Atherosclerosis is attenuated by acacetin via Sirt1-mediated activation of AMPK/Sirt3 signals in diabetic ApoE-/- mice

The strategy of decreasing cardiovascular disorder is imperative to reduce premature death and improve quality of in patients with mellitus. The present study was designed to investigate whether the could improve


Background
Decreasing atherosclerotic cardiovascular disorder is imperative to reduce premature death and improve quality of life in patients with diabetes mellitus, since atherosclerotic cardiovascular disease is the leading cause of increased mortality and an important cause of morbidity in patients with Type 2 diabetes worldwide [1][2][3]. Sustained hyperglycemia (i.e. elevated blood glucose level) leads to macroand microvascular complications [4][5][6]. Experimental and clinical studies show that hyperglycemia accelerated the formation of atherosclerosis in diabetics [7][8][9][10][11]. Atherosclerosis results from diabetic macroangiopathy and characterized by endothelial injury and dysfunction followed by formation of new intra-plaque vessel due to excessive/abnormal neovasculogenesis and angiogenesis, increased vascular permeability of the capillary vessels and tissue edema followed by atherosclerotic plaque hemorrhage and plaque rupture [12].
Although the detailed mechanisms responsible for the accelerated formation of atherosclerosis lesion observed in diabetes are not fully understood [13][14][15],it is generally recognized that mitochondrial dysfunction and increased ROS production are involved in endothelial impairment and acceleration of atherosclerosis in diabetes [16][17][18]. The therapeutic strategies to treat diabetic atherosclerosis include better approaches to prevent, inhibit or reverse diabetic cardiovascular complications. Previous studies have demonstrated that endothelial protection is important against vascular disorders in diabetes mellitus [19][20][21]. Enhancing mitochondrial biogenesis and reducing mitochondrial ROS have emerged as crucial therapeutic approaches to ameliorate diabetic atherosclerosis injury [22].
We have previously found that the natural avone acacetin (5,7-dihydroxy-4'-methoxy avone), in addition to its atrial-selective anti-atrial brillation property [23,24], is cardioprotective against ischemia/reperfusion or hypoxia/reoxygenation injury by its anti-oxidation, anti-in ammation, and antiapoptosis properties [25,26]. The present study was designed to investigate whether acacetin is protective against vascular injury in diabetic ApoE −/− mice induced by streptozotocin (STZ) and in cultured human umbilical vein endothelial cells (HUVECs) exposed to high glucose conditions. Our results suggested that acacetin slowed the development of atherosclerosis in STZ-diabetic ApoE −/− mice and improved the injury of high glucose-cultured HUVECs by Sirt1-mediated activation of AMPK/Sirt3 signals.

Animal experiments
The animal experimental protocol was approved by the Animal Care and Ethics Committee of Xiamen University. Male ApoE -/mice were obtained from Beijing Vital River Laboratory Animal Technology week-old ApoE -/mice by intraperitoneal injection of STZ (daily 50 mg/kg, Sigma-Aldrich, MO, United States) or vehicle citric acid (control) for 5 consecutive days. Random blood glucose was determined 2 weeks after streptozotocin injection with the Accu-Chek Performa glucometer (Roche, United States), and only animals with blood glucose >16.7 mM were classi ed as diabetic. Experiments were assigned as control, control with acacetin treatment, STZ-diabetes, STZ-diabetes with treatment. Animals with acacetin treatment received acacetin prodrug subcutaneously at 20 mg/kg twice daily (the prodrug can be metabolized into acacetin) [24,25], and other animals received subcutaneous equivolume vehicle (0.9% saline). All animals were maintained at room temperature (23±2°C) with a 12 h light/dark cycle and free access to basic diet and water for additional 12 weeks. Bodyweight and blood glucose were measured every four-week. When the animals were sacri ced at end of experiments, the blood was collected in a centrifuged tube with 25 l of heparin. After centrifugation, plasma was collected to analyze the amount of cholesterol and triglycerides and high-density lipoprotein, low-density lipoprotein, lipoprotein A, lipoprotein B, β-OHB with In nity reagent (Thermo Fisher Scienti c, Waltham, MA, United States). Aortas were isolated for immunohistochemistry, immuno uorescence, and western blot analyses.
High-frequency ultrasound imaging.
Vascular lesions were measured non-invasively under anesthetization with a High-frequency Ultrasound Imaging System (Vevo 2100 system with a MicroScan MS550 40-MHz transducer, Visualsonics, Toronto, Canada) as described previously [27] and in Supplemental Methods.

