Effects Of Paeoniflorin On Bilirubin-Induced Apoptosis And NF-ΚB Activation Of Hippocampal Neurons

Objective:To observe the effects of paeoniflorin (PF) on bilirubin-induced apoptosis and nuclear transcription factor-κB (NF-κB) activation of hippocampal neurons.Methods:Primary cultures of hippocampal neurons from 17-19 days old embryo rats were used to study. At 7th day of incubation, the hippocampal neurons were randomly divided into normal control group (added DMSO), bilirubin group (added bilirubin of 10μg/ml), and bilirubin with PF preteatment groups which were divided into PF 1 group,PF 2.5 group and PF 5 group. The PF groups were respectively pretreated with different dose of PF (1, 2.5, 5μg/ml) at 24 h before 10μg/ml bilirubin was added. We employed MTT assay and Hoechst33342 DNA staining to detect the effects of paeoniflorin on cytoactivity and apoptosis of hippocampl neurons, and figuring out the appropriate dose of PF used in later experiments. Then, Annexin V-FITC/PI double staining was used to investigate the impact of PF (2.5μg/ml) on apoptosis of hippocampl neurons. Finally, immunocytochemistry was used to dectect the expression of NF-κB protein in hippocampal neurons at different time points (3, 6, 12, 24 h) after bilirubin stimulation.Results:(1) Compared with normal control group, the hippocampl neuronal activity significantly decreased and the neuronal apoptosis rate increased in bilirubin group (P≤0.001).(2) Compared with bilirubin group, PF (1, 2.5, 5μg/ml) pretreating for 24 h significantly decreased the neuronal apoptosis rate to (14.33±3.22)%, (11.00±2.00)% and (15.67±2.31)%, respectively (P<0.01). PF (2.5μg/ml) increased the neuronal activity to (84.32±17.42)% (P <0.01), whereas PF (1, 5μg/ml) pretreating didn't affect on the cytoactivity.(3) Bilirubin induced significant activation of NF-κB in hippocampal neurons (P<0.05), especially when acting 3-6 h (P<0.05). PF (2.5μg/ml) pretreatment significantly suppressed NF-κB activation of hippocampal neurons at 3-6 h after bilirubin adding (P<0.05).Conclusions:(1) Bilirubin-induced NF-κB activation was in a time-dependent manner and reached a peak at 3-6 h, which might be associated with bilirubin-induced apoptosis of hippocampal neurons. (2) PF (2.5μg/ml) preconditioning 24 h inhibited bilirubin-induced apoptosis of hippocampal neurons, which might result from suppression of NF-κB activation at 3-6 h after bilirubin's stimulation.