Oleanolic Acid Inhibits Ox-LDL Induced Cells Proliferation Of Rat Artery Vascular Smooth Muscle Via FGF2/JNK/PON2 Signaling Pathways

Objective: The aim of this study was to investigate the effects of oleanolic acid(OA)on the proliferation of vascular smooth muscle cells(VSMCs)and the expression of FGF2、JNK、PON2,and observe the correlation with the proliferation of VSMCs.To verify that OA inhibits ox-LDL-induced proliferation of VSMCs through FGF2/JNK/PON2 signaling pathways.Methods: The proliferation model was established by ox-LDL,and then VSMCs were randomly divided into different treatment groups: normal group,model group,OA dosage groups(7.5 μM,15 μM,30 μM).The VSMCs with OA pretreatment for 12 h,ox-LDL was added to the cells for 24 h except for normal cells,then the CCK-8 was used to test the proliferation rate.After incubating with ox-LDL at different time point(0,6,12,24,48 h),m RNA levels of FGF2,JNK and PON2 were detected via RT-PCR seperately.Western Blot and RT-PCR were used to detect each groups the expression levels of FGF2 、JNK and PON2 at both protein and m RNA.NO detection kit was used to measure the intracellular NO level.In order to verify upstream and downstream relationships,we used the Stealth si RNA interference technology.The cells were divided into: ox-LDL model group,ox-LDL+ si-FGF2 group,OA group(30 μM),OA + si-FGF2 group,OA + SU5402 group and OA+ SP600125 group.Western Blot was used to detect the gene silencing efficiency and target gene protein expression.Statistical analysis was performed using SPSS 17.0.Results: The proliferation model in VSMCs were successfully induced by ox-LDL at the concentration of 50μg/ml.Compared with the model group,the proliferation rate of VSMCs was significantly decreased in a dose-dependent manner after 12 h of OA incubation(p < 0.05).The proliferation rate of ox-LDL + si-FGF2 group was lower than that of the model group,and OA + si-FGF2 was lower than that of the simple drug group(p< 0.05).This suggesting that FGF2 may be positively correlated with the proliferation of VSMCs.With the weakening of FGF2 expression,OA antiproliferative activity increased.The proliferation rate of VSMCs decreased after adding the inhibitors SU5402 and SP600125 to the OA group,indicating that the proliferation of FGF2 and JNK was related to the proliferation of VSMCs.The results of RT-PCR showed that the expression of FGF2 m RNA in the cells was significantly higher than that in 0h(p < 0.05),while the expression of PON2 m RNA was decreased(p < 0.05),indicating that the expression of these two gene were negatively correlated.The results of RT-PCR and Western Blot showed that the levels of JNK phosphorylation,FGF2 m RNA and protein in the model group were higher than those in the normal group(p < 0.05),while the m RNA and protein expression of PON2 was decreased(p < 0.05).Compared with the model group,JNK phosphorylation,FGF2 m RNA and protein expression in the OA group decreased(p < 0.05),and PON2 expression increased(p < 0.05).With the increase of OA concentration,the trend is more obvious,showing a dose-dependent relationship.The expression of FGF2 and PON2 was consistent with the protein expression in the m RNA.However,there was no significant change in the expression of JNK m RNA,indicating that p-JNK is the active expression form of JNK.This suggesting that OA can inhibit ox-LDL-induced proliferation of VSMCs by down-regulating FGF2 and p-JNK and up-regulating the expression of PON2.After silencing and inhibition of FGF2 using liposome transfection and inhibitor SU5402,Western blot was used to detect the expression of related proteins.The results showed that the expression of JNK phosphorylation was significantly decreased(p < 0.05),indicating that FGF2 can regulate the phosphorylation of JNK,and OA inhibits the expression of JNK by FGF2.After the addition of JNK inhibitor SP600125,the protein level of PON2 was significantly increased(p < 0.05),suggesting that PON2 is regulated by JNK,an upstream molecule.It shows that OA can upregulate PON2 by inhibiting the expression of JNK.Compared with the normal group,the level of NO in the model group was decreased(p <0.05).The level of NO in the OA group was higher than that in the model group(p < 0.05)and was positively correlated with the concentration,suggesting that the OA could be activated NO levels inhibit the proliferation of VSMCs.Conclusions: This study showed that 50 μg/ml ox-LDL significantly promoted the proliferation of VSMCs,and FGF2 and JNK participated in the proliferation of smooth muscle cells,while PON2 inhibits its proliferation.OA can inhibit ox-LDL-induced abnormal proliferation of VSMCs by regulating FGF2/JNK/PON2 signaling pathways.