The Study Of Antiproliferative Effect Of Esculetin On Vascular Smooth Muscle Cells And The Signal Transduction Pathways

[Objective]To study the effects of esculetin on the proliferation of cultured rat vascular smooth muscle cells(VSMCs) and to screen a safe, effective and cheap drug for prevention and treatment of vascular proliferative disorders.[Methods]Rat VSMCs were cultured in vitro with different concerntration of esculetin. There were three esculetin treating groups(low, moderate and high dose) and two control groups(solvent and positive control). The effects of esculetin on the growth and proliferation of VSMCs were investigated by light microscopy, cell counting and MTT assay in different time. Western Blot was used to detect ERK1/2, Akt and Caspase-9 in Ras signaling pathway.[Results](1) The effects of esculetin with different dose or in different time on the growth of VSMCs were observed by the light microscope.With the increase of concerntration and time, esculetin caused a marked dose-dependent and time-dependent proliferation inhibitory effect on VSMCs in all the three groups. In addition to this, cell apoptosis was observed in the high-dose group. (2) The effects of esculetin with different dose or in different time on the proliferation of VSMCs confirmed by the cell counting.Compared with the solvent control group, three esculetin groups and the positive control group showed significantly proliferation inhibitory effects. The proliferation inhibition rate of VSMCs in positive control group at 24h, 48h and 72h was 31.8%,91.8%,79.6%, respectively.In low-dose group was 19.9%,52.5%,54.0%, in moderate-dose group was 48.7%,64.6%,89.7%, in high-dose group was 76.9%,87.8%,95.5%. The antiproliferative effect of middle and high-dose drug group were more significant than the positive group(P<0.05)at 24,72h.(3) MTT assay: Compared with the solvent control group, three groups of the drug and the positive group showed significantly proliferation inhibitory effects. The proliferation inhibition rate of VSMCs in positive control group at 24h, 48h and 72h was 33.5%, 89.9%, 75.9%, respectively. In low-dose group was 15.6%, 47.9%, 49.3%, in moderate-dose group was 43.0%, 62.3%, 93.1%, in high-dose group was 69.7%, 82.3%, 97.5%. The antiproliferative effect of middle and high drug group were more significant than the positive group(P<0.05)at 24,72h. 4) ERK1/2, Akt and Caspase-9 in Ras signaling pathway were tested by western blot.Esculetin may strongly inhibit the activation of ERK1/2, Akt and Caspase-9 in a dose-dependent manner.[Creation]1) Compared with the rapamycin control group, Esculetin could prevent restenosis through inhibiting the proliferation of rat aortic VSMCs.2) The antiproliferative effect of esculetin possibly inhibit the activation of Akt and ERK1/2. It contributes esculetin to utilize in clinic.3) Esculetin may activate caspase 9 which can lucubrate promoting apoptosis effect of esculetin.[Conclusion]1) Esculetin effectively inhibit the proliferation of rat aortic VSMCs in vitro with dose and time dependent manner. 2) The antiproliferative effect of esculetin was possibly mediated by inhibiting the activation of Ras-PI3K-Akt and Ras-ERK1/2 signal pathways.