AIM: To study whether caspase-3,8 is activated during azurin-induced apoptosis in U2OS cells. METHODS: AnnexinV /PI method was used to detect apoptosis. The changes of procaspase-3 were analyzed by Western blot, the changes of caspase-3 mRNA were detected by semi-quantitative RT-PCR, and caspase-3 relative activity was determined by colorimetric assay. RESULTS: After U2OS cells were treated with 0, 25, 50, 100, 200, 500 mg/L azurin for 24 h, respectively, the level of procaspase-3 protein decreased and the level of caspase-3 mRNA increased as azurin concentration increased. When the cells were treated with 100 mg/L azurin for 6, 12, 24, 48 h , respectively, the caspase-3 activity began to rise from 6 h,reached the peak at 24 h,and was still higher than the control group at 48 h ( P

OBJECTIVE Disturbance of K+ ion balance in inner ear is associated in age-related hearing loss. Our study is to investigate the role of NKCC1 and Na-K-ATPase in cochlea and auditory function regulated by with different expression of NKCC1 and Na-K-ATPase. METHODS Auditory threshold of young or old C57BL/6J mice was measured by auditory brainstem response(ABR). The expression of NKCC1 and Na-K-ATPase in mice cochlea were evaluated by reverse transcription polymerase chain reaction(RT-PCR) and western blotting. Furosemide and Ouabain were applied in vivo to inhibit NKCC1 and Na-K-ATPase in C57BL/6J mice. RESULTS C57BL/6J mice developed hearing loss at 12M by ABR threshold shifting to (75±10), (78±26) and (81±14)dB SPL at frequencies of 8, 16 and 32 kHz; PCR showed that the relative expression of NKCC1 and Na-K-ATPase mRNA in the aged group decreased, which were 0.52±0.06 and 0.35±0.04 times higher than those in the young control group, the difference was statistically significant(t =7.466 and 16.11, all P＜0.05). WB showed that relative expression of NKCC1 and Na-K-ATPase protein level in the aged group decreased by 0.79±0.02 and 0.68±0.05 times as much as that of the young control group, the difference was statistically significant(t =8.857 and 6.771, P all＜0.05). After applied with Furosemide and Ouabain to suppress the two ion transporters, the ABR threshold increased to (50±17), (53±21), (55±17)dB SPL and (56±6), (70±17), (73±6)dB SPL at frequencies of 8, 16 and 32 kHz. CONCLUSION In vivo experiment of C57BL/6J suggested that NKCC1 and Na-K-ATPase might be related to age related hearing loss.