Effect Of Nf-?B Pathway Activated On 5-hydroxytryptamine Receptor 1B Gene Diversity Expression Of Vascular Smooth Muscle Cells

Objectives: To study the underlying relationship between 5-Hydroxytryptamine and activation of NF-?B pathway on vascular smooth muscle cells (VSMCs), we investigate the effect of activation of NF-?B pathway on vascular smooth muscle cells (VSMCs) and their reproduction after transfection of 5-HT1B restructuring plasmids.Methods: Restructure 5-HT1B plasmids, then achieve cell models of which the 5-HT1B expression could be regulated. The VSMCs were divided into 4 groups: control group with complete medium?high dose of lipofectamine 2000(20ul) group?low dose of lipofectamine 2000 (5ul) group and restructuring 5-HT1B plasmids group. PKC?IKK-?protein expression and p42/44MAPK protein phosphorylation level were detected with western blot after addition of 5-HT1B restructuring plasmids or different lipofectamine 2000 into certain groups. Tanon GIS Image Analysis Software is used to analysis those image results.Results: (1) Results of culturing vascular smooth muscle cells (VSMCs) and transfecting 5-HT1B restructuring plasmids into vascular smooth muscle cells (VSMCs): green fluorescence protein(GFP) was observed in cytoplasm under fluorescent microscope(×400),after restructuring 5-HT1B plasmids transfect into vascular smooth muscle cells( VSMCs) for 48 hours, proving that the recombinant plasmid transfect into VSMCs successfully. (2) Western-Blot showed that compared to the control group,the protein expression level of Inhibitor of Nuclear Factor Kappa-B Kinase Alpha (IKK-a) in high dose of lipofectamine 2000(20ul) group was sharply decreased(p<0.001), whereas the protein expreesion level of IKK-a in low dose of lipofectamine 2000 had no statistic difference (P>0.05); ( 3 ) The western-blot analysis of Protein Kinase C(PKC)showed that the protein expression level of PKC in low dose of lipofectamine 2000 group was elevated(p<0.01), and high dose of lipofectamine 2000 had no significant effect on the protein expression level of PKC (P>0.05); (4) In contrast, the MAPK p42/44 protein (EKR1/2)expression level of phosphorylation in restructuring 5-HT1B plasmids group?high dose of lipofectamine 2000 group and low dose of lipofectamine 2000 group were much lower than those in the control group (p<0.001).Moreover, the MAPK p42/44 protein (EKRl/2)expression level in restructuring 5-HT1B plasmids group dropped much more than those in high dose of lipofectamine 2000 group and low dose of lipofectamine 2000 group..Conclusions: (1) The data revealed that the MAPK p42/44 protein (EKR1/2)expression and the level of phosphorylation in restructuring 5-HT1B plasmids group were significantly much lower than those in other groups, suggesting that 5-Hydroxytryptamine receptor 1 B gene expression may influence the activation of the MAPK pathway on vascular smooth muscle cells. (2) No change of the IKK-? and PKC protein expression level in each group is found. This raised a possibility that 5-Hydroxytryptamine receptor does not take part in activation of NF-?B pathway in vascular smooth muscle cells .(3)The protein level of IKKa and MAPK in high dose lipofectamine 2000 group were both inhibated dramaticly, indicating that lipofectamine 2000 can affect the activation of both NF-?B pathway and MAPK pathway in vascular smooth muscle cells.