STAT3/NF‑κB decoy oligodeoxynucleotides inhibit atherosclerosis through regulation of the STAT/NF‑κB signaling pathway in a mouse model of atherosclerosis

Abstract

Atherosclerosis is a progressive chronic inflammatory condition that is the cause of most cardiovascular and cerebrovascular diseases. The transcription factor nuclear factor‑κB (NF‑κB) regulates a number of genes involved in the inflammatory responses of cells that are critical to atherogenesis, and signal transducer and activator of transcription (STAT)3 is a key transcription factor in immunity and inflammation. Decoy oligodeoxynucleotides (ODNs) bind to sequence‑specific transcription factors and limit gene expression by interfering with transcription in vitro and in vivo. The present study aimed to investigate the beneficial functions of STAT3/NF‑κB decoy ODNs in liposaccharide (LPS)‑induced atherosclerosis in mice. Atherosclerotic injuries of mice were induced via intraperitoneal injection of LPS and the mice were fed an atherogenic diet. Ring‑type STAT3/NF‑κB decoy ODNs were designed and administered via an injection into the tail vein of the mice. To investigate the effect of STAT3/NF‑κB decoy ODNs, electrophoretic mobility shift assay, western blot analysis, histological analysis with hematoxylin and eosin staining, Verhoeff‑Van Gieson and Masson's trichrome staining were performed. The results revealed that STAT3/NF‑κB decoy ODNs were able to suppress the development of atherosclerosis by attenuating morphological changes and inflammation in atherosclerotic mice aortae, and by reducing pro‑inflammatory cytokine secretion through inhibition of the STAT3/NF‑κB pathway. In conclusion, the present study provided novel insights into the antiatherogenic molecular mechanism of STAT3/NF‑κB decoy ODNs, which may serve as an additional therapeutic intervention to combat atherosclerosis.

Keywords: atherosclerosis; decoy oligodeoxynucleotides; inflammation; nuclear factor‑κB; signal transducer and activator of transcription 3.

Figure 1

STAT/NF-κB decoy ODNs suppress histological changes and inflammation in atherosclerotic mice aortae. (A) Structure of STAT/NF-κB synthetic decoy ODNs. (B) Electrophoretic mobility shift assay was performed to analyze the effects of STAT/NF-κB synthetic decoy ODNs on STAT and NF-κB-binding activity in atherosclerotic mice (n=5). (C) Histopathological alterations were determined by hematoxylin and eosin staining; representative images from each group are shown (n=5). Scale bar, 50 µm. (D) Western blotting was performed to detect the expression levels of inflammatory cytokines in aorta tissues. The graph summarizes the semi-quantification of protein expression normalized to GAPDH (n=3). ^*P<0.05 vs. NC group; ^†P<0.05 vs. STAT/NF-κB group; ^‡P<0.05 vs. LPS + AD + Scr group. AD, atherogenic diet; IFN-γ, interferon-γ; IL, interleukin; NC, normal control; NF-κB, nuclear factor-κB; ODN, oligode-oxynucleotide; LPS, lipopolysaccharide; Scr, scramble; STAT, signal transducer and activator of transcription.

Figure 2

STAT/NF-κB decoy ODNs diminish structural damage in the aortae of atherosclerotic mice. Histopathological alterations in slides stained with (A) Verhoeff-Van Gieson. Representative images from each group are shown (n=5). Scale bar, 50 µm. (B) Western blotting was performed to detect the expression levels of collagen in aorta tissues. The graph summarizes the semi-quantification of molecules of protein expression normalized to GAPDH (n=3). ^*P<0.05 vs. NC group; ^†P<0.05 vs. STAT/NF-κB group; ^‡P<0.05 vs. LPS + AD + Scr group. (C) Histopathological alterations in slides stained with Masson's trichrome. Representative images from each group are shown (n=5). Scale bar, 50 µm. AD, atherogenic diet; NC, normal control; NF-κB, nuclear factor-κB; ODN, oligodeoxynucleotide; LPS, lipopolysaccharide; Scr, scramble; STAT, signal transducer and activator of transcription.

Figure 3

STAT/NF-κB decoy ODNs attenuate morphological change in atherosclerotic mouse hearts and improve cholesterol metabolism. (A) Histological images of hematoxylin and eosin staining of atherogenic mouse hearts. Representative images from each group are shown (n=5). Scale bar, 50 µm. (B) Western blotting was performed to detect the expression levels of ABCA1 expression in aorta tissues. The graph summarizes the semi-quantification of molecules of protein expression normalized to GAPDH (n=3). ^*P<0.05 vs. NC group; ^†P<0.05 vs. STAT/NF-κB group; ^‡P<0.05 vs. LPS + AD + Scr group. ABCA1, ATP-binding cassette transporter A1; AD, atherogenic diet; NC, normal control; NF-κB, nuclear factor-κB; ODN, oligodeoxynucleotide; LPS, lipopolysaccharide; Scr, scramble; STAT, signal transducer and activator of transcription.

Figure 4

STAT/NF-κB decoy ODNs alleviate atherosclerotic mice aortae injury. Immunohistochemistry images of (A) ICAM-1. Representative images from each group are shown (n=5). Arrows indicate areas of respective adhesion molecule expression. Scale bar, 50 µm. (B) Western blotting was performed to detect the protein expression levels of adhesion molecules in aorta tissues. The graph summarizes the semi-quantification of molecules of protein expression normalized to GAPDH (n=3). ^*P<0.05 vs. NC group; ^†P<0.05 vs. STAT/NF-κB group; ^‡P<0.05 vs. LPS + AD + Scr group. (C) Immunohistochemistry images of VCAM-1. Representative images from each group are shown (n=5). Arrows indicate areas of respective adhesion molecule expression. Scale bar, 50 µm. AD, atherogenic diet; ICAM-1, intercellular adhesion molecule-1; NC, normal control; NF-κB, nuclear factor-κB; ODN, oligodeoxynucleotide; LPS, lipopolysaccharide; Scr, scramble; STAT, signal transducer and activator of transcription; VCAM-1, vascular cell adhesion molecule-1.

Figure 5

STAT/NF-κB decoy ODNs inhibit the expression levels of fibrosis-related proteins and the STAT/NF-κB pathway in atherosclerotic mice. (A) Western blotting was performed to detect the protein expression levels of fibronectin, α-SMA and MCP-1. The graph summarizes the semi-quantification of molecules of protein expression normalized to GAPDH (n=3). ^*P<0.05 vs. NC group; ^†P<0.05 vs. STAT/NF-κB group; ^‡P<0.05 vs. LPS + AD + Scr group. (B) Immunohistochemistry images of the macrophage marker MOMA-2. Representative images from each group are shown (n=5). Arrows indicate areas of MOMA-2-positive cells. Scale bar, 50 µm. (C) Western blotting was performed to detect the protein expression levels of proteins in the STAT and NF-κB pathways (n=3). α-SMA, α-smooth muscle actin; AD, atherogenic diet; MCP-1, monocyte chemoattractant protein-1; NC, normal control; NF-κB, nuclear factor-κB; ODN, oligodeoxynucleotide; LPS, lipopolysaccharide; P-, phosphorylated; Scr, scramble; STAT, signal transducer and activator of transcription; TLR4, Toll-like receptor 4.