Involvement of TLR-4 signaling pathway in shear stress induced IL-8 gene expression in ECV304 cells / 中国病理生理杂志

ABSTRACT

AIM: To examine the role of TLR-4 signaling pathway in laminar low shear stress-induced IL-8 gene expression in ECV304 cells. METHODS: RT-PCR and PCR were used to amplify a TLR4 mutant (lacking the 155 COOH terminal amino acids of the wild type TLR4 ) and-102-+61 bp 5′-flanking region of IL-8 gene (IL8 USCS)from endothelial cells. These two DNA fragments were cloned into pcDNA3 and pEGFP1, respectively, and the recombinant plasmid pcDNA3-mTLR4 and pEGFP1-IL8USCS were obtained. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by using Dosper liposomal transfectional reagent and selected by G418, and then stimulated by 4 2 dyne/cm 2 shear stress for 3 hours. The green fluorescent protein expression was analyzed by Flow Cytometry. Immunoblotting of the cell lysates and NF-?B p65 immunocytofluorescent staining were used to determine I?B phosphorylation, degradation and NF-?B activation. RESULTS: Flow Cytometric analysis showed that when exposed to 4 2 dyne/cm 2 shear stress for 3 hours, there was a marked increase in the enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation. Western blot analysis showed that a markedly increased p-I?B of cell lysates occurred at 10 min of exposure and the blot density almost dropped down to the baseline after 60 min of exposure. The density of I?B blot dropped down with increasing exposure time. NF-?B p65 immunocytofluorescent staining of ECV304 cells showed that when exposed to the same flow shear stress for 0 5,1 hours, the cell nuclei became staining, and after 1 5 or 2 hours, the staining was very strong. CONCLUSION: These results suggested that the inflammatory TLR-4/NF-?B signaling pathway may be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.