Magnetic resonance imaging (MRI)
The MRI was performed under anesthetization with a 9.4T Bruker Micro MRI (Pharma Scan, Ettlingen, Germany) to determine diameter of carotid artery as described in Supplemental Methods.

Atherosclerotic assay
Aortic root lesion and enface lesion areas of whole aorta were xed with 4% paraformaldehyde and stained with Oil Red O as described previously [28]. Aortic root sections (10 μm thickness) of 4% paraformaldehyde-xed, OCT-embedded frozen hearts were cut from the aortic valve lea ets at 150-200 μm following the valve lea et. Sections were concurrently stained with 0.5% w/v Oil Red O and hematoxylin and eosin (HE) to assess atherosclerotic lesions as described previously [29]. The images were captured with an Olympus BX40 microscope (20x magni cation). All image quanti cations were analyzed as described previously [30].

Immunofluorescence analysis
Immuno uorescence analysis was used to identify Sirt3 expression levels on aortic root sections and cultured HUVECs with different treatment. Brie y, aortic root sections were stained with anti-Sirt3 and anti-CD31 antibody (Abcam, Cambridge, MA, United States). Following an overnight incubation with primary antibodies, aortic root sections or cells were washed three time with PBS, followed by a 1-h incubation with Alexa Fluor-conjugated secondary antibodies (Alexa Fluor® 488, A-11001 or A-11034; Alexa Fluor® 568, A-11004 or A-11011; Alexa Fluor® 633, A-21052 or A-21071) (Thermo Fisher Scienti c, Waltham, MA, United States) at room temperature, then mounted on DAPI-containing mounting media (Solarbio Technology, Beijing, China).

Flow cytometry analysis
Flow cytometry analysis was employed to assay the viability, apoptosis, ROS production and mitochondrial transmembrane potential in HUVECs using a ow cytometer (Beckman Coulter, United States) as described in Supplemental Methods.

Mitochondrial oxidative stress and functional evaluation
The mitochondrial oxidative stress proteins (i.e. SOD activity and MDA content) were measured using commercially available kits (Jiancheng Institute of Bioengineering, Nanjing, China) as described previously [31]. The intracellular ATP level was determined using ATP Bioluminescence Assay Kit (Beyotime Technology, Shanghai, China).

NAD + /NADH determination
The harvested cells were rinsed with PBS twice and centrifuged at 4 × 1000 rpm for 10 min, and NAD + and NADH levels were quanti ed using an EnzyChrom TM NAD + /NADH assay kit (Bioassay Systems, Hayward, CA, United States) following the manufacturer's instruction.
The siRNA technique was used to silence speci c genes in HUVECs. The cells with 40%-50% confluence were transfected with speci c siRNA duplexes (Santa Cruz Biotechnology, CA, United States) using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scienti c, Waltham, MA, United States) following the manufacturer's instruction. After 48 h transfection of control siRNA (sc-37007), Sirt1 siRNA (sc-40986), or Sirt3 siRNA (sc-61555) (Santa Cruz Biotechnology, Dallas, TX , United States), and then incubated with 5.5 mM or 33 mM glucose culture medium in the absence or presence of 3 mM acacetin for 5 days. The cells transfected with siRNA were collected for western blot analysis.

