Effects Of Oleanolic Acid On Proliferation Of Rat Arterial Smooth Muscle Cells By SIRT1 / PML / AP-1 Signaling Pathway

Objective To observe the proliferation of rat vascular smooth muscle cells(VSMCs) induced by 10% serem and explore the mechanisms of Oleanolic Acid(OA) on inhibiting the proliferation of VSMCs induced by 10% serem through SIRT1/PML/AP-1 signaling pathway. Methods The culture of rat thoracic aorta vascular smooth muscle cells were done by tissue-piece inoculation.The cultured cells were identified by immunofluorescence. The cell toxicity of OA with different concentrations(10, 20, 30, 40, 50, 60 umol/l) is detected by Lactate dehydrogenase(LDH). CCK-8 was used to detecte the proliferation index of VSMCs induced by serem(10%, 20%)and inhibition rate of OA(10, 20, 30 umol/l) on VSMC in different time(6, 12, 24 h)seperately. Cells were divided into four groups: control group, 10% serem model group, treatment group(OA, 10, 20, 30 μmol/L) and positive control group(AG, 50 mmol/L), SIRT1 activator group(SRT1720, 5 umol/L). The expression levels of SIRT1, PML, c-fos, c- jun m RNA was detected by Real-time PCR. After incubating with 10% serem at different time point(0, 3, 6, 12, 24, 48 h) and preincubating with OA(10、20、30 μmol/L)in 12 h, protein levels of SIRT1, PML, c-fos, c-jun were detected by western blot seperately. The expression of c-fos and c-jun was deteccted by gene silence technology for first time after PML gene was suppressed. Results Cells was developed into peak-valley-shaped cultures. The positive expression of intracellular smooth muscle actin was confirmed with immunofluorescence staining, which proved VSMCs was isolated successful. The toxicity of OA on VSMC is 40 umol/L(P < 0.05). Cell proliferation in model group was significantly higher than the control group after incubating 12 h with 10% serem(P < 0.05), so it was decided to choose 10% serem in 12 h to make the model. The proliferation in OA experimental group(10, 20, 30 umol/L) and AG positive control group(50 mmol/L) after incubating 12 h was significantly lower than model group(P < 0.05), the inhibitory effect of OA in each dose on proliferation was dose dependent in manner. The proliferation in SRT1720 group(50 umol/L) after incubating 6 h was significantly lower than model group(P < 0.05). Compared with normal group, SIRT1 m RNA level was significantly decreased(P < 0.05), the m RNA levels of PML, c-fos and c-jun was significantly increased(p < 0.05), compared with model group, SIRT1 m RNA level in OA treatment group(10, 20, 30 μmol/L) was higher, PML、c-fos、c-jun in OA treatment group(10, 20, 30 μmol/L) was lower(p < 0.05). Protein expression of SIRT1 in 10% serem with 3 h, 6 h, 12 h, 24 h, 48 h was significantly decreased, it reached the lowest in 12 h(p < 0.05), protein expression levels of PML, c-fos and c-jun was significantly increased and reached the lowest in 12 h(p < 0.05). Compared with normal group, protein levels of SIRT1 in model group decreased significantly(P < 0.05), however, protein levels of PML, c-fos, c-jun increased significantly(P < 0.05); Compared with model group, the protein expression of SIRT1 in OA treatment group(10, 20, 30 μmol/L), positive control group(AG, 50 mmol/L) and activator SRT1720 group was higher(P<0.05). Compared with model group, protein levels of PML、c fos and c-jun in PML-si RNA group decreased significantly(P < 0.05). Conclusion 10% serum can induce the proliferation of VSMCs; The inhibition effect of oleanolic acid(OA) on smooth muscle cell proliferation to produce resistance to atherosclerosis is through the SIRT1 / PML/AP-1 signaling pathway.