Acacetin ameliorated alterations of lipid profiles without reducing blood glucose in STZ-diabetic ApoE−/− mice
Lipid profiles changed significantly in STZ-diabetic ApoE−/− mice (Table 1). Triglyceride, total cholesterol, low-density lipoprotein, lipoprotein A, and lipoprotein B (in mM) were increased from 1.95 ± 0.13, 14.22 ± 0.82, 0.09 ± 0.03, 14.10 ± 1.40, and 0.075 ± 0.011 in control respectively to 3.99 ± 0.39, 21.72 ± 1.13, 6.39 ± 0.49, 70.33 ± 9.60, and 0.154 ± 0.013 (n = 8, P < 0.01 vs. control), while high-density lipoprotein was decreased from 0.73 ± 0.05 in control to 0.49 ± 0.03 in STZ-diabetic ApoE−/− mice. Acacetin treatment did not alter the basal lipid profiles in control animals (Table 1); however, it reversed increases in triglyceride, total cholesterol, low-density lipoprotein, lipoprotein A, and lipoprotein B to respectively 2.69 ± 0.2, 16.29 ± 0.95, 4.39 ± 0.53, 27.93 ± 4.59, and 0.078 ± 0.013 mM (n = 8–10, P < 0.01 vs. STZ-diabetes), and reversed the decrease in high-density lipoprotein to 0.66 ± 0.04 mM in STZ-diabetes treated with acacetin (n = 8–10, P < 0.05 vs. STZ-diabetes). However, the random blood glucose level was not significantly reduced in STZ-diabetes treated with acacetin (23.1 ± 2.4 mM vs. 20.4 ± 2.1 mM of STZ-diabetes, P = NS) and final bodyweight showed no differences between STZ-diabetic animals (27.87 ± 1.47 g) and STZ-diabetic animals treated with acacetin (25.91 ± 1.24 g) (Table 1). These results suggest that acacetin ameliorates the alterations in lipid profiles without reducing blood glucose level in STZ-diabetic ApoE−/− mice.
Table 1
Changes in bodyweight and lipid profiles in ApoE−/− mice
Group | Control (n = 8) | Acacetin (n = 8) | STZ (n = 8) | STZ + acacetin (n = 8) |
Initial body weight (g) | 25.13 ± 1.36 | 25.01 ± 1.47 | 24.44 ± 1.68 | 24.69 ± 1.81 |
Final body weight (g) | 31.22 ± 1.77 | 29.35 ± 1.82 | 25.91 ± 1.24 | 27.87 ± 1.47 |
Weight change (g) | 6.09 ± 1.56 | 4.34 ± 1.65 | 1.47 ± 1.46 | 3.18 ± 1.64 |
FBG (mmol·L− 1) | 6.2 ± 1.8 | 7.5 ± 1.6 | 18.4 ± 1.8** | 17.8 ± 1.7** |
RBG (mmol·L− 1) | 7.5 ± 1.4 | 9.1 ± 1.8 | 23.1 ± 2.4** | 20.4 ± 2.1** |
βOHB (mmol·L− 1) | 0.111 ± 0.012 | 0.100 ± 0.005 | 0.147 ± 0.014* | 0.106 ± 0.013# |
TG (mmol·L− 1) | 1.95 ± 0.13 | 2.18 ± 0.15 | 3.99 ± 0.39** | 2.69 ± 0.2## |
TC (mmol·L− 1) | 14.22 ± 0.82 | 14.98 ± 1.02 | 21.72 ± 1.13** | 16.29 ± 0.95# |
HDL (mmol·L− 1) | 0.73 ± 0.05 | 0.78 ± 0.08 | 0.49 ± 0.03** | 0.66 ± 0.04# |
LDL (mmol·L− 1) | 0.09 ± 0.03 | 0.11 ± 0.03 | 6.39 ± 0.49** | 4.39 ± 0.53**,## |
LP(A) (mmol·L− 1) | 14.10 ± 1.40 | 18.25 ± 2.10 | 70.33 ± 9.60** | 27.93 ± 4.60## |
LP(B) (mmol·L− 1) | 0.075 ± 0.011 | 0.076 ± 0.010 | 0.154 ± 0.013 | 0.078 ± 0.013 |
FBG, fasting blood glucose; RBG, random blood glucose;βOHB,β-hydroxybutyrate༛TG༌triglyceride; TC, total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein; LP (A), lipoprotein A; LP (B), lipoprotein B. *P < 0.05, **P < 0.01 vs. control; #P < 0.05, ##P < 0.01 vs. STZ. |
Acacetin attenuated carotid artery injury in STZ-diabetic ApoE −/− mice
The carotid artery injury was determined with MRI (Fig. S1) in STZ-diabetic ApoE−/− mice. T1WI, T2WI and walls of left and right common carotid arteries in non-diabetic ApoE−/− mice showed even and slightly high signal without obvious abnormal signal, while in STZ-diabetic ApoE−/− mice, there were uneven high signal areas on T1WI and T2WI, and artery lumen was clearly narrowed (vascular stenosis). These symptoms were significantly improved in STZ-diabetic ApoE−/− mice treated with acacetin (Table 2).