Western blot analysis
Western blot analysis was employed to determine the expression of speci c proteins in aortic tissues and cultured HUVECs. Proteins of aortic tissue homogenate lysates or HUVECs lysates prepared in SDS lysis buffer were extracted with RIPA buffer supplemented with protease and phosphatase inhibitors on ice, and protein concentration was determined using the BCA protein assay kit (Solarbio, Beijing, China) as described previously [25,26]. The proteins of HUVEC mitochondria were isolated using the Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scienti c, Waltham, MA, United States) following the manufacturer's instruction. SDS-PAGE and transferring PVDF membranes (Bio-Rad, Hercules, CA, United States) were applied to the separation of proteins samples. The membranes were blocked and incubated with primary antibodies (1:1000) overnight at 4°C overnight. After washout, membranes were incubated with secondary antibody (1:10000) for 1 h at room temperature. Blots were visualized with ECL TM reagents (Advansta, Menlo Park, CA, United States), and the protein signals were captured with FluorChem E chemiluminescence detection system (ProteinSimple, San Jose, CA, United States). All cellular western blots were repeated at least ve times, and the signal intensity of the immunoreactive bands was quanti ed using Image J software (NIH, Bethesda, MD, United States) and normalized to that of β-actin in each sample. The primary antibodies are followed: anti-pAMPKα (#2535) and anti-AMPKα (#2532) antibodies were from Cell Signaling (Danvers, MA, United States); anti-β-actin (sc-47778) was from Santa Cruz; anti-Sirt1 (ab32441) anti-Sirt3 (ab217319), anti-Bcl-2 (ab182858), anti-Bax (ab32503), anti-PGC-1α (ab54481), anti-SOD1 (ab16831), anti-SOD2 (ab16956) antibodies were from Abcam (Cambridge, United Kingdom).

Statistical Analysis
Statistical analyses were performed with GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, United States). Results are presented as means ± SEM. One-way ANOVA followed by Bonferroni post hoc test were used for comparison among groups. P values <0.05 were considered statistically significant.
Ultrasound was used for determining the wall thickness of aortic arch and blood ow velocity of left and right carotid arteries (Fig. S2) in ApoE −/− mice. The wall thickness of aortic arch and the artery distensibility measured by ultrasound biomicroscopy showed that the intimal thickness of aortic arch was increased and the carotid artery distensibility was decreased in STZ-diabetic mice, these effects were signi cantly countered in STZ-diabetic mice treated with acacetin ( Table 2).
An earlier study revealed that carotid blood ow velocity was elevated as the artery stenosis became marked [32]. Here we also found that the diastolic and systolic blood ow velocities of left and right carotid arteries were signi cantly increased due to the artery stenosis, while the radial strain and tangential strain of left and right carotid arteries were reduced in the STZ-diabetic ApoE −/− mice. These alterations were countered in diabetic ApoE −/− mice treated with acacetin ( Table 2). These results indicate that acacetin could improve the carotid artery stenosis thereby increasing the vascular radial strain and tangential strain and improving the movement ability of vascular walls.

