Tanshinone II A stabilizes vulnerable plaques by suppressing RAGE signaling and NF-κB activation in apolipoprotein-E-deficient mice

Abstract

Tanshinone II A (TSIIA) is a diterpene quinone extracted from the roots of Salvia miltiorrhiza with anti-inflammatory and anti‑oxidant properties that is used to treat atherosclerosis. In the current study, morphological analyses were conducted to evaluate the effects of TSIIA on atherosclerotic vulnerable plaque stability. Additionally, receptor of advanced glycation end products (RAGE), adhesion molecule, and matrix‑metalloproteinases (MMPs) expression, and nuclear factor-κB (NF‑κB) activation were examined in apolipoprotein E (apoE)‑deficient mice treated with TSIIA. Eight‑week‑old apoE-/- mice were administered TSIIA and fed an atherogenic diet for 8 weeks. TSIIA exhibited no effects on plaque size. Analysis of the vulnerable plaque composition demonstrated decreased numbers of macrophages and smooth muscle cells, and increased collagen content in apoE‑deficient mice treated with TSIIA compared with untreated mice. Western blotting revealed that TSIIA downregulated the expression levels of vascular cellular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule‑1 (ICAM‑1), and MMP‑2, ‑3, and ‑9, suppressed RAGE, and inhibited NF‑κB, JNK and p38 activation. The present study demonstrated that the underlying mechanism of TSIIA stabilization of vulnerable plaques involves interfering with RAGE and NF‑κB activation, and downregulation of downstream inflammatory factors, including ICAM‑1, VCAM‑1, and MMP‑2, ‑3 and ‑9 in apoE-/- mice.

Figure 1.

TSIIA exhibits no effect on atherosclerotic lesion size within the brachiocephalic arteries in apolipoprotein E^−/− mice. (A) The percent of aortic lesion area (lesion area compared to total arch area) was measured by quantitative histomorphology of Oil Red O-stained en face specimens (n=8; P=0.3443). The lesion sizes of the descending aortic lesion areas and (B) BCA were analyzed in the TSIIA and model groups as demonstrated by Movat staining (n=8; P=0.3998). Data represent the mean ± standard error. Con, control; TSIIA, Tanshinone II A.

Figure 2.

TSIIA decreases the counts of macrophages, major harmful cellular sources in advanced lesions, within the BCA lesions. (A) Immunohistochemical staining for MAC-3 (CD107b) and α-actin, picrosirius staining and polarization microscopy were performed on BCA samples from the model and TSIIA group. The amounts of (B) macrophages, (C) SMC and (D) collagen in the lesions within the BCA were determined by immunohistochemical and picrosirius staining (n=6; P=0.0327, 0.0934 and 0.0111). Data represent the mean ± standard error. *P<0.05, **P<0.01 vs. model group. BCA, brachiocephalic artery; TSIIA, Tanshinone II A; SMC, smooth muscle cell.

Figure 3.

TSIIA suppresses NF-κB activation and downregulates inflammatory factors, adhesion molecules and MCP-1. (A) Expression levels of macrophage markers, galectin-3 and CD68, in descending arteries of apoE^−/− mice was detected by western blotting (P=0.0157 and P=0.0274). (B) Expression levels of VCAM-1, ICAM-1 and MCP-1 in the descending arteries were detected using western blotting (P=0.0218, 0.0223 and 0.0018). (C) NF-κB activation in the descending arteries was analyzed by measuring NF-κB, p-NF-κB, IκBα, and p-IκBα levels (P=0.2743, 0.0234, 0.00428 and 0.034). Data represent the mean ± standard error (n=4). *P<0.05, **P<0.01 vs. con. Con, control; TSIIA, Tanshinone II A; VCAM-1, vascular cell adhesion molecule; ICAM-1, intercellular adhesion molecule; MCP-1, monocyte chemoattractant protein-1; p-, phosphorylated; NF-κB, nuclear factor-κB; IκBα, NF-κB inhibitor α.

Figure 4.

TSIIA decreases the expression of MMP-2, −3, and −9, and RAGE, and inhibits the activation of proteins involved in the MAPK signaling pathways. (A) TSIIA suppressed active MMP-2, −3, and −9, and RAGE expression in the descending arteries (P=0.0351, 0.0292 and 0.0127). (B) Activations of ERK1/2, JNK and p38 were determined by the analysis of ERK1/2, p-ERK1/2, JNK, p-JNK, p38, and p-p38 protein levels (P=0.0022, 0.0233 and 0.0199). (C) TSIIA inhibited RAGE expression (P=0.0051). Data represent the mean ± standard error (n=4). *P<0.05, **P<0.01 vs. con. Con, control; TSIIA, Tanshinone II A; MMP, matrix metalloproteinase; p-, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; RAGE, receptor for advanced glycation endproducts.