Formononetin attenuates atherosclerosis via regulating interaction between KLF4 and SRA in apoE -/- mice

Figure 1. Formononetin inhibits atherosclerosis and enhances plaque stability. ApoE-/- mice in 2 groups (15/group) received the following treatment for 16 weeks: Control, HFD; FNT, HFD containing formononetin (FNT) (10 mpk). (A) After treatment, aortas were isolated for determination of lesions in en face aortas by Oil Red O staining and quantified by a computer assisted image analysis protocol. Lesion areas were expressed as μm2, n=15. The following assays were performed in aortic root cross sections: (B) Haematoxylin and eosin staining followed by quantitative analysis of sinus lesions (upper), necrotic core area and fibrous cap area (bottom) in aortic root cross sections. nc: necrotic cores marked by black dotted line; fc: fibrous cap marked by blue dotted line; Lesion areas were expressed as μm2, n=15. (C-F) Representative photomicrographs (left) and quantification (right) of aortic root sections stained with Oil Red O (C), Verhoeff-Van Gieson (D), CD68 (E) and αSMA (F) in atherosclerotic plaque, n=15. (G) Alizarin Red S staining for calcification (indicated by black arrows) and quantification of calcification positive areas, n=15. (H) Evaluation of total calcium extracted from whole…

Figure 2. Formononetin inhibits lipid accumulation in HASMCs and PMs. (A) Evaluation of the dose-dependent lipid-reducing effects and cytotoxicity of formononetin in peritoneal macrophages (PMs) and HASMCs using Oil Red O (ORO) staining and MTT assay, n=5. (B) Images (Left) and quantitation (Right) of the extracted ORO dyes from the stained PMs and HASMCs that were treated with oxLDL (O), formononetin (FNT, F) and oxLDL together with FNT (O/F), n=5. (C) PMs isolated from mice used in Figure 1 were stained with ORO to assess formation of foam cells (>10 lipid droplets/cell, >10 fields/sample), n=5. (D, E) PMs isolated from untreated apoE-/- mice (D) and HASMCs (E) were treated with oxLDL or/and FNT, followed by ORO staining to assess formation of foam cells, n=5. (F) Evaluation of CD68 and αSMA expression after incubation with oxLDL or/and formononetin by immunofluorescence staining, n=5. (G, H) Cholesterol uptake of PMs and HASMCs were determined by Dil-oxLDL after treatment as indicated, n=5. Data are presented as mean ± SEM, *P<0.05, significantly different as indicated.

Figure 3. Function of SRA in formononetin-inhibited lipid overload in HASMCs and macrophages. (A, B) Expression of SRA, CD36, SRBI, LOX1, ABCA1, and ABCG1 in protein of PMs after formononetin or siSRA treatment by Western blot, followed by quantification, n=5. (C, D) After knockdown of SRA by siRNA, PMs and HASMCs were treated with oxLDL (100 μg mL-1) and followed by ORO staining (left) and corresponding quantitative analysis (right), n=5. (E, F) Cholesterol uptake of PMs and HASMCs were determined by Dil-oxLDL after treatment as indicated, n=5. (G, H) Aortic root cross sections from mice used in Figure 1 were conducted co-immunofluorescent staining with anti-SRA and CD68 or αSMA antibodies with quantitative analysis of CD68+SRA+ or αSMA+ SRA+ area, n=5. (I) CD68, SRA and αSMA in HASMCs were evaluated by Western blot after treatment as indicated, n=5. (J) Cholesterol efflux of PMs and HASMCs were assessed after treatment as indicated, n=5. Data are presented as mean ± SEM, *P<0.05, significantly different as indicated; ns: not significantly different. Ctrl or C: control; FNT or F: formononetin; O: oxLDL.

