Involvement Of ERK/ NF-КB Signal Pathway In Regulation Of Bilirubin-induced Apoptosis Of Hippocampal Neurons

Objective:To investigate the role of extracellular signal-regulated protein kinase (ERK)1/2 and nuclear transcription factors (NF-кB) in the process of bilirubin-induced apoptosis of hippocampal neurons.Methods:Primary cultured neurons were obtained from 17-19-day-old rat embryo hippocampus. When cultured for 8 d, the hippocampal neurons were randomly divided into control group (added DMSO), treated group (added bilirubin) and inhibited group (added PD98059 and then bilirubin). Neuronal viability and morphology were investigated by modified MTT test and fluorescent staining with Hoechst33342. Flow cytometric DNA analysis was performed in order to evaluate the percentage of apoptotic cells. The expression of NF-кB protein in hippocampal neurons were detected by immuocytochemistry.Results:(1) Neuronal viability in treated group is (85.418±1.406)%, significantly lower than that of control group (99.990±2.782)% (P<0.01), but higher than that in inhibited group (63.951±1.148)% (P<0.01).(2) The percentage of apoptotic neurons in treated group is (20.767±1.662)%, significantly higher than that of control group (4.2±1.058)%, but lower than that in inhibited group (35.167±1.041)% (P<0.01).(3) Cells with subdiploid DNA content in treated group is (4.15%), which significantly higher than that of control group (1.62%), but lower than that in inhibited group (5.38%).(4) Moreover, the light density value and positive cell rate of NF-кB protein in control group are (0.157±0.037) and (0.986±0.795)% ,respectively, whereas those in inhibited group are (0.249±0.059) and (2.622±1.552)%,both of which are lower than those of treated group (0.306±0.072) and (6.882±2.626)%, respectively (P<0.01).Conclusion(1) ERK/NF-кB protein signal pathway of hippocampal neuron cultured in vivo is actived by bilirubin.(2) Moreover, inhibition of ERK/NF-кB pathway deteriorates neuron apoptosis induced by bilirubin.