An Investigation of the Effects of an Aqueous Extract of Radix Salvia Miltiorrhiza-Radix Pueraria Lobata Mixture on Atherosclerotic Events and the Underlying Biochemical Mechanisms.

摘要

Atherosclerosis is a major form of cardiovascular disease characterized by the thickening of the arterial wall and narrowing of the artery lumen. The aim of the present study is to investigate the anti-atherosclerotic effects of the aqueous extract containing Radix Salvia miltiorrizha (Danshen) and Radix Pueraria lobata (Gegen) (DG).;In the anti-inflammatory study, DG inhibited lipopolysaccharide (LPS)-induced production of inflammatory mediators by macrophages, including nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), but not tumor necrosis factor-alpha (TNF-alpha). Further study revealed the underlying mechanism was associated with inhibition of NFkappaB pathway.;The anti-foam cell formation study indicated DG was able to decrease cellular acLDL content in macrophages, and thus subsequently inhibited macrophage foam cell formation. Further mechanistic study demonstrated the disruption of LDL influx pathway, by down-regulating the two major scavenger receptors for acLDL, CD 36 and SR-A. This anti-foam cell formation effect of DG was related to activation of peroxisome proliferator-activated receptor (PPAR) family, including PPARalpha, PPARgamma and LXRalpha.;In the study investigating the effect of DG on TNF-alpha-induced endothelial dysfunction, DG inhibited the two markers for endothelial dysfunction, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These inhibitory effects were associated with activation of nuclear factor 2 (Nrf2), which resulted in up-regulation of heme oxygenase-1 (HO-1).;Balloon injury of rat carotid was developed to study the in vivo holistic effect of DG. Intima-media thickening (IMT) of rat carotid arteries was induced by balloon injury. Histological analysis revealed proliferation of underlying vascular smooth muscle cells (VSMCs) that constitutes the neointima and contributes to IMT. DG exerted significant reduction on thickness of injured carotid arteries, implying possible anti-VSMC proliferative effect. Molecular study of artery tissue indicated down-regulation of inflammatory genes, such as ICAM-1, VCAM-1, TNF-alpha and MCP-1, but a protective gene, HO-1, was up-regulated. Bilirubin levels were increased in blood of rats with DG treatment. The elevated bilirubin levels are in agreement with the up-regulation of HO-1 by DG both in endothelial cells and rat carotid arteries, which might explain the protective effect of DG against IMT.;The anti-VSMC proliferative effect of DG was substantiated by inhibition of DNA synthesis in PDGF-BB-induced proliferating VSMC. Microarray study identified 1,419 differentially expressed genes with p ≤ 0.05 by t-test and fold change ≥ 1.2. Cell signaling pathways, including receptor-mediated pathways, MAPK/Erk pathways and NFkappaB pathway, as well as functional genes encoding enzymes for DNA synthesis and cell cycle progression were implicated in inhibition of VSMC proliferation. Furthermore, DG down-regulated a group of genes for a proliferative, synthetic phenotype, but up-regulated the gene for a quiescent, contractile phenotype.;The interaction effects of the two component herbs of DG were interpreted with the application of statistical methods. This study indicated three modes of interactions, including synergistic, additive and antagonistic effects, on anti-inflammation, anti-foam cell formation and anti-VSMC proliferation, respectively.;In conclusion, the present comprehensive study provided concrete evidence for the anti-atherosclerotic effects of DG, including anti-inflammation, anti-foam cell formation, anti-endothelial dysfunction and anti-VSMC proliferation.