The Effects And Mechanisms Of NF-κB-mediated JNK Pathway On Cognitive Impairment In A Rat Model Of Sleep Apnea

Objective:The aim of this study was to determine the effects and mechanisms of NF-κB-mediated JNK pathway in chronic intermittent hypoxia-induced cognitive impairment in a rat model of sleep apnea.Methods:96 male Sprague–Dawley rats were randomly assigned to Sham group,chronic intermittent hypoxia（CIH）group,sustained hypoxia（SH）group,CIH+melatonin group,CIH+vitamin E group,CIH+normal saline（NS）group,CIH+Bay11-7082 group and CIH+DMSO group.Rats were exposed to normoxia,CIH（21%O₂for 60 s and 10%O₂ for 60 s,cyclically repeated for 10 h/day）or SH（10%O₂ for 10h/day）with melatonin,vitamin E or Bay 11-7082 pretreatment for 14 days.Afterwards Morris Water Maze Test was conducted for cognitive function evaluation.Levels of oxidative stress in rat hippocampus were tested.Immunoblotting was used to evaluate the activation and inhibition of nuclear factor-κB（NF-κB）and c-Jun N-terminal kinase（JNK）pathway signals.Real-time PCR and ELISA were used to evaluate the expression of tumor necrosis factor-α（TNFα）,IL-6 and inducible nitric oxide synthase（iNOS）.Caspase-3 activity assay and TUNEL assay were used to detect apoptosis.Results:We successfully established a rat model of sleep apnea.In this rat model of sleep apnea we found chronic intermittent hypoxia caused significant oxidative stress in rat hippocampus.Malondialdehyde（MDA）and 8-ISO-PGF2αin hippocampus were significantly increased in the CIH group（P<0.01）;superoxide dismutase（SOD）and glutathione（GSH）level were decreased.Sustained hypoxia did not cause oxidative stress.Antioxidants of melatonin and Vitamin E administrated to rats prior to intermittent hypoxia exposure significantly reduced the increases of MDA and 8-ISO-PGF2αand the decreases of SOD and GSH（P<0.05）.NF-κB inhibitor Bay 11-7082 had on effect on oxidative stress.All rats had improved performance during acquisition according to the lowering in the escape latency over the four training days.On day 1 and day 2,there was no significant difference in escape latency among all the groups,which meant all rats had similar motor and visual capabilities.Compared with the sham group,the escape latency of the rats in the CIH group was significantly longer for days 3 and day 4（P<0.01）.We also found significant differences between CIH and Sham groups with regard to staying time in the target quadrant（P<0.01）.Rats pretreated with melatonin,Vitamin E,or Bay 11-7082 had significantly shorter escape latency for days 3 and day 4 and longer staying time in the target quadrant compared with CIH group（P<0.05）.No statistically significant difference was detectable between Sham group and SH group.To evaluate the activation of NF-κB by chronic intermittent hypoxia,we performed immunoblotting analysis to detect the expression level of P65 in the cytoplasm and nucleus.We found that both chronic intermittent hypoxia and sustained hypoxia could promote the translocation of P65 into the nucleus in rat hippocampus,which was statistically more significant in CIH group.Along with the activation of NF-κB the phosphorylation of JNK and its downstream signaling molecule including c-Jun,ATF2,and JunD were also detected in both CIH and SH group,but the degree of phosphorylation was statistically more significant in CIH group.Pretreatment of vitamin E and melatonin partially inhibited nuclear translocation of P65 induced by chronic intermittent hypoxia and the phosphorylation of JNK and its downstream signaling molecule were lower.Pretreatment of Bay 11-7082 greatly reduced the migration of P65into the nucleus under chronic intermittent hypoxia condition and almost totally suppressed the phosphorylation of JNK and its downstream signaling molecules of c-Jun,ATF2,and JunD.Consistent with the validated activation of NF-κB by chronic intermittent hypoxia,the expression of TNF-αmRNA,TNF-αprotein,IL-6 mRNA,and iNOS mRNA was increased（P<0.01）.Pretreatment of antioxidants of either melatonin or Vitamin E administration prior to intermittent hypoxia exposure significantly reduced these increments suggesting that the activation of NF-κB was induced by ROS from the oxidative stress（P<0.05）.Pretreatment of NF-κB inhibitor Bay 11-7082 reduced TNF-α,IL-6,and iNOS expressions even more（P<0.01）,which had no significant difference with those in sham group.Sustained hypoxia caused the nuclear translocation of P65 as shown by immunoblotting,and it also increased the expressions of TNF-αmRNA,TNF-αprotein,IL-6,and iNOS（P<0.05）,but these increments were significantly lower than those in CIH group（P<0.01）.Since there was no evidence of oxidative stress in SH group,this activation of NF-κB by sustained hypoxia did not appeared be mediated by ROS.Chronic intermittent hypoxia caused apoptosis of hippocampus neurons as proved by the increased activity of caspase-3 and a large number of neurons positive for TUNEL staining.Sustained hypoxia did not induce apoptosis.Pretreatment of vitamin E and melatonin could inhibit the increase of caspase-3 activity induced by chronic intermittent hypoxia and protect hippocampus neurons from apoptosis（P<0.05）.Pretreatment of Bay11-7082 also significantly reduced caspase-3 activity and protected hippocampus neurons from apoptosis.Conclusion:1.Hippocampus had severe oxidative stress in the rat model of sleep apnea.2.Oxidative stress activated NF-κB,caused inflammatory reaction and induced caspases-regulated hippocampus neuron apoptosis.3.Hippocampus neuron apoptosis is responsible for the cognitive impairment in the rat model of apnea.4.T NF-κB-mediated JNK pathway with the involvement of c-Jun,JunD and ATF-2 is at least partially implicated in CIH-induced hippocampus neuron apoptosis and cognitive impairment.5.Inhibition of NF-κB activation provided a therapeutic potential for cognitive impairment in sleep apnea.