Research Of NOD2Up-regulates The Gene Expression Of Type Ⅰ Interferons In HAECs And TLR3Induces Apoptosis In HUVECs

Charpter1MDP up-regulates the gene expression of type I interferons in human aortic endothelial cellsBackground:Muramyldipeptide (MDP) is a pathogen-associated nolecular patterns (PAMPs). MDP has been shown to be recognized by a:ytoplasmic PRP nucleotide-binding oligomerization domain2(NOD2). NLRs were newly found molecules of innate immunity in animals. The first NLRs had been reported to respond to PAMPs stimulus and-ecognize microbes were NOD1(CARD4) and NOD2(CARD15), NLRs was found to be related to the pathogenesis of atherosclerosis. It was reported that type I Interferon has been involved in the unstabilityty of itherosclerotic plaque. MDP has been repored to induce the production of:ytokines such as type I Interferon and to regulate the reaction of mmunity and inflammation, we have observed that MDP can induce the production of type I Interferon in Human aortic endothelial cells HAECs), but the mechanism is unknown.Objecive To observe the influence of MDP on Biological function of HAECs. Investigate the mechanism of MDP up-regulates the gene expression of type I interferons in human aortic endothelial cells, and)bserve the influence of MDP on the gene expression of)ro-inflammatory cytokines in HAECs.Methods:Above all, according to the requirements of experiment, HAECs were treated with MDP, the gene expression of IFN-α, IFN-β, IL-1β, IL-8and MCP-1were measured by RT-PCR. qRT-PCR. Next, the gene expression of NOD1, NOD2, TNF-a and IRFs related to the expression of type I interferon were measured by RT-PCR. Finally, we used Western-blot technology investigated the mechanism of MDP up-regulates the gene expression of type I interferons in human aortic endothelial cells.Results:1. HAECs were treated with indicated concentrations (0.0.1.0.5.1.5.10μg/ml) of MDP for24h, RT-PCR and qRT-PCR showed that MDP induced dose-dependent up-regulation of interferon gene expression.2. HAECs were treated with10μg/mL MDP for indicated hours (0,1,3.6.12.24h). RT-PCR and qRT-PCR showed that MDPinduced time-dependent up-regulation of interferon gene expression.3. HAECs were treated with indicated concentrations (0,0.1,0.5,1,5.10μg/ml) of MDP for24h. RT-PCR and qRT-PCR showed that MDP up-regulated the gene expression of NOD2.4. HAECs were treated with indicated concentrations (0,0.1,0.5,1,5.10μg/ml) of MDP for24h. RT-PCR and qRT-PCR showed that MDP down-regulated the gene expression of NOD1.5. HAECs were treated with indicated concentrations (0,1,5,10,50,100ng/ml) of TNFα for24h. RT-PCR and qRT-PCR showed that TNFa induced the up-regulation of interferon gene expression.6. HAECs were treated with the indicated concentrations (0,0.1,0.5,1,5,10μg/ml) of MDP for24h. RT-PCR showed that no gene expression of TNFa was found within24h. 7. HAECs were pre-treated with TNFα neutralizing antibody, the induction of IFNs induced by MDP re-stimulation was not decreased.8. The gene expression of IRFs in HAECs were measured by RT-PCR, The results showed that HAECs expressed IRF1,2,3,9, but not IRF4,5,6,7,8.9. HAECs were treated with the indicated concentrations (0,0.1,0.5,1,5,10μg/ml) of MDP for24h, and found that MDP induced the activation of IRF3at time points5,10and30minutes. The semi-quantitation data showed the significant up-regulation of phosphorylated IRF3compared with the non-treated group.10.HAECs were treated with the indicated concentrations (0,0.1,0.5,1,5,10μg/ml) of MDP for24h, RT-PCR and qRT-PCR showed that MDP induced dose-dependent up-regulation of pro-inflammatory cytokines, including IL-1β, IL-8and MCP-1.Conclusion:MDP Up-Regulates the Gene Expression of Type Ⅰ Interferons in Human Aortic Endothelial Cells by the phosphorylation of IRF3, MDP induced dose-dependent up-regulation of pro-inflammatory cytokines, including IL-1β, IL-8and MCP-1. Charpter2Poly(I-C) Induces Apoptosis in Human Umbilical Vein Endothelial CellsBackground:Atherosclerosis is a kind of inflammatory diseases. It was reported that there were close relationship between TLRs, such as TLR1,TLR2,TLR4and TLR9,and Atherosclerosis. Toll like receptor3 (TLR3) was the first member of the TLR family known as a pattern recognition receptor and has been demonstrated to detect a conserved viral molecular pattern, doublestranded RNA (dsRNA), TLR3was expressed in a variety of cells. Recently, it has been reported to elicit apoptosis in several melanoma cells. In this study, we found that TLR3can induce vascular endothelial cell apoptosis through extrinsic and intrinsic apoptosis pathways,and account for endothelial dysfunction.Objecive:To observe the influence of Polyinosinic-polycytidylic acid (poly(I-C)), a synthetic analog of dsRNA, on biological function of HUVECs, investigate the function of Pathogen recognition receptors (PRRs) in the process of Poly(I-C) Inducing apoptosis in HUVECs and investigate the mechanism of poly(I-C) Induces apoptosis by the activation of TLR3in Human Umbilical Vein Endothelial Cells (HUVECs)Methods:According to the requirements of experiment, Primary HUVECs and/or Immortalized HUVECs were treated with poly(I-C), Cell apoptosis was measured by flow cytometry analysis. The gene or protein expression of TLR3and Inflammatory factors were measured by RT-PCR, qRT-PCR or flow cytometry analysis. Next, we used Western blot, ELISA, RT-PCR and RNA interference technology investigated the expression of cytokine in the extrinsic and intrinsic pathways.Results:Pretreated with μig/ml poly(I-C) for24h, were re-stimulated with the indicated concentrations of poly(I-C) for24h. Flow cytometry analysis showed that early apoptotic and late apoptotic were increased. TLR3down-regulation by RNA interference abrogated poly(I-C)-induced cell apoptosis. P63down-regulation repressed poly(I-C)-induced cell apoptosis. RT-PCR showed HUVECs express low level of TRAIL, DR4, and DR5, poly(I-C) up-regulated the gene expression of TRAIL, DR4, and DR5, down-regulated the gene expression of Bcl-2, up-regulated the gene expression of BH3, up-regulated the gene expression of TAp63a, p63RNA interference inhibited the expression of TRAIL, DR4, and DR5. Western blot results showed poly(I-C) up-regulated the expression of TLR3protein, TLR3levels were low in5-10μg/ml poly(I-C) treated cells, poly(I-C) down-regulated the expression of Bcl-2, up-regulated the expression of BH3, up-regulated the expression of TAp63a, TLR3down-regulation by TLR3shRNA transient transfection inhibited the expression of TAp63αin primary HUVECs, inhibited the down-regulated of Bcl-2, inhibited the up-regulated of noxa, inhibited the activation of caspases8and9and PARP. qRT-PCR results showed poly(I-C) significantly up-regulated the gene expression of TLR3. ELISA result showed poly(I-C) up-regulated the protein expression of TRAIL.Conclusion Poly(I-C) Induces Apoptosis in HUVECs, Toll-like Receptor3(TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the TAP63a.