| Aim: The aim of this study was to establish high oxidative low density lipoprotein(high ox-LDL) induced oxidative damage model in human umbilical vein endothelial cells(HUVECs) and investigate the role of ROCK/PPAR--γ/CXCL16 signaling pathway in the protective effects of ursolic acid(UA). Methods: The optimal concentration for24-hour high ox-LDL exposure were determined, then HUVECs were assigned into different treatment groups, including control group, high ox-LDL group, UA + high ox-LDL group, ROCK inhibitor Y27632 group, Y27632 + high ox-LDL group, UA +Y27632+high ox-LDL group, PPAR-γ agonist rosiglitazone, rosiglitazone + high ox-LDL group, UA + rosiglitazone + high ox-LDL group,PPAR-γ antagonist GW9662 group,GW9662+ high ox-LDL group and UA + GW9662 + high ox-LDL group. CCK-8 assay was used to assess cell viability in different groups. Molecular biomedical techniques were used to detect the phosphorylation level of ROCK in different groups(expressed as the ratio of p-ROCK to ROCK) and the expression levels of PPAR-γ and CXCL16 at both m RNA and protein levels. Specific inhibitors/antagonists/agonists were used to elucidate the order of signaling pathway. Amplite ROS Green method was used to measure the intracellular ROS level. Statistics were performed with SPSS 17.0. Results:The oxidative damage model in HUVECs were successfully established at 35 μg/ml high ox-LDL exposure for 24 hour, with intracellular ROS level raised to 2.4 times as of control cells. The ratio of p-ROCK to ROCK, the m RNA and protein expression level of CXCL16 were significantly increased, whereas the m RNA and protein expression level of PPAR-γ decreased(P<0.05). Comparing to the high ox-LDL group, 100 μM Y27632 exposure for one hour upregulated the expression of PPAR-γ at protein level(P<0.05), on the other hand, 100 μM Y27632 or 100 μM rosiglitazone exposure for one hour decreased the protein expression levels of CXCL16 as well as intracellular ROS level(P<0.05). Additionally, 20 μM GW9662 exposure for 30 minute further increased the expression level of CXCL16 and intracellular ROS level(P<0.05). Our data suggests that the activation of ROCK/PPAR-γ/CXCL16 is associated with high ox-LDL induced oxidative cytotoxicity. On the other hand, pretreatment with 5, 10 or 20 μM UA for 16 hour dose dependently improved cell viability in high ox-LDL exposed HUVECs(P<0.05). Co-treatment of UA and Y27632 or rosiglitazone further enhanced the cytoprotective effect of UA(P<0.05), while co-treatment with GW9662 abolished such effects(P<0.05). Computer aided drug design(CADD) indicated that the unsaturated carboxyl oxygen group could bind to the lysine and tyrosine residues by hydrogen bonding at the active site of ROCK. Western blotting results demonstrated that the phosphorylation level of ROCK and expression level of CXCL16 were decreased, while expression level of PPAR-γ was increased following different doses of UA treatment.Elevated PPAR-γ m RNA level and decreased intracellular ROS level were also observed.Co-treatment of UA and Y27632 up-regulated expression of PPAR-γ and down-regulated CXCL16 and ROS generation compared to high ox-LDL group(P<0.05), but not statistically different from UA or Y27632 alone groups(P>0.05). Co-treatment of UA and GW9662 abolished the down-regulation of CXCL16 and ROS level(P<0.05),suggesting the role of UA mediated down-regulation of ROCK/ PPAR-γ/CXCL16 in its cytoprotective effects. Conclusion: Oxidative damage model in HUVECs was successfully established with 35 μg/ml high ox-LDL exposure for 24 hour, in which exposure to exogeneous high ox-LDL upregulated CXCL16 expression in HUVECs. UA stabilizes vascular endothelium via down-regulation of ROCK/PPAR-γ/CXCL16 signaling pathway. |
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