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.