Combination of tanshinone IIA and astragaloside IV attenuate atherosclerotic plaque vulnerability in ApoE(-/-) mice by activating PI3K/AKT signaling and suppressing TRL4/NF-κB signaling

Abstract

Tanshinone IIA (TS IIA) and Astragaloside IV (AS IV) are natural herbal products which exert anti-inflammatory and anti-oxidant effects in order to eliminate unstable plaque in atherosclerosis. However, the combined effect of these two drugs on atherosclerotic plaque vulnerability and its molecular mechanism remains unclear. In the current study, we evaluate the effects of TS IIA and AS IV on atherosclerotic unstable plaque stability, and then further explore the mechanism of TS IIA and AS IV intervention on unstable plaque in vivo and in vitro. Histological characterization of atherosclerotic plaques was measured by Hematoxylin-Eosin (HE), Masson's Trichrome and Oil Red O staining. Cellular lipid droplet was measured by Oil Red O staining. The size of atherosclerotic lesion areas and content of lipids and collagen in the right common carotid arteries of apoE-/- mice were examined by Hematoxylin-Eosin (HE), Oil-red O, and Masson staining, respectively. The protein expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and C-reactive protein (CRP) in ApoE^-/- mice and RAW264.7 cells were determined by enzyme-linked immunosorbent assay. The protein expression levels of matrix metalloproteinase-9 (MMP-9) and endothelial nitric oxide synthase (eNOS) in ApoE^-/- mice and RAW264.7 cells were determined by western blotting. In addition, the PI3K/AKT and TRL4/NF-κB signaling were determined by western blotting. Our results revealed that the combination of TS IIA and AS IV significantly decreased lipid areas, increased collagen content and thickened fibrous cap in the right common carotid arteries compared with ApoE (-/-) mice model group. TS IIA and AS IV visibly reduced the cytoplasmic lipid droplet accumulation induced by oxLDL in RAW 264.7 macrophages. The ApoE^-/- mice model group and oxLDL -stimulated RAW 264.7 macrophages treated with TS IIA and AS IV showed a downregulation in IL-6, MMP-9, TNF-α and CRP protein expression and upregulation in eNOS protein expression. Furthermore, TSIIA and AS IV may activate PI3K/AKT signaling and suppress TLR4/NF-κB signaling in vivo and in vitro. Additionally, blocking the PI3K/Akt signaling enhanced the translocation of NF-κB to the nucleus, TLR4, IL-6, MMP-9, TNF-α and CRP expression and inhibited eNOS expression in TS IIA and AS IV-treated RAW 264.7 macrophages. Therefore, the present study implicates that TS IIA and AS IV reinforces plaque stability via PI3K/AKT and TLR4/NF-κB signaling. TS IIA and AS IV administration may provide the basis for a potential therapeutic approach for the inhibition of vulnerable atherosclerotic plaques.

Keywords: Astragaloside IV; Atherosclerotic plaque vulnerability; PI3K/AKT signaling; TRL4/NF-κB signaling; Tanshinone IIA.