Acacetin prevents the viability reduction and increases in apoptosis and oxidative stress induced by high glucose exposure in HUVECs
To investigate the potential molecular mechanism that vascular protection of acacetin against diabetic atherosclerosis, the related effects of acacetin were tested in HUVECs cultured with normal glucose concentration (5.5 mM) medium or a high glucose concentration (33 mM) medium. Acacetin (0.3-3 µM) had no effect on viability in HUVECs cultured with normal glucose medium (Fig. 3A), while it reversed high glucose-induced viability reduction in a concentration-dependent manner (Fig. 3B). Flow cytometry analysis revealed that the viability reduction was related to high-glucose-induced increase in apoptosis (Fig. 3C), and acacetin signi cantly decreased the apoptosis ( Fig. 3C and 3D). Results of western blot analysis showed that pro-apoptotic protein Bax was increased, while anti-apoptotic protein Bcl-2 was decreased in HUVECs cultured with 33 mM glucose medium. Acacetin treatment reversed the Bax increase and enhanced the Bcl-2 reduction, and increased the reduced Bcl-2/Bax ratio in a concentration dependent manner (Fig. 3E-3G). These results suggest that acacetin protects HUVECs against high glucose injury by inhibiting apoptosis.
It is generally believed that ROS overproduction is involved in endothelial apoptosis in diabetic cardiovascular complications. ROS production and the expression of antioxidant proteins SOD1 and SOD2 were therefore determined in HUVECs cultured with 33 mM glucose medium (Fig. 4). High glucose culture induced an increase of ROS production in HUVECs,acacetin (3 µM) signi cantly impeded the ROS production ( Fig. 4A & 4B). It reversed the increase in malondialdehyde (MDA) content (Fig. 4C) and reversed the reduction in SOD activity (Fig. 4D) in HUVECs cultured with 33 mM medium in a concentration-dependent manner. Moreover, high glucose-induced reductions of SOD1 and SOD2 proteins were also reversed in HUVECs treated with acacetin ( Fig. 4E & 4F),

Acacetin Reduced High Glucose-induced Mitochondrial Injury In Huvecs
To investigate the potential protection of acacetin against high glucose-induced endothelial mitochondrial injury, we determined mitochondrial transmembrane potential, ATP production and apoptosis-related proteins Bax and Bcl-2 in HUVECs (Fig. 5). The HUVECs cultured with 33 mM glucose medium showed signi cant decrease of mitochondrial transmembrane potential and ATP production, and the reduction of mitochondrial transmembrane potential and ATP production was countered in cells treated with 3 µM acacetin. Moreover, mitochondrial Bax (mitoBax) was increased, while mitochondrial Bcl-2 (mitoBcl-2) and Sirt3 (mitoSirt2) were reduced in HUVECs cultured with 33 mM glucose medium (P < 0.01 vs. 5.5 mM glucose). The decreased ratio of mitoBcl-2/mitoBax and mitoSirt3 were counted (P < 0.01 vs. 33 mM glucose alone) in cells treated with 3 µM acacetin (Fig. 5D-5G). Immunocytochemistry analysis also revealed the high glucose-induced decrease of mitoSirt3 was reversed in cells treated with 3 µM acacetin (Fig. 5H). These results indicate that vascular protection of acacetin against high glucose injury is resulted from preserving mitochondria function.

Silencing Sirt3 abolishes the protective effects of acacetin against high glucose-induced injury in HUVECs
To determine the potential role of Sirt3 in acacetin protection against high glucose-induced injury, siRNA molecules targeting Sirt3 were employed in HUVECs. Figure 6 illustrates the effects of acacetin on high glucose-induced apoptosis, ROS production, and ATP reduction in HUVECs cultured with 33 mM glucose medium and transfected with control siRNA or Sirt3 siRNA in the absence or presence of 3 µM acacetin. Acacetin signi cantly decreased high glucose-induced apoptosis, ROS production in cells transfected with control siRNA, but not in cells transfected with Sirt3 siRNA. Also, acacetin increased ATP content in cells transfected with control siRNA, but not in cells transfected with Sirt3 siRNA. These results indicate that Sirt3 plays an important role in vascular protection against high glucose-induced injury, and also mediates mitochondrial ATP production.
Western blot analysis revealed that acacetin not only reversed the high glucose-induced decrease of Sirt3 in a concentration-dependent manner, but also the high glucose-induced downregulation of Sirt1, PGC-1α, and pAMPK (Fig. 7). These results indicate that vascular protective effect of acacetin is related to upregulating Sirt1, Sirt3, pAMPK, and PGC-1α reduced by high glucose-culture in HUVECs or in STZdiabetic ApoE −/− mice.

