Mechanism Of Rb/E2F1Signaling Pathway In B[a]P-induced Neuronal Apoptosis

[Objective] To study the role of Rb/E2F1-related signaling pathway in B[a]P-induced corticalneuronal apoptosis,[Methods]1. Establishment of infected model in vitro: Neuron cell of1-3day’s SD rats were separatedand cultured in Neurobasal/B27free serum culture system for5days. The primary corticalneuronal cells were exposed to eight groups: the blank control, the DMSO control, the S9control, the Ros control,0.5μmol/L B[a]P,1μmol/L B[a]P,2μmol/L B[a]P.2. Neuronal viability was detected by CCK-8assay.3. Annexin-V/PI double staining was used to detect neuronal apoptosis rats.4. The expression levels of protein of Rb p-Rb[Ser780], p-Rb[Ser807/811], p53, CDK5, E2F1in the primary cortical neuronal cells were detected by Western-blot.[Results]1The result of cortical neuronal activity detected by CCK-8showed, compared with normalcontrol group, the viability of cells exposed to B[a]P decreased14.84%,23.75%and44.48%. Compared with2μmol/L B[a]P group, the activity of2μmol/L B[a]P+Ros groupincreased0.26times.2According to the results of neuron apoptosis rate detected by Annexin-V/PI double staining,we found that, the rate of neuron apoptosis increased in a dose-dependent manner.Compared with blank control groups,the rate of neuron apoptosis in the0.5μmol/L B[a]P,1μmol/L B[a]P,2μmol/L B[a]P group increased3.45,6.79,13.33times. The difference wasstatistically significant. The rate of neuron apoptosis in the2μmol/L B[a]P+Ros groupdecreased42.66%compared with2μmol/L B[a]P group.3The expression of cell cycle-related protein increase depend on the dose of B[a]P. Aftertreated with the dose B[a]P at1μmol/L,2μmol/L the expression of Rb protein increased0.67times and1.83times respectively compared with the blank control group. The level ofRb protein in the2μmol/L B[a]P+Ros group decreased34.03%compared with the2μmol/L group. After exposed to the dose B[a]P at1μmol/L,2μmol/L, the level ofp-Rb[Ser780] is higher than the control group and0.5μmol/L B[a]P group, the levelincreased0.51times and0.62times, respectively. p-Rb[Ser780] in the2μmol/L B[a]P+Ros group decreased16.85%compared with2μmol/L group. The expression ofp-Rb[Ser807/811] proteins in the1μmol/L group,2μmol/L group increased0.48times,0.54times, compared with control group and the difference was statistically significant.Compared with2μmol/L B[a]P group, the level of p-Rb[Ser807/811] proteins decreased3.61%in the2μmol/L B[a]P+Ros group. Compared with blank control group, the level ofE2F1protein increased0.49times,0.62times and0.98times at doses of0.5μmol/L,1μmol/L and2μmol/L, and the difference was statistically significant. The expression ofE2F1protein in2μmol/L B[a]P+Ros group decreased23.21%compared with2μmol/LB[a]P group.4The result of the level of CDK5protein detected by western blotting showed that theexpression of CDK5protein in the1μmol/L B[a]P group,2μmol/L B[a]P group increased0.58times and1.19times respectively compared with blank control group.5The result of the level of P53protein detected by western blotting showed that theexpression of P53protein in the0.5μmol/L group,1μmol/L group,2μmol/L group weresignificantly increased by0.93times,0.98times and1.38times, compared with blankcontrol group. The expression of P53protein in the2μmol/L B[a]P+Ros group decreased20%compared with2μmol/L B[a]P group.[Conclusion]1B[a]P could decrease the vitality of cortical neurons, and induced the cell cycle reentrywhich would lead cortical neurons to apoptosis.2CDK5can regulate transcription factor E2F1by phosphorylation Rb protein and makeneurons to cell cycle reentry under the action of B[a]P. The neuronal apoptosis startedthrough P53-dependent apoptosis signal pathway.3Ros reduce B[a]P-induced neuronal apoptosis by inhibiting phosphorylation of Rb andtranscription factor E2F1in vitro.