The Study Of TNF-α-NF-κB Signaling Pathway On Cerebral Ischemia In Rats

ObjectiveTo explore the role of TNF-α-NF-κB signaling pathway on cerebral ischemia(Cerebral ischemia,CI)model in rats.Normobaric oxygen(NBO)therapy was also used as an intervention method to provide theoretical and experimental basis for the new programs of the prevention and treatment.Methods1.MCAO was used to establish a CI model of SD rats to simulate clinical acute cerebral infarction of human.The rats were randomly divided into blank control group(Cont),sham operation group(Sham),cerebral ischemia model group(CI),CI+NF-κB inhibitor BAY11-7082 group(CI+BAY),CI+solvent control 1%DMSO group(CI+DMSO),CI+ normobaric oxygen therapy group(CI+NBO),n=24 in each group.2.Animal behavior evaluation and 2% TTC staining method were used for model verification,and the neurobehavioral ability and cerebral infarction area of each group were compared.3.Using HE staining method and Nissl staining method to observe the morphology and distribution of neuron in the hippocampus CA1 area of each group.4.Using real-time fluorescent quantitative PCR and Western-blot to detect the expression of TNF-α,TNFR1,IκB,NF-κB in the hippocampus of each group of rats,and combined with immunohistochemical staining method to observe and compare the expression and localization of TNF-α in the top cortex and hippocampus CA1 area of each group.Results1.Animal behavioral test results: Compared with the Cont group and Sham group,every model group showed that cerebral ischemia can cause neuromotor function damage(p<0.05).The limb symmetry experiment and bilateral front the paw grasping experiment showed that NF-κB inhibitor intervention can aggravate the contralateral nerve motor function damage of the rat lesion,resulting in further loss of muscle strength(p<0.05),and normobaric oxygen therapy can reduce the damage of the contralateral nerve function of the lesion and promote the recovery of muscle strength(p<0.05).2.2% TTC staining results: Compared with the Cont group and the Sham group,every model group had white infarcts,and there was no significant difference in the area size of infarcts between each model groups(p>0.05),it showed that the cerebral ischemia model was successfully established,and the area size of cerebral ischemia was relatively stable;and neither oxygen therapy nor inhibitor intervention resulted in the significant changes of cerebral infarct size.3.Histomorphology test results: HE staining and Nissl body staining results in the hippocampus CA1 were consistent,both showed: Compared with Cont group and Sham group,the neuron of each model group appear disordered,and some of the cell volume shrinkage,irregular nuclear pyknosis,deep staining of the cytoplasm with glial scar formation,the number of Nissite was reduced,and lighter staining;compared with the CI+DMSO group,the layer number of neuron was obviously reduced in the CI+BAY group,the arrangement was scattered,most of the neuron were degenerated and necrotic,the nucleus were severely deformed and triangular,the cytoplasm was densely stained,the structure were unclear,and the Nissl body were missing;compared with the CI group,the neuron of the CI+NBO group were relatively neatly arranged,a small part of the cells became smaller,the number of cells with unclear cytoplasmic structure and eosinophilic changes were significantly reduced,the number of Nissl bodies increased,and the dyeing were relatively deepened.4.Results of immunohistochemical staining methods: The expression level of TNF-α protein in the parietal cortex and hippocampus CA1 area showed consistent changes.The number of TNF-α immunopositive cells in every model group increased significantly(p<0.05);compared with the CI+DMSO group,the number of TNF-αimmunopositive cells in the hippocampal CA1 area of the CI+BAY group was significantly increased(p<0.05);compared with the CI group,the number of TNF-αimmunopositive cells was significantly reduced(p<0.05)in CI+NBO group.5.Western blot detection results: Compared with Cont group and Sham group,the expression of TNF-α,TNFR1,IκB and NF-κB of protein levels in the hippocampus of each model group were significantly higher(p<0.01);compared with the CI+DMSO group,the expression of TNF-α,TNFR1,IκB and NF-κB of the CI+BAY group were significantly higher(p<0.05);compared with the CI group,the expression of TNF-α and NF-κB of CI+NBO group were significantly reduced(p<0.05),and the TNFR1 expression was significantly increased(p<0.05).6.Real-time fluorescent quantitative PCR detection results: Compared with the Cont group and the Sham group,the expression of TNF-α,TNFR1,IκB and NF-κB of m RNA level in the hippocampus of each model group were significantly higher(p<0.05);compared with the CI+DMSO group,the TNF-α expression of CI+BAY group was significantly reduced(p<0.05),and the expression of TNFR1,NF-κB and IκB were significantly increased(p<0.05);compared with the CI group,the expression of TNF-α,TNFR1 and IκB in the CI+NBO group were significantly higher(p<0.05).Conclusions1.Thread bolt method MCAO can successfully establish a CI model with stable cerebral infarct size in rat.2.The ischemic injury of hippocampal neurons and neurobehavioral dysfunction in CI rats may be related to the high expression level of TNF-α,TNF-α is an important inflammatory effect molecule in the pathological mechanism of CI,and the main expression effect is related to TNF-α-TNFR1-IκB-NF-κB pathway,NF-κB signaling pathway plays an important role in the pathological process of CI inflammation.3.NBO therapy has a certain protective effect on the pathological process of CI.This effect may be achieved by reducing the high expression of the inflammatory factor TNF-α and inhibiting the activation of the TNF-α-NF-κB inflammatory signal pathway.