Protection of acacetin against hyperglycemia-induced injury is related to Sirt1-mediated activation of AMPK/Sirt3 signals in endothelial cells
To further identify the molecule target of acacetin for the protection against high glucose-or hyperglycemia-induced vascular injury, siRNA molecules targeting to Sirt1 or Sirt3 and the AMPK inhibitor Compound C were utilized in HUVECs cultured with 33 mM glucose medium to determine the effects of the siRNA molecules or AMPK inhibitor on acacetin-induced upregulation of Sirt1, Sirt3, pAMPK and PGC-1α ( Fig. 8A & 8B). It is interesting to note that silencing Sirt1 abolished the acacetin-induced increase of Sirt1, Sirt3, pAMPK and PGC-1α, while silencing Sirt3 only inhibited the upregulation of Sirt3, but not the

Discussion
In the present study, we demonstrated that acacetin antagonized hyperglycemic atherosclerosis with association of an improved intimal thickness increase of aortic arch and carotid artery distensibility and a reduced stenosis of carotid arteries, and an attenuated vascular lesion progression without decreasing blood glucose in STZ-diabetic ApoE −/− mice. The vascular protection of acacetin against high glucose insult is related to ameliorating mitochondrial oxidative stress and mitochondrial dysfunction. Sirt1mediated activation of AMPK/Sirt3 signals are proved to play a key role in mediating endothelial protective and anti-atherosclerotic action of acacetin in STZ-diabetic ApoE −/− mice. Notably, this study is the rst to demonstrate the anti-atherosclerotic effect and potential mechanisms of acacetin against hyperglycemic injury, suggesting that acacetin may be a potential therapeutic drug candidate for reducing cardiovascular complications in diabetic patients.
Our previous studies have reported that acacetin is effective in treating atrial brillation by selectively blocking atrial potassium currents (I Kur , I KACh , and sK Ca ) [23,24,38,39], and is cardioprotective against ischemia/reperfusion injury via increasing AMPK/Nrf2 and anti-oxidation and inhibiting apoptosis and in ammation [25,26]. Other groups demonstrated that acacetin has anticancer [40,41], anti-peroxidation [42] and anti-neuronal in ammation [43,44] effects. In addition, acacetin may reduce E-selectin expression in endothelial cells by regulating MAP kinase [45]. The present study provides the novel information that acacetin provides signi cant vascular protection against atherosclerosis in STZ-diabetic ApoE −/− mice by Sirt1-mediated activation of AMPK, Sirt3 and PGC-1α signals.
acacetin-induced increase of Sirt1, pAMPK and PGC-1α. The AMPK inhibitor Compound C decreased the expression of pAMPK, PGC-1α, and Sirt3, and slightly reduced the Sirt3 increase by acacetin, whereas it fully abolished the increase of pAMPK and PGC-1α, but not Sirt1. These results indicate that protective effect of acacetin against hyperglycemia-induced vascular injury is related to Sirt1-mediated activation of AMPK, Sirt3 and PGC-1α signals in endothelial cells.
We therefore determined the potential effects of acacetin on the NAD + /NADH ratio and expression of Sirt1 and Sirt3 in the absence or presence of the NAMPT inhibitor GMX-1778 (CHS-828) [37]. The NAD + /NADH ratio was reduced in HUVECs cultured with 33 mM glucose medium, and the reduction was countered in cells treated with 3 µM acacetin, but not in cells co-treated with 10 nM GMX-1778 (Fig. 8C).
Moreover, GMX-1778 not only induced a further decrease of Sirt1 and Sirt3 proteins, but also prevented the acacetin induced increase of Sirt1 and Sirt3 proteins (Fig. 8D & 8E) in HUVECs cultured with 33 mM glucose medium. These results indicate that the vascular protection of acacetin against high glucose-or hyperglycemia-induced vascular injury is related to increasing NAMPT and NAD + followed by Sirt1mediated activation of AMPK/Sirt3 signals, thereby elevating cellular oxidation and decreasing apoptosis.