Figure 4. Formononetin inhibits monocyte adhesion to ECs and endothelium. (A) HUVECs in 24-well plates and THP-1 cells were pretreated with oxLDL (100 μg mL-1) for 2 h followed by addition of formononetin overnight. After incubation with oxLDL, THP-1 cells were labeled with CFSE, and then added to HUVECs and co-incubated for 1 h. The image of adherent THP-1 cells were captured with a microscope. The number of adherent THP-1 cells in control group (treated with oxLDL alone) was defined as 1. The fold changes were obtained by calculating the ratio of adherent cells in the corresponding group to that in the control group, n=5. (B) Aorta isolated from untreated apoE-/- mice and THP-1 cells were pretreated with oxLDL (100 μg mL-1) for 2 h followed by addition of formononetin overnight. CFSE-labeled THP-1 cells were then added to HUVECs and co-incubated for 1 h. The evaluation of adherent THP-1 cells was performed as described in (A), n=5. (C, D) After knockdown of SRA, THP-1 cells were labeled with CFSE and then incubated with oxLDL-pretreated HUVEC or aortic endothelium. The evaluation of adherent THP-1 cells was conducted as described in (A), n=5. (E) Expression of VCAM-1, ICAM-1 and PECAM-1…

Figure 5. Formononetin inhibits inflammation and promotes the macrophage M2 transition. (A, B) Aortic root cross sections from mice used in Figure 1 were performed co-immunofluorescent staining with anti-CD68 and iNOS or Arg1 antibodies with quantitative analysis of CD68+iNOS+ or CD68+ Arg1+ area, n=5. (C) Expression of Arg1 and iNOS mRNA was determined by q-RT-PCR, n=5. (D) expression of IL-1β, TNFα and IL-6 mRNA was determined by q-RT-PCR, n=5. (E) Serum IL-1β, TNFα and IL-6 was determined by Elisa, n=15. (F) After SRA knockdown in PMs, expression of IL-1β, TNFα and IL-6 mRNA was determined by q-RT-PCR, n=5. (G) After SRA knockdown in PMs, expression of Arg1 and iNOS was determined by immunofluorescent staining, n=5. Data are presented as mean ± SEM, *P<0.05, significantly different as indicated; ns: not significantly different.

Figure 6. Formononetin increases KLF4 expression and nuclear translocation in PMs and HASMCs. (A, B) After overexpression of KLF4 by infection with Ad-KLF4, PMs and HASMCs were exposed to oxLDL in presence or absence of formononetin. Expression of KLF4, SRA, CD36 and LOX-1 protein in PMs or KLF4, SRA in HASMCs was determined by Western blot (left) followed by quantification of band density correspondingly (right), n=5. (C, D) PMs and HASMCs were stained with Oil Red O after overexpression of KLF4, n=5. (E, F) PMs and HASMCs were treated with formononetin at the indicated concentrations overnight. And then expression of KLF4 protein in nuclear extracts was determined by Western blot, respectively, n=5. (G, H) Expression of KLF4 protein in PMs and HASMCs was determined by immunofluorescent staining, n=5. (I, J) Expression of KLF4 protein in macrophages and VSMCs within plaque was determined by co-immunofluorescent staining with KLF4 and CD68 or αSMA antibodies, n=5. Data are presented as mean ± SEM, *P<0.05; significantly different as indicated; ns: not significantly different.

Figure 7. KLF4 acts as a transcriptional suppressor of SRA. (A) 293T cells were transfected with pGL4.10 vector containing SRA promoter-linked Firefly gene (pSRA) together with pGL4.70 vector containing Renilla gene, and then treated with formononetin for 12 h. The transcriptional activity of SRA was measured by a dual luciferase assay system and normalized to that of Renilla, n=5. (B) 293T cells were infected with Ad-KLF4, and then transfected with pSRA together with pGL4.70 vector, followed by treatment with formononetin for 12 h. The transcriptional activity of SRA was measured by a dual luciferase assay system and normalized to that of Renilla, n=5. (C) 293T cells were transfected with pGL4.10 vector containing mutant SRA promoter-linked Firefly gene (pSRA-KLFREmut) together with pGL4.70 vector, and then treated with formononetin for 12 h. The transcriptional activity of SRA was measured as described in (B), n=5. (D) 293T cells were firstly infected with Ad-KLF4, and then transfected with pSRA-KLFREmut together with pGL4.70 vector, followed by treatment with formononetin for 12 h. The transcriptional activity of SRA was measured as described in (B), n=5. (E) After treatment of…