Procyanidin B1Inhibit Endotoxin LPS-stimulated Inflammatory Response Through TLR/MD2

Objective:Endotoxic lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, leading to responses that are injurious to the host, produced pro-inflammatory mediators and may rapidly result in severe sepsis, or MODS. Innate immune cells such as Macrophages recognizes a common ’pattern’ in structurally diverse LPS molecules by Toll-like receptor (TLR)4. Macrophages received LPS by its Toll-like receptor on the surface membrane, which activates macrophages to produce a variety of pro-inflammatory cytokines such as tumor necrosis factor (TNF-a). Crystal structures of human Myeloid differential protein-2(MD-2) and its complex with the anti-endotoxic tetra-acylated lipid A core of LPS have been determined, shows a deep hydrophobic cavity sandwiched by two b sheets, in which four acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. Myeloid differentiation primary response protein88(MyD88) binds to the TLR4, which triggers the activation of NF-B and MAPK signaling pathway. Procyanidins consist of one or more aromatic rings with one or more hydroxyl groups, was found to have a anti-inflammatory effect. Procyanidin B1have been shown to reduce inflammation, however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized. This research was carried out to clarify the potential role and molecular mechanisms of procyanidin B1in the anti-inflammatory effect.Methods:Human monocytes cell line THP-1Cells were cultured in RPMI1640supplemented with10%FBS, at37℃in a humidfied atmosphere of5%CO2in air. The medium was replaced every3days. The cell intensity was maintained at5～10×105/ml. Cell viability was assessed by CCK-8assay. Cells were cultured in96-well plates and stimulated with Procyanidins (50～200μg/ml) for18h. The supernatant was harvested and mixed with an equal volume of CCK-8reagent, the absorbance at450nm was measured using a micro plate reader. THP-1cells were cultured in96-well plates. Cells were treated with LPS (1ug/mL) with or without Procyanidins for18h. Culture medium was then collected for the assay of TNF-a. TNF-a secreted in the culture medium were measured by specific ELISA using monoclonal anti-human TNF-a antibody as a capture antibody. The absorbance at450nm was measured using a micro plate reader. Expression of MyD88and MD2mRNAs were measured by Real-time PCR. We extracted total RNA from treated THP-1cell as before, The cycle threshold (Ct) values of the interested genes were first normalized with GAPDH from the same sample, and then the relative differences between the groups were calculated and expressed as relative increases, setting the blank control group as1. Each sample was tested in triplicate.Results:CCK-8:Procyanidins100ug/ml group and Procyanidins50ug/ml group suggested that the cell viability was above96%. But when the Procyanidins concentration is above150ug/ml, the cell viability will be impaired. ELISA:Be compared to the group of control, the LPS stimulated alone Group shows that express of TNF-a level in THP-1cell was significant elevated. But these inductions were significant inhibited by treatment with Procyanidins in a concentration dependent manner. Real-time PCR:We observed a significant up-regulation of MyD88mRNA and MD-2mRNA in LPS stimulated alone Group when compared with the control group. And also the up-regulation of MyD88mRNA and MD-2mRNA was inhibited by Procyanidins.Conclusions:Treatment with procyanidin B1resulted in decrease in MD2level, without changing TLR4level. procyanidinBl also inhibit MyD88induced signaling pathways. These three factors play a major role in molecular mechanisms of inflammatory activity. ProcyanidinB1inhibited the release of pro-inflammatory cytokines tumor necrosis factor-a, which associated with concentration in lipopoly-saccharide (LPS)-induced THP-1. Procyanidin B1inhibit the action of the inflammatory response in dose correlation, and MD2expression decrease may be associated with such above. Procyanidin B1plays a potent role in with MD2or TLR4/MD2in molecular, in turn, resulted in decrease in MD2level and regulate intracellular signaling pathways of LPS, may be one of the mechanisms of regulation of inflammatory response, and is expected to become novel treatment of excessive inflammation.