We investigated whether ginsenoside Rb1 (Rb1) could block tumor necrosis factor-alpha (TNF-a a)-induced over-expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HU-VECs) and human lung microvascular endothelial cells (HMVECs-L). Cells were treated with various concentrations of TNF-a a with or without Rb1 pre-treatment for 16 h. The mRNA and protein levels of VCAM-1 were determined with real-time polymerase chain reaction (PCR) and flow cytometry,

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... respectively. Human monocytic THP-1 cells labeled with fluorescent dye (Calcein-AM) was used for the adhesion assay on HUVEC monolayers. Dihydroethidium (DHE) was used to demonstrate in situ levels of superoxide production. JC-1 dye was used to measure changes in mitochondrial membrane potential. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-k k B (NF-k kB) was determined by Bio-Plex immunoassay. TNF-a a treatment significantly increased the mRNA and protein levels of VCAM-1 in HUVECs in a dose dependent manner. Rb1 pre-treatment effectively blocked the TNF-a a-induced expression of VCAM-1 mRNA or protein by 80% and 43%, respectively (pϽ0.01). THP-1 adhesion was also blocked. Furthermore, Rb1 reduced the TNF-a a-induced increase of superoxide anion production by 41% and inhibited the TNF-a a-induced decrease of mitochondrial membrane potential by 44% in HUVECs. Rb1 also effectively blocked TNF-a a-induced activation of p38, c-Jun N-terminal protein kinase, extracellular signal-regulated kinase 1/2 and Ik kBa a. In conclusion, Rb1 effectively blocked the TNF-a a-induced over-expression of VCAM-1, increased THP-1 adhesion and over-production of superoxide anion. Furthermore, Rb1 inhibited TNF-a a-induced MAPKs and NF-k kB activation. These data suggested that Rb1 might have potential therapeutic effects in controlling inflammation in vascular diseases. # These authors contributed equally to this work. Activation of three major MAPKs (ERK1/2, JNK, and p38) was determined using Bio-Plex immunoassay system. HUVECs were treated with TNF-a (1 ng/ml) and/or Rb1 (10 mM) for different durations (0, 2, 5, 10, 15, 30, 45, 60 min). The phosphorylated and total proteins of each MAPK were detected by Bio-Plex immunoassay system. Rb1 treatment reduced the phosphoprotein/total protein ratio of p38 (Fig. 5A) , JNK (Fig. 5B ) and ERK1/2 (Fig. 5C ) at 30-45, 45-60 and 10 min after treatment, respectively.