Role Of NF-κB-MMP-9 Signaling Pathway On Delayed Encephalopathy After Acute Carbon Monoxide Poisoning

Objective To establish a rat model of delayed encephalopathy(DEACMP)caused by acute carbon monoxide poisoning.Dynamic observation of morphological changes of hippocampal neurons,the ultrastructure of synapses and the behavioral changes of rats.Investigate the role of nuclear factor-kappa B(NF-kappa B)and matrix metalloproteinase-9(MMP-9)signaling pathways in DEACMP.Explore the possible drug treatment of acute carbon monoxide poisoning besides hyperbaric oxygen therapy,and to provide a new theoretical basis for the prevention and treatment of DEACMP.Method 1.150 healthy(non-cognitive impairment)male SD rats were randomly divided into air group(AC group),CO poisoning group(CO group),pyrrolidine dithiocarbamate(PDTC)+CO poisoning group(PC group),50 rats in each group.Each group was divided into five subgroups according to the first,third,seventh,fourteenth and twenty-first day after exposure,10 rats in each subgroup.2.The DEACMP model was duplicated by modified intraperitoneal injection.The rats in AC group were injected with the same amount of air.The rats in PC group were injected with PDTC 100 mg/kg 1 hour before the duplication of DEACMP model,and intraperitoneally once a day at the same time after the duplication of DEACMP model,until they were executed.3.Morris water maze test was used to detect the learning and memory ability of rats.HE staining was used to observe the morphology of hippocampal CA3 cells.The expressions of NF-kappa B and MMP-9 were detected by immunofluorescence and Western Blot methods.The ultrastructure of synapses was observed by transmission electron microscopy.Result 1.Establishment of rat DEACMP model:(1)Symptoms of CO poisoning: Most of the rats in CO group and PC group suffered from shortness of breath,irritability,wandering around,bumping into rat cages(some rats suffered from lip and nose bruises),and a few of them suffered from depression and limb paralysis.With the addition of CO,some rats suffered from limb convulsions,feces and urinary incontinence,and some of them were retarded,drowsy,even coma and death.The above findings were not found in AC group.(2)Changes of HbCO concentration in rats: the results of dynamic tail vein blood sampling showed that the concentration of HbCO in rat blood could be maintained above 50% within 16 hours.(3)Morris water maze test results(changes in cognitive function): there was no significant difference in average escape latency and number of times of platform crossing between CO group and AC group and PC group at 1d,3d and 7d(P > 0.05).At 14 and 21 days,the average latency of rats in CO group was significantly longer than that in AC group and PC group(P < 0.05),and the number of crossing plateau was significantly reduced(P < 0.05).There was also significant difference between PC group and AC group(P < 0.05).2.Immunofluorescence method: in CO group,the expression of NF-kappa B increased(1d)-rapidly increased(3d)-slowly increased.The expression of MMP-9 increased first after exposure(peak value of 3d)and then decreased.The expression of MMP-9 increased and then decreased(3d to peak)after exposure(7d).The expression of NF-kappa B and MMP-9 in PC group was restricted,and there was significant difference between PC group and CO group and AC group(P < 0.05).3.Western Blot: the expression of NF-kappa B and MMP-9 in hippocampus of rats in AC group at different time points was very little,and there was no significant change(P > 0.05).The expression of NF-kappa B in hippocampus of CO group tended to increase(day 1)-rapidly(day 3).The expression of MMP-9 protein increased first after exposure(peak value of 3 days)and then decreased.The expressions of NF-kappa B and MMP-9 in PC group were lower than those in CO group(P < 0.05),but still higher than those in AC group(P < 0.05).4.HE staining: the apoptotic peak in CO group was delayed to 7-14 days after exposure,and the apoptotic cells in PC group were significantly reduced,which was obvious on the 14 th day.The morphology of neurons in AC group was basically normal at each time point.5.Transmission electron microscope: there was no significant abnormality of neuronal synapses at each time point in AC group.CO group: The synapses of hippocampal neurons did not change significantly in 1-3 days,but swelling of the pre-synaptic and post-synaptic membranes,narrowing and blurring of synaptic space appeared gradually in 7-21 days,most obvious in 14 days.The degree of pathological changes in PC group was between the other two groups.Conclusion 1.The delayed encephalopathy model of acute carbon monoxide poisoning was prepared by modified intraperitoneal injection.The mechanism of poisoning was similar to that of clinic.The success rate of the model was high.The operation process was simple,easy,controllable and reproducible,and CO gas was saved.2.According to the behavioral manifestations of rats after poisoning,the changes of HbCO concentration in vivo,the Morris water maze test results before and after poisoning,and the histopathological changes of hippocampus,we can judge whether the model is successful or not.3.NF-kappa B and its downstream factor MMP-9 participate in the occurrence of DEACMP,play a protective role in the early stage of exposure,and have a damaging effect when overexpressed,which provides a new theoretical basis for the pathogenesis of DEACMP.4.PDTC inhibits the NF-kappa B pathway,although it only partially inhibits the expression of MMP-9 protein,but it can significantly improve the cognitive function of DEACMP rats,which provides a new experimental basis and direction for future research on clinical targeted therapy of DEACMP.