidity. All animals had access to food and water ad libitum.
To establish an atherosclerosis model, all ApoE -/mice were fed a HFD, consisting of 21% fat from lard and 1.25% (wt/wt) cholesterol for 8 weeks. Subsequently, the ApoE -/mice were randomly divided into 2 groups (n=12/group), a PBS and RV group and received an intraperitoneal injection of PBS or RV (50 mg/kg/day), respectively, for 5 weeks. At the end of experiment, all the mice were sacrificed following anesthesia with sodium pentobarbital (35 mg/kg) and cervical dislocation.
Immunohistochemistry (IHC) staining. Following euthanasia, the heart, aortic trunk and right apex of the left ventricular myocardium were dissected from the mice. The tissues were rinsed with pre-cooled 0.9% saline, fixed in 4% paraformaldehyde for 2 h at room temperature and embedded in paraffin. Paraffin-embedded tissue samples were cut into 5-µm serial sections. The tissue sections were subsequently deparaffinized in xylene at room temperature and rehydrated in a descending ethanol series. Sections were then blocked with 3% hydrogen peroxide for 10 min at 25˚C to inhibit endogenous peroxidase activity. Tissue sections were incubated with the following primary antibodies for 12 h at 4˚C: Anti-MMP-9 (1:4,000; cat. no. ab38898; Abcam) and anti-CD40L (1:4,000; cat. no. ab2391; Abcam). Following the primary antibody incubation, the sections were incubated with an Alexa Fluor 488-conjugated secondary antibody (1:2,000; ab150077; Abcam) at 25˚C for 2 h. The slides were observed under an Olympus IX73 microscope (magnification, x100; Olympus Corporation). The quantification of expression levels was performed using Quantity One version 3.2 software (Bio-Rad Laboratories, Inc.).
Oil Red O staining. To quantify the area of the atherosclerotic lesions in the aortic sinus, Oil Red O staining was used. Slices (5 µm) were fixed with formalin for 10 min, rinsed with 60% isopropanol for 5 min at room temperature and stained with freshly prepared Oil Red O (1.0%) at room temperature. Subsequently, slides were stained with alum hematoxylin at room temperature. The average area of the atherosclerotic lesions in the aortic sinus were quantified by determining the percentage of positively-stained Oil-Red O areas using Image-Pro Plus software Version 1.1 (Media Cybernetics, Inc.).
LDH and CPK activity measurement. Serum (200 µl) from mice was obtained as previously described above. LDH and CPK activity was estimated using a fully automated XL-640 clinical chemistry analyzer (Transasia Bio-Medicals).
Cell transfection. DNA sequences of PI3K were amplified and cloned into the pcDNA3.1 plasmid expression vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate PI3K overexpressing (PI3KOR) plasmids. Empty plasmids were used as the control. In brief, 1x10 6 UVECS were cultured in 6-well plates for 24 h at 37˚C and subsequently transfected with 100 nM PI3KOR or the empty plasmid using a Lipofectamine ® 2000 transfection kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cells were collected for subsequent experimentation following 72 h of transfection.
Statistical analysis. All experiments were repeated at least three times. Statistical analysis was performed using SPSS version 13.0 software (SPSS Inc.). Statistical differences among groups were determined using Student's t-test or one-way ANOVA followed by Tukey's post hoc analysis. All data are presented as the mean ± SEM. P<0.05 was considered to indicate a statistically significant difference.

Pathological characteristics of ApoE -/atherosclerosis model mice.
To verify the role of RV treatment in ApoE -/mice, the characteristics of the arteriosclerotic lesions were investigated. Fibrous caps of atherosclerotic lesions were observed in the aortic sinus of ApoE -/mice fed the high fat diet (Fig. 1A); however, no significant lesion was observed in the aortic sinus intima of ApoE -/mice without high fat diet. RV treatment significantly decreased the formation of atherosclerotic plaques in atherosclerosis model mice compared to the control group (Fig. 1B). These results indicated that RV may improve the pathological characteristics of atherosclerosis in ApoE -/mice.

RV treatment increases body weight and improves blood lipid levels in atherosclerosis model mice.
Changes in the body weight of atherosclerosis model mice were analyzed in the RV and control treatment group. The body weight of mice was significantly decreased from 2 weeks to the end of experiment in the RV group compared with the control group ( Fig. 2A). Furthermore, RV treatment significantly reduced the TC, TG, LDL-c and HDL-c levels compared with the PBS group in atherosclerosis model mice following 5 weeks of treatment (Fig. 2B-E).

RV treatment decreases serum levels of inflammatory cytokines in atherosclerosis model mice.
The anti-inflammatory effects of RV were investigated in atherosclerosis model mice. RV treatment significantly decreased the serum levels of TNF-α and CRP compared with the control group ( Fig. 3A and B). In addition, RV treatment significantly decreased MMP-9 and CD40L expression levels in arterial lesion tissue compared with the control group (Fig. 3C).
RV treatment demonstrates anti-atherosclerotic activity in atherosclerosis model mice. RV treatment significantly decreased HMG-CoA reductase activity compared with the control group (Fig. 4A). LDH and CPK activities were also significantly decreased by 5 weeks post-treatment in the RV group compared with the control group ( Fig. 4B and C). In addition, serum levels of ALT, AST and ALP were significantly decreased in the RV group compared to the control group (Fig. 4D-F). These results indicated that RV treatment may exhibit anti-atherosclerotic activity in atherosclerosis model mice.

