The effect of the nuclear factor-κB inhibitor on the focal cerebral ischemia reperfusion injury in the hippocampal CA1 region of rats

Abstract

Objective To observe the effect of the nuclear factor (NF)-кB inhibitor on the inflammatory injury and the secondary remote damage in remote areas of the CA1 region in the right hippocampus of the focal cerebral ischemic/reperfusion rats, and the NF-кB essential modifier binding domain (NBD) peptide was used to inhibit the activation of NF-кB signaling pathway to explore the function and mechanism of the NBD peptide in restraining inflammatory injury and reducing secondary remote damage in the hippocampus CA1 region. Methods According to the random number table, the male Sprague-Dawley (SD) rats were randomly divided into a sham group (n=24), an ischemia/reperfusion (I/R) group (n=38), a NBD group (n=38) and a modified type peptide ( MT-NBD ) group (n=38), then at the time point of 24 h and 7 d after reperfusion, the above 4 groups were divided into 2 subgroups. The experimental models were made by middle cerebral artery occlusion (modified line plug method) for 2 hours. The NBD peptide and the MT-NBD peptide were respectively injected into the right hippocampus of the experimental groups. The injury of neurons was examined by the methods of H&E and Fluoro-Jade B-(FJB) staining. The levels of interleukin-1β (IL-1β) and IL-1Ra were detected by using enzyme-linked immunosorbent assay. The protein expressions of NF-κB p65 and IκBα were analyzed by Western blotting and the double-labelling immunofluorescence. Results Compared with the NBD group (24 h 0.206±0.013, 7 d 0.090±0.012) and the sham group (24 h 0.120±0.007, 7 d 0.100±0.014), the NF-κB p65 protein expression in the I/R group (24 h 2.340±0.101, 7 d 2.440±0.081) was increased significantly (q=64.431, 66.704, 67.747, 56.624, all P<0.05). The level of IL-1β was remarkably increased in the I/R group (24 h (1.850±0.192) ng/ml, 7 d (1.000±0.178) ng/ml) compared with the NBD group (24 h (1.250±0.211) ng/ml, 7 d (0.560±0.183) ng/ml, q=10.730, 9.710, P<0.05). The percent of survival neurons was significantly lower in the I/R group (24 h 27.50%±3.59%, 7 d 28.10%±4.46%) and the MT-NBD group (24 h 27.30%±4.53%, 7 d 26.30%±5.03%)than the NBD group (24 h 58.90%±3.46%, 7 d 68.40%±4.20%, q=19.949, 19.731, 2.139, 22.249, all P<0.05). The FJB staining showed that the number of neuron degeneration in the I/R group (24 h 28.10±2.13, 7 d 29.50±2.45) was higher than the NBD group (24 h 12.50±2.41, 7 d 9.30±2.52, q=3.211, 4.521, P<0.05). Compared with the other three groups (sham group: 24 h 0.130±0.008, 7 d 0.150±0.010; I/R group: 24 h 1.340±0.213, 7 d 1.750±0.119; MT-NBD group: 24 h 1.250±0.114, 7 d 1.620±0.097), the IκBα protein expression in the NBD group (24 h 1.680±0.148, 7 d 2.010±0.085) was significantly increased (q=6.348, 9.139, 9.414, 1.711, 5.277, 5.555, all P<0.05). Compared with the I/R group (24 h (0.570±0.028) ng/ml, 7 d (0.430±0.039) ng/ml) and the MT-NBD group (24 h (0.490±0.042) ng/ml, 7 d (0.380±0.018) ng/ml), the level of IL-1Ra in the NBD group (24 h (1.390±0.055) ng/ml, 7 d (1.250±0.043) ng/ml) was remarkably increased (q=4.577, 6.205, 9.683, 6.389, all P<0.05). The results between the I/R group and the MT-NBD group were not significantly different. Conclusions The research shows that NBD peptide treatment contributes to altering the NF-κB p65/IκBα expression in nucleus effectively. And it directly regulates the NF-κB activation to alleviate the inflammatory injury in the hippocampus CA1 region after the secondary remote damage. Key words: Brain ischemia; Reperfusion injury; Hippocampus; Peptides; Transcription factor RelA; I-κB proteins