Biphasic activation of nuclear factor-κB and expression of p65 and c-Rel following traumatic neuronal injury

Abstract

The transcription factor nuclear factor-κB (NF-κB) has been shown to function as a key regulator of cell death or survival in neuronal cells. Previous studies indicate that the biphasic activation of NF-κB occurs following experimental neonatal hypoxia-ischemia and subarachnoid hemorrhage. However, the comprehensive understanding of NF-κB activity following traumatic brain injury (TBI) is incomplete. In the current study, an in vitro model of TBI was designed to investigate the NF-κB activity and expression of p65 and c-Rel subunits following traumatic neuronal injury. Primary cultured neurons were assigned to control and transected groups. NF-κB activity was detected by electrophoretic mobility shift assay. Western blotting and immunofluorescence were used to investigate the expression and distribution of p65 and c-Rel. Reverse transcription-quantitative polymerase chain reaction was performed to assess the downstream genes of NF-κB. Lactate dehydrogenase (LDH) quantification and trypan blue staining were used to estimate the neuronal injury. Double peaks of elevated NF-κB activity were observed at 1 and 24 h following transection. The expression levels of downstream genes exhibited similar changes. The protein levels of p65 also presented double peaks while c-Rel was elevated significantly in the late stage. The results of the trypan blue staining and LDH leakage assays indicated there was no sustained neuronal injury during the late peak of NF-κB activity. In conclusion, biphasic activation of NF-κB is induced following experimental traumatic neuronal injury. The elevation of p65 and c-Rel levels at different time periods suggests that within a single neuron, NF-κB may participate in different pathophysiological processes.

Figure 1

NF-κB DNA-binding activity in cultured neurons was determined by EMSA in the control group and at 1, 6, 12, 24, 36 and 48 h following transection. (A) EMSA results and (B) quantitative analysis reveal that NF-κB activity in the transection groups was significantly increased compared with that in the control group, presenting double peaks at 1 and 24 h post-injury, ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control group. Bars represent the mean ± standard error of the mean (n=6 per group). NF-κB, nuclear factor-κB; EMSA, electrophoretic mobility shift assay.

Figure 2

Western blotting was performed to detect the nuclear protein levels of p65 and c-Rel in cultured neurons. (A) Representative western blots show the p65 and c-Rel protein levels of the neurons in all groups. Histone 3 was used as the loading control. Nuclear protein levels of (B) p65 and (C) c-Rel in the cultured neurons were markedly increased in the transection groups compared with those in the control group, showing different activated phases. The p65 protein levels exhibited two peaks at 1 and 24 h, while c-Rel levels were significantly elevated during the later stage post-injury. Data are presented as mean ± standard error of the mean (n=6 per group). ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control group.

Figure 3

Representative photomicrographs of the fluorescent double staining of NeuN (red), p65 (green) or c-Rel (green). Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. The colocalization of NeuN with p65 or c-Rel was demonstrated. In the control groups, p65 and c-Rel displayed staining mostly in the cytoplasm but weak or no staining in the nuclei. In the 1 and 24 h groups, p65 accumulated in the nuclei, whereas c-Rel was observed to accumulate in the nuclei in the 24 h group. NeuN, neuronal nuclei.

Figure 4

The mRNA levels of NF-κB downstream genes were detected by reverse transcription-quantitative polymerase chain reaction. (A) TNF-α mRNA expression was increased significantly in the 6 and 24 h groups, showing a biphasic increase. (B) IL-1β mRNA was markedly elevated in the 12 and 36 h groups. (C) Caspase-3 mRNA expression also exhibited two peaks in the 1 and 24 h groups. (D) Bcl-2 mRNA expression was markedly increased in the 1 and 24 h groups. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control group. Bars represent the mean ± standard error of the mean (n=6 per group). NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; Bcl-2, B-cell lymphoma 2.

Figure 5

LDH release assay was performed for the assessment of neuronal injury. (A) Significant increases in LDH release were observed in all groups following transection injury compared with the control group. During the late stages, LDH release exhibited no marked further increase. (B) Trypan blue staining was performed to evaluate neuronal death. Following transection injury, the viability of the neurons initially decreased markedly while in the later stages, neuronal viability decreased only very slowly. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control group. Bars represent the mean ± standard error of the mean (n=6 per group). LDH, lactate dehydrogenase.