Activation Of Tie2 Signaling Pathway In Vasculature Protection

Background: The peritubular capillaries injury plays the same important roles in the pathogenesis of both acute kidney injury and progression to chronic kidney disease.Compared with the renal tubular epithelial cells, renal microvascular endothelial cells lack an efficient regeneration ability. When renal function recovers to normal levels, the reduction of peritubular microvessel density persists for a long time, which results in chronic kidney disease. Tie2 signaling pathway is thought to be important in vasculature protection, in most studies Ang-1 was used as the agonist of Tie2 signaling pathway. However, in some reports, Ang-1 shows its effects in promoting incidence of AKI and inflammatory cellular infiltration, even promoting renal fibrosis in the later stage. So we focus on the vasculature protection effect of Tie2 signaling pathway.Objectives:In this research, we use LPS to induce endothelial cell injury to explore whether activation of Tie2 signaling pathway has endothelial cell protection effects, in order to provide experimental and theoretical basis for the further researches.Methods:Part I:Human umbilical vein endothelial cells were incubated respectively at 96 well Plates with densities of 4×104/m L,200 u L/well.Different concentrations of LPS was used in the experiment: 0ug/ml,0.125 ug/ml,0.25 ug/ml,0.5 ug/ml,1 ug/ml,2 ug/ml,5 ug/ml,10 ug/ml.After 24 h of LPS stimulation, cell proliferation activity wasdetected by cck8. A suitable concentration of LPS was chosed to establish the cell injury model according to the results.Part II:According to the first Part of the experiment,l0ug/m L LPS and 5ug/m L Tie2 agonist antibody was choosed for this Part. Human umbilical vein endothelial cells were randomly divided into four groups: Normally cultured cells group(control), Normally cultured cells with LPS group(group LPS),Normally cultured cells with LPS and Tie2 agonist antibody(group LPS+Tie2 agonist antibody).s VCAM-1 was detected by enzyme linked immunosorbent assay(ELISA) after 24 hours of incubation.VCAM-1 m RNA and apoptosis-related genes Bax, Bcl-2 were detected by Polymerase Chain Reaction(PCR) after 6 hours of incubation.α-SMA was detected by WESTERN BLOT after 24 and 48 hours of incubation.Results:Part I:Cck8 results show that, after stimulated by LPS, cell growth was restrained by all concentrations of LPS compared with control group(P<0.05).Part II:After stimulated by LPS of 10ug/ml, compaired with control group, the expressions of s VCAM-1 and VCAM-1m RNA of group LPS were significantly increased(P<0.05), and decreased after Tie2 agonist antibody administration(P<0.05). After 6h incubation, Baxm RNA and Baxm RNA/Bcl-2m RNA of group LPS were significantly increased than control group(P<0.05), and decreased after Tie2 agonist antibody used(P<0.05). There was no statistically significant of Bcl-2 m RNA among 3 groups(P>0.05). After 24 h and 48 h incubation, compaired with control group, the expressions of α-SMA of group LPS were significantly increased(P<0.05), and deceased after Tie2 agonist antibody administration(P<0.05).Conclusions: 1. LPS could induce endothelial damage, the increase of Baxm RNA, Baxm RNA/Bcl-2m RNA, s VCAM-1, VCAM-1m RNA and α-SMA suggests a promotion of apoptosis, inflammatory and endothelial – mesenchymal transition. 2.After the administration of Tie2 agonist antibody,it shows a decrease of both s VCAM-1 and VCAM-1m RNA and a decrease of Bax/Bcl-2, which implies that the activation of Tie2 signaling pathway could promote an anti-in?ammatory and anti-apoptosis effects. 3.Tie2 signal pathway could decrease endothelial – mesenchymal transition by studying the expression of α-SMA.