Table 2
Ultrasound and MRI parameters in ApoE−/− mice
Group | Control (n = 8) | Acacetin (n = 8) | STZ (n = 8) | STZ + acacetin (n = 8) |
Ultrasound parameters |
Intimal thickness (mm) | 0.0559 ± 0.0048 | 0.0582 ± 0.0047 | 0.0893 ± 0.0045** | 0.0610 ± 0.0054## |
LCCA EDV (mm/s) | 38.58 ± 3.26 | 39.02 ± 1.61 | 62.69 ± 5.33** | 50.35 ± 2.61# |
LCCA PSV (mm/s) | 278.22 ± 15.53 | 272.94 ± 11.57 | 408.67 ± 17.89** | 316.01 ± 19.67## |
LCCA resistance index | 0.7577 ± 0.0109 | 0.7613 ± 0.0091 | 0.8665 ± 0.0077** | 0.7965 ± 0.0014**,## |
Distensibility (1/MPa) | 146.87 ± 12.95 | 140.51 ± 11.34 | 55.99 ± 8.91** | 110.16 ± 11.10## |
Tangential Strain near (%) | 3.52 ± 0.25 | 3.31 ± 0.24 | 1.74 ± 0.17** | 2.85 ± 0.22## |
Tangential Strain far (%) | 0.93 ± 0.09 | 0.95 ± 0.04 | 0.26 ± 0.04** | 0.72 ± 0.08### |
Radial strain (%) | 43.56 ± 3.09 | 42.92 ± 4.37 | 15.19 ± 1.48** | 33.18 ± 2.92## |
LCCV EDV (mm/s) | 41.21 ± 2.92 | 40.97 ± 2.02 | 76.63 ± 4.26** | 48.10 ± 4.67## |
RCCA PSV (mm/s) | 262.87 ± 10.75 | 256.93 ± 11.38 | 403.38 ± 14.34** | 324.33 ± 16.42**,## |
RCCA resistance index | 0.7681 ± 0.0111 | 0.7749 ± 0.0077 | 0.8611 ± 0.0100** | 0.8084 ± 0.0143*,## |
Distensibility (1/MPa) | 136.60 ± 8.21 | 137.28 ± 9.29 | 71.64 ± 7.24** | 97.92 ± 9.22**,# |
Tangential Strain near (%) | 3.75 ± 0.31 | 3.66 ± 0.28 | 1.87 ± 0.15** | 2.63 ± 0.16**,# |
Tangential Strain far (%) | 0.92 ± 0.04 | 0.94 ± 0.05 | 0.39 ± 0.05** | 0.75 ± 0.05*,### |
Radial strain (%) | 46.52 ± 3.67 | 44.67 ± 3.07 | 20.42 ± 1.39** | 37.56 ± 3.97## |
MRI parameters |
Diameter of LCCA (cm) | 0.475 ± 0.013 | 0.468 ± 0.009 | 0.340 ± 0.019** | 0.418 ± 0.024## |
Diameter of RCCA (cm) | 0.480 ± 0.006 | 0.472 ± 0.011 | 0.358 ± 0.023** | 0.425 ± 0.012## |
LCCA EDV,left common carotid artery end-diastolic velocity; LCCA PSV༌left common carotid artery peak systolic velocity; RCCA EDV, right common carotid artery end-diastolic velocity; RCCA PSV, right common carotid artery peak systolic velocity. *P < 0.05, **P < 0.01 vs. control; #P < 0.05, ##P < 0.01 vs. STZ. |
Ultrasound was used for determining the wall thickness of aortic arch and blood flow 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 significantly countered in STZ-diabetic mice treated with acacetin (Table 2).
