Roles Of MD-2 In LPS-induced Endothelial Cell Activation And Its Regulation

Sepsis with subsequent septic shock and multiple organ dysfunction syndrome(MODS) is the most common cause of mortality in the critical ill,including severe trauma,burns,shock,major operation,etc.More and more researches have indicated that endothelial cells play a crucial role in the pathogenesis of endotoxic shock and MODS.Therefore,it is necessary to explore the activated machanism of endothelial cells induced by LPS in sepsis and septic shock.Endothelial cells do not express membrane CD14,and its activation induced by LPS needs LBP and soluble CD14(sCD14).LPS is transferred to Toll-like receptor 4(TLR4) of endothelial cells by sCD14,but the resent studies have discovered that MD-2 is involved in LPS signal transduction. Most researches used gene transfection to study MD-2,while few scientific reports dealt with roles of MD-2 in LPS-induced endothelial cell activation and its signal transduction.The purpose of this study is provide sufficient experimental materials, elucidate the roles of MD-2 in LPS-induced endothelial cell activation and its signal transduction,find new target and new step to interrupt endothelial cell activation induced by LPS at the membrane receptor level,and prevent and treat sepsis and septic shock.Based on cultured human umbilical vein endothelial cell(HUVEC),the expression of MD-2 and the effect of LPS on the expression were observed.Meanwhile,the roles of MD-2 in LPS-binding with endothelial cell and LPS-induced cell activation were investigated.Finally,the expression of TLR4 and MD-2 regulated by interferon-γ(IFN-γ) was studied.The main results and conclusions were summarized as follows:1.HUVEC could express MD-2.LPS could obviously upregulate the expression levels in dose- and time-dependent manner with RT-PCR and westernblot analysis,suggesting that MD-2 may play an important role in LPS-induced This work was supported by the Major State Basic Research Development Program of China(No.G1999054203)endothelial cell activation.2.Blood serum could obviously promote LPS-bingding with endothelial cells.Anti-TLR4 and anti-MD-2 monoclone antibody significantly inhibited LPS-binding with cells in dose-dependent manner,indicating that TLR4 and MD-2 are necessary for LPS-binding with endothelial cell.3.Transfection of non-signaling mutant forms of MD-2 significantly inhibited LPS-induced NF-кB activation and IL-8 secretion in endothelial cells in dose-dependent manner,further indicating that MD-2 is necessary for LPS-induced cell activation.4. IFN-γ coule obvously upregulate the expression of TLR4 and MD-2 in dose- and time-dependent manner, suggesting that IFN-γ may promote LPS-induced endothelial cell activation through upregulating the expression of TLR4 and MD-2.