It is generally recognized that atherosclerosis is a multifactorial vascular progressive disorder involving alteration of several cellular and molecular events in diabetes [12,46]. These include the impairment of mitochondrial morphology and function associated with downregulation of SOD, pAMPK and PCG-1α, etc., leading to decrease of ATP product and increase of ROS production [12,46]. In the present study, diabetic neointima hyperplasia and atherosclerotic lesions were associated with downregulation of signaling molecules (i.e. SOD, Bcl-2, PGC-1α, pAMPK, Sirt3 and Sirt1) in artery tissues in STZ-diabetic ApoE −/− mice and also in HUVECs cultured with high glucose medium.
Sirt1 is widely studied from crossroads of nutrient (energy) sensing to various adaptive pathways. Sirt3 located mainly in mitochondria regulates metabolism and oxidative stress [48,49]. The present study demonstrated that mitochondrial Sirt3 was reduced with association of decrease of Sirt1, pAMPK and PGC-1α in artery tissues in STZ-diabetic ApoE −/− mice and also in high glucose-induced injury in cultured HUVECs. It appears that Sirt3 plays a crucial role in acacetin-mediated regulation of mitochondrial function including oxidation balance and ATP generation. Silencing Sirt3 abolishes the protective effect of acacetin against high glucose-induced ROS production and apoptosis, and ATP reduction in HUVECs, but does not affect acacetin-induced upregulation of Sirt1, pAMPK, and PGC-1α. This suggests that Sirt3 is not involved in regulation of Sirt1, pAMPK and PGC-1α.
However, silencing Sirt1 signi cantly decreased expression of Sirt3, pAMPK and PGC-1α and completely abolishes acacetin-induced upregulation of these molecules in HUVECs cultured with high glucose medium, whereas the AMPK inhibitor Compound C reduced expression of Sirt3, pAMPK and PGC-1α and fully inhibited acacetin-induced upregulation of pAMPK and PGC-1α (partially inhibited Sirt3 upregulation by acacetin). AMPK inhibition does not affect Sirt1 expression or acacetin-induced upregulation. These results indicate that protection of acacetin against vascular hyperglycemic injury is related to Sirt1mediated activation of Sirt3 and AMPK,followed by AMPK-dependent PGC-1α activation for regulating biosynthesis [50,51], thereby slowing down atherosclerotic progression via inhibiting oxidation and apoptosis in STZ-diabetic ApoE −/− mice.
The clinical drug metformin [22] and a number of natural bioactive compounds [52][53][54] have been reported to have anti-atherosclerotic effect, including curcumin, quercetin, puerarin, resveratrol, etc. by activating Sirt1 and/or AMPK. However, most of these promising natural compounds face a problem of druggability due to the poor solubility or bioavailability. The present study showed that acacetin, in addition to activating pAMPK as previously reported [26], stimulates Sirt1, Sirt3, and PGC-1α and therefore is a novel activator of Sirt1 with lower concentration range (0.3-3 µM) than other previously reported activators with similar activation mechanism, i.e. increasing NAD + /NADH ratio. Importantly, the solubility and bioavailability issues of acacetin have been solved by synthesis of the water-soluble prodrug, which can be used clinically in the future to not only treat atrial brillation, myocardial ischemia/reperfusion injury, but also diabetic atherosclerosis.
We observed that acacetin reversed the alterations of lipid pro les in STZ-diabetic ApoE −/− mice, including the decrease of the elevated triglyceride, total cholesterol, low-density lipoprotein, lipoprotein A, and lipoprotein B and the increase of reduced high-density lipoprotein; the related mechanisms of these phenomena were not explored in the present study, and is a study limitation which remains clari ed in the future. However, this limitation will not affect the conclusion of the present study.

Conclusions
This study demonstrates that novel information that acacetin treatment effectively lessens

Declarations
The authors declare no con ict of interest.

Supplementary Files
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