Effect of Acteoside on the Cell Cycle Synchronization, Cytoprotection and SCNT Efficiency of Canine Fetal Fibroblasts.

Abstract

Acteoside [2-(3,4-dihydroxyphenylethyl)-1-O-a-L-rhamnopyranosyl-(1/3)-b-D-(4-O-caffeyl)-glucopyranoside] is a phenylpropanoid glycoside compound isolated from the violet flowers of Syringa vulgaris, which is widely distributed in plants. This compound has various biological activities in vitro and in vivo such as anti-oxidative activity, anti-apoptotic, cytotoxicity against various tumor cells and cell cycle synchronization as CDK inhibitor. In this study, we were investigated the effect of acteoside on the cell cycle synchronization, cytoprotection and SCNT efficiency of canine fetal fibroblasts. A primary fetal fibroblast cell line has been isolated and various volume of acteoside (10, 30, 50 micromol/liter) and treatment time (24, 48, 72h) for synchronization of the cell cycle (Exp.1) have been tested. The effect of acteoside in canine fetal fibroblast as ROS scavenger and anti-apoptotic activity (Exp. 2) were estimated. Also, this study was performed to improve the efficiency of Somatic cell nuclear transfer (SCNT) in dogs by treatment with acteoside on the canine fetal fibroblasts (Exp. 3). In Exp.1, no significant differences were found in all groups of various volume and treatment time, but the highest percentage of cells in G0/G1 stage was obtained after 48h of 30 micrcomolar acteoside-treated group (78.3%). This group was higher than contact-inhibition group (74.6%) and similar to serum starvation (79.2%). In Exp. 2, analyze of cell apoptosis and anti free-radical effects using the reactive oxygen species (ROS) by flow cytometry. The results showed that serum starvation group was induced a significantly higher level of ROS compared to the contact-inhibition group and acteoside treated group. In apoptosis production, serum starvation group was generated a significantly more (21.8%) than to the contact-inhibition group and acteoside-treated group (2.6 and 2.9%, respectively). The results indicated that canine fetal fibroblasts can be arrested at G0/G1 stage of the cell cycle and reduced ROS and apoptosis formation by acteoside. In Exp. 3, SCNT efficiency was compared between the contact inhibition group and the 30 micromolar acteoside-treated group. Cleavage rate of acteoside-treated group was 36.1%, which was higher than that of contact inhibition group (20.6%). The percentage of 4-cell and 8-cell embryos observed in acteoside-treated group were 29.5% and 14.8%, respectively, while those observed in contact inhibition group were 11.1% and 3.2%, respectively. These data suggested that canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage of cell cycle. Also, acteoside showed the effect of reducing the formation of ROS and apoptosis, which protects the canine fetal fibroblasts from oxidative stress. The cytoprotective effect of acteoside combined with cell cycle synchronization effect may contribute to positive outcomes on the overall efficiency of canine SCNT. (poster)