Oleanolic Acid Inhibited The Proliferation Of Vascular Smooth Muscle Cells Through UCP2/FGF-2/p53/TSP-1 Singaling Pathway

Objective: To simulate the oxygenized low density lipoprotein(ox-LDL) induced damage in vascular smooth muscle cells(VSMCs) in rat, and to study the inhibitive effect of oleanolic acid(OA) on the ox-LDL induced proliferation in VSMCs and to explore its mechanism involved in the UCP2/FGF-2/p53/TSP-1 signaling pathway. Methods: Filter out the toxic concentration of OA by LDH cytotoxicity detection method. Screen out the appropriate concentration of ox-LDL to establish the model and administration concentrations by CCK-8. UCP2 inhibitor Genipin, FGF-2 si RNA and p53 activator PRIMA-1 were used to analyze the UCP2/FGF-2/p53/TSP-1 signaling pathway in VSMCs. The cells were divided into five groups including control group, ox-LDL damaged group, low concentration of OA pretreated group, medium concentration of OA pretreated group and high concentration of OA pretreated group. Extract RNAs from the designed groups, and check RNA purify and concentration before RT-PCR.The transcription of UCP2、FGF-2、p53、TSP-1 m RNA were detected by RT-PCR. Extract proteins from the designed groups and check their concentrations before western-Blot. The expression of UCP2 、FGF-2、p53、TSP-1 were analyzed by western-Blot. Give genipin together with 30μM OA to determine whether OA targeted the UCP2/FGF-2/p53/TSP-1 signaling pathway. Results: The toxic concentration of OA screened by lactate dehydrogenase cell toxicity testing method was 40μM. The result of CCK-8indicated that 50μg/ml ox-LDL could induce the maximum proliferation in VSMCs. Therefore 50μg/ml ox-LDL was chosen to induce the damage in the model. The expression of FGF-2 was decreased, p53 and TSP-1 were increased after genipin was added to the ox-LDL damaged group, which indicated that UCP2 was the upstream of FGF-2, p53 and TSP-1. The expression of UCP2 kept unchanged, while the expression of p53 and TSP-1 were enhanced after FGF-2 si RNA was given to the ox-LDL damaged group,which demonstrated that FGF-2 was the downstream of UCP2 and the upstream of p53 and TSP-1. The expression of TSP-1 was elevated but no obverse change were found in UCP2 and FGF-2 after the p53 activator PRIMA-1 was administrated in the ox-LDL damaged group, which showed that p53 was the upstream of TSP-1 and the downstream of UCP2 and FGF-2. The results of RT-PCR showed the transcription of UCP2 and FGF-2 were increased while p53 and TSP-1 were decreased in the ox-LDL damaged group compared to the control group. After the pretreatment of OA, the transcription of UCP2 and FGF-2 were decreased while p53 and TSP-1 were increased, which was an obvious reverse to the effect of ox-LDL. The result of western blot was in accordance with the result of RT-PCR abovementioned. Furthermore, give genipin together with 30μM OA, the expression of the proteins had no significant change compared with given genipin or OA only. Conclusion: 50μg/ml ox-LDL was the appropriate concentration to establish the VSMCs model in rat. The toxic concentration of OA was 40μM.OA could inhibit the proliferation induced by ox-LDL and the inhibitive effect was closely related to the modulation of UCP2/FGF-2/p53/TSP-1 signaling pathway.