An earlier study revealed that carotid blood flow velocity was elevated as the artery stenosis became marked [32]. Here we also found that the diastolic and systolic blood flow velocities of left and right carotid arteries were significantly 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.
The Oil Red O staining of enface aorta showed that atherosclerotic lesion was greater in STZ-diabetic ApoE−/− mice than nondiabetic ApoE−/− mice (control), and the lesion was reduced in STZ-diabetic ApoE−/− mice treated with acacetin (Fig. 1A). The analysis of Oil Red O stained area (Fig. 1B) exhibited that acacetin treatment significantly decreased the aorta lesion area from 11.0 ± 1.12% (n = 11, P < 0.01) in non-treated STZ-diabetic ApoE−/− mice to 7.0 ± 0.6%.
The aortic root sections were stained with hematoxylin and eosin (Fig. 1C) or Oil Red O (Fig. 1E) to show the neointimal thickness and the lesion burden, in aortic roots of animals with different treatment. The increase in neointimal thickness (Fig. 1D) and lesion burden (Fig. 1F) were significantly reduced in STZ-diabetic ApoE−/− mice treated with acacetin (n = 8, P < 0.01 vs. STZ-diabetes). These results suggest that acacetin provides effective vascular protection against hyperglycemia-induced injury in STZ-diabetic ApoE−/− mice.
Acacetin reversed the downregulation of related protective molecule signals in aortic tissues of STZ-diabetic ApoE −/− mice
It is well documented that diabetic atherosclerosis is associated with downregulation of a series of signaling molecules involved in energy metabolism, anti-oxidation and anti-apoptosis, including Sirt1, AMPK, PGC-1α, SOD1, SOD2, Bcl-2, etc. [33, 34]. To investigate whether the vascular protection of acacetin is mediated by upregulating these signaling molecules, we determined the expression of Sirt1, pAMPK, PGC-1α, SOD1, SOD2, Bcl-2, and also Sirt3 in aortic tissues in non-diabetic mice, STZ-diabetic ApoE−/− mice, and STZ-diabetic ApoE−/− mice treated with acacetin (Fig. 2). As reported previously, the signaling molecules Sirt1, PGC-1α, SOD1, SOD2, and also Sirt3 were remarkably reduced (Fig. 2A & 2B), pAMPK was decreased (Fig. 2D), the anti-apoptotic protein Bcl-2 was downregulated, and the pro-apoptotic Bax was increased (Fig. 2A & 2C) in STZ-diabetic ApoE−/− mice (n = 8, P < 0.01 vs. control). The alterations in the expression of these molecules were reversed in STZ-diabetic ApoE−/− mice treated with acacetin (Fig. 2A-2D) (P < 0.01 vs. STZ-diabetes). These results suggest that the natural flavone acacetin confers vascular protection against diabetic atherosclerosis by reversing the downregulation of Sirt1, PGC-1α, SOD1, SOD2, Sirt3, pAMPK, and ratio of Bcl-2/Bax. Moreover, we first reported the reduced Sirt3 is involved in diabetic atherosclerosis. The immunohistochemical results showed that Sirt3 remarkably decreased on vascular wall, especially on endothelium stained with CD31, in STZ-diabetic ApoE−/− mice, which partially reversed in animals treated with acacetin (Fig. 2E).
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 significantly 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) significantly 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 significant 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 significantly 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 STZ-diabetic 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 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.
It is well recognized that sirtuins are nicotinamide adenine dinucleotide- (NAD+-) dependent deacetylases that controls metabolism, and biosynthesis of NAD+ is mediated by nicotinamide phosphoribosyltransferase (NAMPT) [35]. The sirtuins activity is correlated to ratio of NAD+/NADH [36]. 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 Sirt1-mediated activation of AMPK/Sirt3 signals, thereby elevating cellular oxidation and decreasing apoptosis.