The Effects Of Human ?-defensin 3 On Inflammation-induced Vascular Injury In ApoE-deficient Mice

Objective:The aim of this study was to investigate the role of human ?-defensin 3(hBD3)in the inflammatory response and the formation of atherosclerotic lesions induced by Porphyromonas gingivalis lipopolysaccharide(Pg-LPS);To evaluate the effect of hBD3 in the inflammatory response of macrophages stimulated by Pg-LPS;To explore the role of hBD3 in endothelial cell activation during the initiation stage of atherosclerosis.Methods:1.The effects of hBD3 in Pg-LPS-induced inflammation and atherosclerotic lesions:In vivo,32 ApoE-deficient mice were randomly divided into four groups,namely NC,Pg-LPS,Pg-LPS+hBD3,hBD3,and the Pg-LPS groups were exposed to chronic Pg-LPS stimulation for 10 weeks via intraperitoneal injection.At the end of the study point,the whole blood of ApoE-deficient mice was collected for blood test.Subsequently,the mice were euthanized and the aortic tissues plus serum was obtained for enzyme-linked immunosorbent assay(ELISA)and quantitative polymerase chain reaction(Q-PCR)analysis.Histopathological examinations,for example,immunohistochemistry,Oil Red O staining,and elastin staining,were used to evaluate the effect of hBD3 on atherosclerotic lesions.2.The effects of hBD3 in Pg-LPS-induced inflammation in macrophages:In vitro,the mouse macrophage cell line RAW 264.7 was stimulated with Pg-LPS and various concentrations of hBD3.Cell culture supernatants were examined with ELISA.Upregulated or downregulated genes and pathways by hBD3 intervention were investigated with mouse whole genome chip.Western blot analysis was used to detect the possible signaling pathways involved in this process.3.The effects of hBD3 upon TNF-a-induced endothelial activation of HUVECs:The primary cultured HUVECs were treated with TNF-a and intervened with various concentrations of hBD3.Inflammatory mediators released by HUVECs were measured with ELISA.Reactive oxygen species(ROS)induced by TNF-a was measured using DCFH-DA.F-actin staining was evaluated with phalloidine.In addition,protein levels of key molecules regarding cell adhesion,signaling pathways and apoptosis were detected using western blot.Results:1.The effects of hBD3 in Pg-LPS-induced inflammation and atherosclerotic lesions:In vivo,hBD3 inhibited serum monocyte chemoattractant protein-1(MCP-1),soluble intercellular adhesion molecular-1(sICAM-1)levels of ApoE-deficient mice exposed to Pg-LPS in a chronic inflammation model.In addition,Q-PCR analysis revealed that hBD3 markedly inhibited the expression of intercellular adhesion molecule-1(ICAM-1)and vascular adhesion molecule-1(VCAM-1)in Pg-LPS-treated ApoE-deficient mice.Blood test demonstrated that hBD3 reversed Pg-LPS-stimulated lymphocyte monocyte ratio(LMR)downregulation in ApoE-deficient mice.While for the atherosclerotic lesions,serum levels oftotal cholesterol(TC)and low-density lipoprotein(LDL)were also markedly reduced with hBD3 intervention.In addition,thinned vascular walls,less macrophage infiltration and the formation of atherosclerotic lesions in the aortic root and arterial tree were observed in the hBD3-treated group.2.The effects of hBD3 in Pg-LPS-induced inflammation in macrophages:In vitro,hBD3 profoundly suppressed the production of TNF-a and interleukin-6(IL-6)in RAW 264.7 cells induced by Pg-LPS in a dose-dependent manner.hBD3 successfullly suppressed elevated production of TNF-a induced by Toll-like receptor(TLR)-2 agonist Pam3CSK4 and TLR4 agonist MPLA in RAW 264.7 cells.Downregulatory effects of inflammatory genes by hBD3 were observed in the genome chip assay.In addition,hBD3 dose-dependently inhibited the phosphorylation of p38 and extracellular-signal regulated protein kinase(ERK)in the mitogen-activated protein kinase(MAPK)pathway within 30 min.3.The effects of hBD3 upon TNF-a-in duced endothelial activation of HUVECs:First,hBD3 reduced the production of IL-6,IL-8,monocyte chemoattractant protein-1(MCP-1)and macrophage migration inhibitory factor(MIF)of HUVECs in a dose-dependent manner.In addition,hBD3 significantly prevented intracellular ROS production of HUVECs.Second,western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of ICAM-1 and VCAM-1 in TNF-a-induced HUVECs.As a result,hBD3 inhibited monocyte adhesion to TNF-a-treated endothelial cells.Additionally,hBD3 suppressed TNF-a-induced F-actin reorganization in HUVECs.Thirdly,hBD3 markedly inhibited NF-?B activation by the downregulation of phosphorylation of IKK-?/?,I?B and the subunit of p65 within 30 min.Moreover,the phosphorylations of p38 and c-Jun N-terminal protein kinases(JNK)of mitogen-activated protein kinase(MAPK)pathway were also inhibited by hBD3 in HUVECs.Finally,hBD3 inhibited TNF-?-stimulated apoptosis of HUVECs.Conclusion:With anti-inflammatory roles in macrophages and inhibitory effects in endothelial cell activation,hBD3 exerts protective role in vascular injury induced by inflammation in ApoE-deficient mice.