| Objective: The aim of this study was to establish oxidized low-density lipoprotein(ox-LDL)induced proliferation in rat vascular smooth muscle cells(VSMCs)and investigate the role of Co Q10/UCP2/ROS/Cyto-C signaling pathway in the protective effects of oleanolic acid(OA).Methods: The cell proliferation model was established with induction of 50 μg/ml ox-LDL for 24-hour,then VSMCs were assigned into different treatment groups,including control group,ox-LDL group,OA(5 μM,20 μM,35 μM)+ high ox-LDL group.To investigate the effect of OA,VSMCs were exposed to 50 μg/ml ox-LDL for 24 hours,with or without OA pretreatment(5 μM,10 μM,20 μM)for 12 hours.Real-time PCR and western blotting were used to investigate the m RNA and protein expression levels of Co Q10,UCP2,Cyto-C.Amplite ROS Green method was used to measure the intracellular ROS level.After incubating with 50 μg/ml ox-LDL at different time point(0,3,6,12,24 h),m RNA level of Co Q10,UCP2,Cyto-C were detected via Real-time PCR separately.To confirm the signaling pathway involved in VSMCs proliferation and the molecular target of OA.VSMCs were assigned into ox-LDL group,ox-LDL+ si Co Q10/si UCP2/Genipin group,OA(35 μM)group,OA(35 μM)+ si Co Q10/ si UCP2/Genipin group.The interference efficacy and target m RNA expression levels were assessed with Real-time PCR.Statistics were performed with SPSS 17.0.Results: CCK-8 results indicated that:(1)50 μg/ml ox-LDL treatment for 24 hour significantly increased cell proliferation rate,while pretreatment with 5,10 or 20 μM UA for 12 hour effectively inhibited the proliferation(P < 0.05).(2)VSMCs were exposed to 200 ng si-RNA for 48 hours,but not statistically different from control groups(p > 0.05).MRNA expression level of Co Q10 and UCP2 in 50 μg/ml ox-LDL with 3 h,6 h,12 h,24 h were significantly increased,but the m RNA level of Cyto-C was significantly decreased,suggesting that Co Q10 and UCP2 m RNA expression was positively correlated,Co Q10,UCP2 and Cyto-C m RNA expression was negatively correlated.Compared with the control group,real-time PCR and western blotting revealed increased expression level of the Co Q10 and UCP2,decreased expression level of Cyto-C with decreased intracellular ROS in model group(p < 0.05).It suggested that Co Q10 and UCP2 were positively correlated with cell proliferation,Cyto-C and ROS were negatively correlated with cell proliferation;compared with the model group,real-time PCR and western blotting revealed decreased expression level of the Co Q10 and UCP2,increased expression level of Cyto-C with increased intracellular ROS in OA group(p < 0.05).It suggested that OA can inhibit proliferation of VSMCs through down-regulating Co Q10,UCP2 and up-regulating ROS,Cyto-C.Meanwhile,si-Co Q10 and si-UCP2 co-treatment further decreased the proliferation rate in both ox-LDL model group and OA group relative to their counterpart without si-Co Q10 and si-UCP2(p < 0.05),suggesting a positive modulation of ox-LDL induced proliferation by Co Q10 and UCP2,which also contributes to the anti-proliferation effects of OA.Real time PCR revealed decreased expression level of UCP2 in si-Co Q10 group,increased phosphorylation of ROS in si-UCP2 group and Genipin group(p < 0.05).Si-Co Q10 treatment silenced the expression of Co Q10 m RNA and decreased the expression level of UCP2(p < 0.05),suggesting the positive modulation on UCP2 by Co Q10.Si-UCP2 treatment silenced the expression of UCP2 m RNA and increased the expression level of ROS(p < 0.05),suggesting the negative modulation on ROS by UCP2.Furthermo-re,ox-LDL+OA+ Genipin treatment increased the expression of intracellular ROS and Cyto-C m RNA while the m RNA level of Co Q10 unchanged relative to ox-LDL model group(p < 0.05),suggesting that Co Q10 was located on the upstream of UCP2.Conclusions: Co Q10 was confirmed as a new drug target modulating VSMCs proliferation,which negatively regulates cell proliferation.OA modulates VSMCs proliferation via Co Q10/UCP2/ROS/Cyto-C signaling pathway. |
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