Figure legends
Figure 1. Arsenic trioxide
(As2O3, ATO) attenuates atherosclerotic
plaque progression and destabilization in ApoE-/-mice. (A)The development of a plaque in the aorta of
ApoE-/- mice treated with
ATO(As2O3) or vehicle.
(B) Representative images of Oil Red O staining of en
face preparations of aortas from the different treatment groups
(injected with As2O3 at
2.5 mg·kg−1/day or Nacl for 4 weeks). (C)
Representative images of hematoxylin and eosin staining of lesion areas
of aortic sinus for
ApoE-/- mice
treated with vehicle or ATO(As2O3).
Bar=100 µm. (D) Quantitation of Oil
Red O+ areas of the entire aorta. Values are mean ±
SEM from 9 mice. (E) Quantitation of lesion areas of the two groups.
Values are mean ± SEM from 9 mice. (F) Quantitation of necrotic core
areas relative to total plague area. Values are mean ± SEM from 9 mice.
(G) Cryosections of the aortic sinus stained with Oil Red O; hematoxylin
was used as a counterstain. Bar=100 µm. (H) Representative histological
findings in masson’s trichrome stained of the aortic root in treated
with vehicle or ATO(As2O3). Collagen
fibers are indicated in blue. Bar=100 µm. (I) Quantitation of necrotic
core areas. Values are mean ± SEM from 9 mice. (J) Quantitation of Oil
Red O+ areas relative to total plague area. Values are
mean ± SEM from 9 mice in each group. (K) Quantification of collagen
content using a masson’s trichrome staining. (L) Representative
photomicrographs of immunofluorescent staining for
macrophage(CD68+) in aortic sinus sections. Bar = 100
μm. (M) Percentage of CD68+-stained area relative to
total lesion area, quantified with ImageJ software. Values are mean ±
SEM from 5 mice in each group. The data are presented as the mean ± SEM.
*p < 0.05, **p < 0.01 vs control
group.
Figure 2. Arsenic trioxide
(As2O3, ATO) regulate serum lipid
metabolism in ApoE-/-mice and decrease CD36-mediated
ox-LDL up-take in macrophages. (A-E) The effects of
As2O3treatment on plasma cholesterol levels. The concentration of total
cholesterol(A), triglycerides(B), HDL cholesterol(C), LDL
cholesterol(D), and ox-LDL(E), values are mean ± SEM from 6 mice in each
group. (F) The mRNA levels of CD36 in the aortic root of
ApoE-/- mice
treated with vehicle or As2O3 were
determined via RT‐PCR analysis. (G) Representative images of CD68
macrophage (green), CD36 staining (red) and CD36 on macrophages
(CD68/CD36 merge) in the aortic root. Bar=200 µm. (H) Quantification of
CD36 on CD68 positive macrophages in the aortic root. n = 5 mice in each
group. (I, L) The protein levels of CD36 in the aortic root of
ApoE-/- mice treated with vehicle or
As2O3 were revealed by western blot
analysis. GAPDH was used as an internal control. (J, K) The protein
levels of CD36 in the RAW264.7 cell were revealed by western blot
analysis after being treated with ox-LDL (50 μg ml-1)
in the presence or absence of As2O3(2.5μM, 5.0 μM) for 24 h. GAPDH was used as an internal control. (M-N)
RAW264.7 cell were treated with ox-LDL (50 μg ml-1) in
the presence or absence of As2O3(2.5μM,5.0 μM) for 24h. Area of the
average size of lipid deposits in RAW264.7 cell. (O) CD36 mRNA
expression in RAW264.7 cells was analyzed by RT-qPCR after being treated
with ox-LDL (50 μg ml-1) in the presence or absence of
As2O3 (2.5μM,5.0 μM) for 24 h. The data
are presented as the mean ± SEM. *p < 0.05,
**p < 0.01 vs control
group. #p <0.05, ##p <0.01 vs oxLDL-stimulated
group.
Figure 3. Arsenic trioxide
(As2O3, ATO) inhibits inflammatory
factor expression and macrophage inflammatory polarization in
ApoE-/- mice. (A) IL-6, (B)TNFα, and (C) IL-10 mRNA
levels in serum of ApoE-/- mice treated with vehicle
or As2O3 were measured by ELISA assays.
(D) IL-6, (E)TNFα, and (F) IL-10 levels in the aortic root of
ApoE-/- mice treated with vehicle or
As2O3 were determined via RT‐PCR
analysis. (G-I) RAW264.7 macrophages were induced by addition of ox-LDL
(50 μg ml-1) without or with
As2O3 (2.5μM,5.0 μM) for 24h. (G) IL-6,
(H)TNFα, and (I) IL-10 mRNA levels in cell lysate were determined via
RT‐PCR analysis. n = 5 mice in each group. The data are presented as the
mean ± SEM. *p < 0.05,
**p < 0.01 vs control
group. #p <0.05, ##p <0.01 vs ox-LDL-stimulated
group.
