To clarify the suppressive effects of astragalus injection (AI) on different stages of early hepatocarcinogenesis induced by weak promotion, SD rats initiated with a single intraperitoneal (i.p.) injection of N-diethylnitrosamine (DEN) at 200 mg/kg body weight and promoted with 0.5% piperonyl butoxide (PBO) in diet were repeatedly administered AI at 5 ml/kg body weight/day in the early postinitiation (EPI) or late postinitiation (LPI) period for 2 or 8 weeks, respectively. The number and area of glutathione S-transferase placental form (GST-P)-immunoreactive (+) foci tended to increase in the DEN+PBO group compared with the DEN-alone group. Among the PBO-promoted groups, number and area of GST-P+ foci did not visibly change in the DEN+PBO+AI-EPI group compared with the DEN+PBO group. In contrast, number and area of GST-P+ foci tended to decrease in the DEN+PBO+AI-LPI group compared with the DEN+PBO group. Number of Ki67+ cells was increased in the DEN+PBO group compared with the DEN-alone group and was decreased in both AI-administered groups compared with the DEN+PBO group. Gene expression analysis revealed that the DEN+PBO+AI-LPI group showed increased transcript levels of Ccne1, Cdkn1b, Rb1, Bax, Bcl2, Casp3, and Casp9 compared with the DEN+PBO group; however, the DEN+PBO+AI-EPI group did not show changes in the transcript levels of any genes examined compared with the DEN+PBO. These results suggest that AI administration during the LPI period caused weak suppression of hepatocarcinogenesis under weak promotion with a low PBO dose by the mechanism involving facilitation of cell cycle suppression causing G1/S arrest and apoptosis via the mitochondrial pathway. In addition, the results suggest that AI administration during the EPI period has no effect on weakly promoted hepatocarcinogenesis.
Keywords: apoptosis; astragalus injection; cell cycle; cell proliferation activity; two-stage hepatocarcinogenesis.