RV decreases PI3K/AKT/mTOR signaling in UVECs.
The potential RV-mediated mechanism was investigated in UVECs isolated from atherosclerosis model mice. RV treatment significantly decreased the mRNA and protein expression levels of PI3K, AKT and mTOR compared with the control group ( Fig. 5A and B). UVECs treated with the PI3KIR demonstrated significantly decreased mRNA and protein expression levels of AKT and mTOR compared with the control group (Fig. 5C and D). In addition, PI3K overexpression in UVECs using PI3KOR was observed to significantly increase the mRNA and protein expression levels of PI3K, AKT and mTOR compared with the control group ( Fig. 5E and F), whereas PI3KOR-transfected UVECs significantly reversed the RV-mediated decrease in mRNA and protein expression levels of AKT and mTOR (Fig. 5G and H).

Discussion
RV was previously demonstrated to reduce the expression levels of inflammatory cytokines in atherosclerosis model rabbits (28). In the present study, the therapeutic effects of RV on atherosclerosis model mice was analyzed and the relationship between RV and the PI3K/AKT/mTOR signaling pathway in UVECS obtained from atherosclerosis model mice was also investigated. The results demonstrated that RV treatment increased the expression levels of PI3K, AKT and mTOR, as well as the ratios between phosphorylated and total proteins in UVECS and increased the serum levels of  The study also observed that RV promoted anti-inflammatory effects, which may contribute to the anti-atherosclerotic activity of RV.
A previous study reported that RV treatment protected against TNF-α-induced injury in human UVECs through promoting sirtuin-1-induced repression of NF-kB and p38 MAPK (29). Verschuren et al (30) demonstrated that RV treatment decreased CRP expression levels, which improved lipid metabolism and reduced atherosclerotic lesion development in female transgenic mice. Results from the present study observed that RV treatment decreased TNF-α and CRP serum levels in atherosclerosis model mice. In addition, HMG-CoA reductase inhibitors reduced chronic subacute inflammation in atherosclerosis induced by dietary cholesterol (31). The present study found that RV treatment decreased the body weight of atherosclerotic mice by measuring pathological improvement of atherosclerosis. A previous study reported that the association between liver function and coronary atherosclerosis may be more complex than appreciated (32), and TG and LDL-c levels were found at higher levels in patients with atherosclerosis (33). In this study, RV treatment decreased the serum levels of TC, TG, LDL-c and HDL-c in atherosclerotic model mice compared to the control group. It was also reported that RV treatment decreased HMG-CoA reductase activity and marker enzymes, including LDH, CPK, AST, ALT and ALP in atherosclerosis model mice, which suggested that atherosclerosis may be associated with liver dysfunction.
RV has demonstrated numerous pharmacological effects, including acting as an antioxidant, an anti-inflammatory agent, eliminating free radicals, exhibiting an anti-tumorigenic role, regulating lipids and regulating glucose metabolism (34)(35)(36). Data from the current study observed that RV treatment improved lipid metabolism compared with the control group in atherosclerosis model mice. MMP-9 serum levels are consistently associated with markers of carotid atherosclerosis and lesion vulnerability (37); and the present study demonstrated that RV treatment could significantly decrease MMP-9 and CD40L expression levels in arterial lesion tissue compared to the control group. Notably, the in vivo experiments demonstrated the protective role of RV against atherosclerotic lesions in atherosclerosis model mice. Clinically, HDL-c is response to statin treatment by improving carotid intima-media thickness, which is closely related to a regression of atherosclerosis (38). Data have supported that serum levels of LDL-c can be used to predict the severity of coronary atherosclerosis (39). Consistently, our data found that RV treatment significantly reduced the degree of atherosclerosis. In addition, RV treatment increased the metabolism of hyperlipidemia in HFD-fed atherosclerosis model mice, which further led to an increase in anti-atherosclerotic activity and may prevent cardiovascular complications. Thus, it was hypothesized that decreasing HDL-c levels in the serum may have a negative effect of RV in the treatment of HFD-fed atherosclerosis; however, further studies should be performed to identify the anti-atherosclerotic mechanism of RV in oxidized (ox)-LDL-induced human endothelial cells.
The mTOR inhibitor, everolimus, has been proven to prevent the development of atherosclerosis in LDLR -/mice, even in the presence of severe hypercholesterolemia (40). A previous study also suggested that the increased activation of the PI3K/AKT signaling pathway attenuated ox-LDL-induced endothelial cell apoptosis (41); and similarly, another study demonstrated that targeting the PI3K/AKT/mTOR signaling pathway in vascular endothelial cells represented a potential therapeutic target for the treatment of atherosclerosis (42). Notably, the activation of the PI3K/AKT/mTOR signaling pathway exerted a protective role against atherosclerosis (43). In addition, previous studies have reported that inhibiting the PI3K/AKT/mTOR pathway alleviated ox-LDL-induced apoptosis of human endothelial cells, which further prevented atherosclerosis development (44)(45)(46). Results in the present study were consistent with the majority of these previous studies; RV treatment downregulated the PI3K/AKT/mTOR signaling pathway in UVECS obtained from atherosclerosis model mice. In addition, it was demonstrated that PI3K overexpression increased and abolished RV-regulated AKT and mTOR expression in UVECS; however, further investigations are required to determine the therapeutic efficacy of RV in patients with atherosclerosis. In addition, future studies should aim to analyze the dissociation constant/inhibition constant (KD/KI) between downstream molecules of RV. In conclusion, the results from the present study indicated that RV may improve atherosclerosis through the PI3K/AKT/mTOR signaling pathway. This study provided evidence for the application of RV in the treatment of atherosclerosis.