Antiulcerogenic Effects of Kolaviron: Inhibition of Apoptosis induced by Indomethacin in the intact Rat Stomach and Rat Gastric Mucosa Cell line

Abstract

The crude extracts of the seed of Garcinia kola (GK) and its constituent biflavonoid, Kolaviron (KV), have been shown to effectively protect the stomach of rats form ulceration induced by HCl/Ethanol mixture as well as indomethacin. However, the anti-ulcerogenic mechanisms are yet to be fully elucidated. Since apoptosis has been identified as a potent factor in ulcerogenesis, the study was designed to examine the anti-apoptotic effect of KV in intact and cultured gastric cells (RGM- 1) of rat. Confluent Rat Gastric Mucosal (RGM-1) cells were challenged with medium containing 500uM indomethacin in the presence or absence of 0.5ug/ml and 5.0ug/ml KV. Cell viability was measured by the MTT [3-(4, 5– dimethylthiazol-2-yl]- 2,5-deiphenyltetrazolium bromide] assay. The effect of the treatments on the cell cycle phases was determined by flow cytometry using a Beckman- Coulter FACScan. Apoptosis was measured as the proportion of cells in the  sub G₀/G₁ phase of the cell cycle. An assessment of the generation of reactive oxygen species (ROS) after treatments was also carried out. Treatment of cells with indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation. Pretreatment of cells with KV significantly suppressed cell death caused by long-term indomethacin treatment. In addition, indomethacin-induced RGM-1 cell apoptosis was evidenced by an increase in the proportion of cells in the sub-G₀/G₁ phase from 6.93 ± 2.56% in the unexposed cells to 30.44 ± 2.56% in cells exposed to 500uM indomethacin (P < 0.001). Treatments with 0.5ug/ml and 5.0ug/ml KV reversed the apoptotic effect of indomethacin. In vivo, pre-treatment of rat with KV significantly reduced indomethacin-induced ulcer formation and total apoptotic score. These results imply that the inhibition of apoptosis may be an important mechanism via which KV protects the stomach from ulcerogenesis.

Keywords: Indomethacin-induced-apoptosis, ulcer, gastric, RGM-1 cell line