Abstract
Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein-HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1 μg/ml LPS for 4 hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound-healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT-qPCR and Western blot, respectively. The release of cytokines interleukin-6 and tumour necrosis factor-α was measured by ELISA. HSPA12B suppressed LPS-induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS-induced up-regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS-induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS-induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway.
Highlights
The inflammatory response of endothelial cells is critical for the pathogenesis of various diseases, such as endotoxic shock, thrombosis, cancer, atherosclerosis and diabetes mellitus [1, 2]
The results demonstrated that the LPS-induced increases in mRNA levels of ICAM-1, VCAM-1, IL-6 and tumour necrosis factor (TNF)-a were significantly exaggerated by 147.6%, 151%, 161.6% and 148.4%, respectively, compared with the LPS-treated human umbilical vein endothelial cells (HUVECs) with normal Heat shock protein A12B (HSPA12B) (Fig. 7, P < 0.05)
Activation of the PI3K/Akt signalling pathway plays an important roles in inflammatory responses [16, 24, 25], we examined the effect of HSPA12B on the levels of p-Akt in HUVECs
Summary
The inflammatory response of endothelial cells is critical for the pathogenesis of various diseases, such as endotoxic shock, thrombosis, cancer, atherosclerosis and diabetes mellitus [1, 2]. Human umbilical vein endothelial cell permeability was determined by measuring FITC-labelled dextran (Sigma-Aldrich) across the monolayer as Hordijk PL described [18]. The cells were washed with serum-free M199 medium, and the transfection mixture was added to the six-well plates and incubated for 6 hrs. Cells were allowed to grow to monolayers and interrupted using a 200 ll pipette tip, washed twice with PBS and challenged with 1 lg/ml of LPS for 4 hrs. Human umbilical vein endothelial cells were seeded at a concentration of 1 9 105 cells/well into 24-well plates (Corning Costar) and incubated for 24 hrs at 37°C.
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