Prevention of apoptosis by toki-shakuyaku-san TJ-23 I in cultured cerebellar granule cells

The effect of TJ-23 on cultured cerebellar granule eel is was examined. In 5mM KC1 BME (5K) , the cells were grown 4 days in vitro (DIV); a rapid neuronal death with DNA fragmentation occurred in the following days. At 8DIV there was only 20% of neuronal survival (PDA/PI stain). While in 25mM KCl BME (25K) cultures, there was 89% neuronal survival without DNA fragmentation. Observations of phase-contrast microscopy and DNA electrophoresis indicated that neuronal death in 5K was through apoptosis. Adding 0.05 mg/ml of TJ23 into the 5K BME at 2DIV brought about 70% neuronal survival and prevented nuclear condensation and DNA fragmentation. To compare the function of DNA in each cultured condition, we examined the production of c-fos protein at 8DIV with SDS-page. 25K cultures demonstrated c-fos protein production; however, 5K cultures did not. Treatment with 0.05 mg/ml of TJ-23 produced c-fos proteins in 5K cultures. Data indicated that TJ-23 promotes neuron maturation and prevented apoptosis. To confirm these results, we designed the following experiment : cultured medium was changed to 5K from 25K at 7DIV, and we observed neuronal death (apoptosis} at 24, 48 and 72h later. Neuronal survival was 35% with nuclear condensation and DNA fragmentation at 72h later. Adding 0.05 mg/ml of TJ-23 to the 5K BHE brought about 65% neuronal survival and prevented nuclear condensation and DNA fragmentation. Results confirmed the anti-apoptosis effects of TJ-23 in maturated neurons. This study inferred the potential of TJ-23 on the prevention of apoptosis and supported the possibility of TJ-23 being a remedy of therapeutics for Alzheimer's disease.