The Effect And Mechanism Of NF-κB Signaling Pathway On Apoptosis In Cerebral Ischemia Reperfusion Injury In Mice

Objective: To investigate the effect and mechanism of NF-κB signaling pathway on apoptosis in cerebral ischemia reperfusion injury(CIRI) in mice. Methods: CIRI models were copied by stopping the blood of common carotid arteries in both sides at the same time with noninvasive arteriole clamps. Mice models were divided into 8 groups: 3h, 6h, 12 h, 1d, 3d, 7d, 14 d, 21 d, according to the reperfusion time. The accurate injured region was confirmed by TTC staining. HE staining was used to observe the changes of cell morphology while the nissl staining was used to observe the changes of neuronal function. The expression of NF-κB p65/Rel A was detected by immunohistochemical(IHC) staining. The number of apoptotic cells was detected by TUNEL and the changes of the target gene/protein Bcl-2 and C-myc in NF-κB signaling pathway in injured region was detected by in-situ hybridization(ISH) detection and western blotting. And the TUNEL and ISH detection were used again for the groups in which the NF-κB signaling pathway was controlled by using PDTC. The groups were also divided into 1 day group(1d-PDTC), 7 day group(7d-PDTC), 14 day group(14d-PDTC), according to the reperfusion time. Results: TTC staining: pale areas appeared in each brain coronal slice of the hippocampus with different degrees in each model group, especially in 7d group. HE staining: compared to the normal group, morphology and arrangement of nerve cells in hippocampal CA3 area had no significant difference in the sham-operation group. Pathological changes were observed in the model group, such as nerve cells swelling(3h), glial cells proliferation and inflammatory cells increased(6h), cytoplasmic vacuoles(12h), tissue edema( appeared in 1d group, become serious in 7d group), arranged in loose and disordered(14d), nucleus deformation and nuclear pyknosis(21d). Nissl staining: with the progress of CIRI, appeared to have: cell swelling(3h), Nissl bodies decreased or disappeared(appeared to decrease in 12 h group and followed by a gradual increase, disappeared inseveral neurons when it come to 21d), tissue edema( appeared in 1d group, serious in 7d group), degeneration and necrosis of neuron into vacuole(21d). IHC staining: The CA3 area of hippocampus had a low expression of NF-κB p65/Rel A in normal group and stam-operation group. It started to increase in 3h group, and gradually increased in the subsequent 7 days to the maximum value. In the 3d group, an increasing displacement of NF-κB p65/Rel A into the nucleus can be observed. NF-κB p65/Rel A expression gradually decreased both in the cytoplasm and cell nuclear in the 14 d group and 21 d group, but was still higher than that in the normal group and sham-operation group, the difference was statistically significant(P < 0.05). After the application of PDTC, the expression of NF-κB p65/Rel A in nerve cell nucleus of hippocampal CA3 significantly reduced at different time points.And the average optical density value significantly decreased compared with the model group at the same time. TUNEL, IHC and ISH staining showed that: the number of apoptotic cells, the expression of NF-κB p65/Rel A,the m RNA and protein of Bcl-2 and C-myc in each group significantly increased compared with the normal group and sham-operation group. The number of Bcl-2 m RNA positive cells decreased compared with each model group at the same time [ 1d:( 43.10±3.712) <(76.05±2.964); 7d:(64.05±4.807) <(79.90±3.508); 14d:(42.00±3.309) <(70.00±4.496)] when PDTC was used to restrain NF-κB, while the number of TUNEL [1d:(46.20±3.205) >(29.90±2.292); 7d:(103.95±3.348) >(65.50±3.411); 14d:(116.10±3.093)>(45.55±1.959)] and C-myc m RNA [1d:(109.00±7.609)>(101.40±8.287); 7d:(126.45±9.572)>(109.25±6.206); 14d:(98.00±5.058)>(89.20±5.836)] positive cells increased. The differences were statistically significant(P < 0.05). Conclusion: By the way of stopping the blood of common carotid arteries in both sides at the same time with noninvasive arteriole clamps, a cerebral ischemia reperfusion injury model in mouse can be copied successfully. NF-κB signaling pathway is involved in the pathological process of cerebral ischemia reperfusion injury in mice. Apoptosis happens in the early(3h) of CIRI and is in a sustainable progress for a longer time(21d). NF-κB signaling pathway plays a suppressive role in the regulation of apoptosis in cerebral ischemia reperfusion injury by controling both of Bcl-2 up-regulation and C-myc down-regulation.