Figure 4. Arsenic trioxide
(As2O3, ATO) inhibits
activation of TLR4/NF-κB inflammatory pathway in macrophages. (A) The
protein level of TLR4 in the aortic root of
ApoE-/- mice treated with vehicle or
As2O3 were revealed by western blot
analysis. GAPDH was used as an internal control. (B) RAW264.7 cell was
induced by addition of ox-LDL (50 μg ml-1) without or
with As2O3 (2.5μM,5.0 μM) for 24h. The
protein level of TLR4 in cell lysate was revealed by western blot
analysis. GAPDH was used as an internal control. (C) RAW264.7 cell was
pretreated with or without BAY-11(5mM, 30min) or
As2O3 (2.5μM,5.0 μM,2-6 h) and then the
cells were incubated with LPS(200 ng ml-1) for 30
min. Nuclear extracts were used to analyze p65 translocation by Western
blot. PCNA was used as an internal control to nuclear fractions. (D)
Quantitative analysis of TLR4 protein levels in the aortic root. GAPDH
was used as an internal control. (E) Quantitative analysis of TLR4
protein levels in RAW264.7 cell lysate. GAPDH was used as an internal
control. (F) Quantitative analysis of p65 protein levels in nuclear
fractions of RAW264.7 cell.PCNA was used as an internal control. (G)
RAW264.7 cell was pretreated with or without BAY-11(5mM, 30min) or
As2O3 (2.5,5.0 μM, 2-6 h) and then the
cells were incubated with LPS (200 ng ml-1) for 30
min. Cytoplasmic extracts were used to analyze p65 translocation by
Western blot. GAPDH and was used as an internal control to cytoplasmic
fractions. (I) Quantitative analysis of p65 protein levels in
cytoplasmic fractions of RAW264.7 cell. GAPDH was used as an internal
control. (H-J) RAW264.7 cell was pretreated with or without As2O3
(2.5,5.0 μM, 2-6 h) and then the cells were incubated with LPS (200 ng
ml-1) for 30 min. Expression of
IκBα in RAW264.7 cells was
determined by western blot (H) followed by quantitative analysis (J).
(K) Schematic diagram of ATO acting on TLR4/ NF-κB signaling pathway.
n = 5 mice in each group. The data are presented as the mean ± SEM.
*p < 0.05, **p < 0.01 vs control
group. #p <0.05, ##p <0.01 vs LPS or
ox-LDL-stimulated group.
Figure 5. Arsenic trioxide
(As2O3, ATO) reduces the
characteristics of pyroptosis in the aorta of ApoE-/-mice and inhibits macrophage pyroptosis. (A-B) The protein levels of
NLRP3, C-IL-1β and C-caspase-1 in the aortic root of
ApoE-/- mice treated with vehicle or
As2O3 were revealed by western blot
analysis. GAPDH was used as an internal control. (C)THP-1 cell was
pretreated with PMA(100μM,12 h) and then the cells were incubated by
addition of ox-LDL (50 μg ml-1) without or with
As2O3 (2.5,5.0 μM) for 24h. The protein
levels of NLRP3, C-IL-1β and C-caspase-1 in the THP-1 cells were
revealed by western blot analysis.(D-E) Quantitative analysis of
NLRP3(D), C-IL-1β and C-caspase-1(E) protein levels in the aortic root.
GAPDH was used as an internal control. (F) The mRNA levels of NLRP3 and
IL-1β in the aortic root of ApoE-/- mice treated with
vehicle or As2O3 were determined via
RT‐PCR analysis. (G-I) Quantitative analysis of NLRP3(G), C-IL-1β(H)and
C-caspase-1(I) protein levels in THP-1 cell lysate. GAPDH was used as an
internal control. (J)The mRNA levels of NLRP3 in Cell lysate were
determined via RT‐PCR analysis. (K-L) Representative images of CD68
macrophage (green),IL-1β staining (red) and IL-1β on macrophages
(IL-1β/CD36 merge) in the aortic root(K). Bar=200 µm. Quantification of
IL-1β on CD68 positive macrophages in the aortic root(L). (M) The mRNA
levels of IL-1β in Cell lysate were determined via RT‐PCR analysis. (N)
THP-1 cell was pretreated with PMA(100μM,12 h) and then the cells were
incubated by addition of ox-LDL (50 μg ml-1) without
or with As2O3 (2.5,5.0 μM) for 24h.
IL-1β levels in culture supernatants were quantified by ELISA. (O)
Schematic diagram of ATO acting on NLRP3 signaling pathway. n = 5 mice
in each group. The data are
presented as the mean ± SEM. *p < 0.05,
**p < 0.01 vs control
group. #p <0.05, ##p <0.01 vs ox-LDL-stimulated
group.