| BackgroundsDelayed encephalopathy after acute carbon monoxide poisoning（DEACMP）is a series of neurological and psychiatric symptoms that appear after（2-60 days）a period of vacation recovery in patients with acute carbon monoxide poisoning（ACMP）.Its pathogenesis is complex and is mostly related to tissue ischemia and hypoxia,oxidative stress injury,and immune-inflammatory cascade reactions.Studies have found that the Rho/ROCK signaling pathway can activate p38 MAPK in central nervous system diseases to regulate pro-inflammatory cytokines,such as IL-6 and TNF-α.It indicated that Rho/ROCK signaling pathway may be related to DEACMP immune dysregulation and inflammatory activation.ObjectivesBy establishing DEACMP rat models,to explore whether ROCK kinase inhibitor H1152 can improve the brain injury caused by acute CO poisoning and the expressions of IL-6 and TNF-α downstream of Rho/ROCK signaling pathway in the brain of DEACMP rats at different time points level.This will provide a new target for the treatment of DEACMP nerve injury.Methods1.A total of 80 SPF male SD rats with a standard body weight of 200-220 g were purchased and randomly divided into a model group（CO group,n=70 rats）and a control group（Control group,n=10 rats）.After 1 week of adaptive feeding in independent ventilated cages（IVC）,the rats in the model group were injected with 100 ml/kg of CO gas by intraperitoneal injection for the first time,and then 3 injections with a volume of 50 ml/kg,with an interval of 4 hours each.Control group used the same method to inject equal volume of air.2.The rats in the model group were randomly divided into two groups（CO group,n=30 rats,CO+H1152 group,n=24 rats）.The CO+H1152 group was intraperitoneally injected with ROCK kinase inhibitor H1152 for 5 consecutive days at a dose of 10 mg/kg.Control group and CO group were intraperitoneally injected with equal dose of 0.9%normal saline for 5 consecutive days.3.At week 1,week 2,week 3 after modeling,behavioral experiments such as Y-maze,new object recognition,open field and runner were carried out to detect the motor ability and cognitive function of rats in each group.Demyelination staining were used to detect the neuropathological changes of brain tissue of rats in each group at different time points.Western blot and immunofluorescence were used to detect ROCK2,RhoA and P-P38 related inflammatory factor TNF-α、IL-6 protein expression in rat brain（hippocampus,frontal lobe and striatum）.4.All data were statistically analyzed by SPSS 25.0 statistical software.Independent sample t-test was used for comparison between different groups,and nonparametric test was used for data that did not conform to normal distribution.P<0.05 means the difference is statistically significant.Results1.Changes of behavior,pathology and protein molecules in DEACMP model rats（1）The behavioral results showed that in the open field experiment,compared with the control group,the total distance of rats in the CO group decreased in the first,second and third weeks after CO poisoning（P<0.05）.At the same time,in the runner fatigue experiment,compared with the Control group,the climbing ability of CO group rats showed a downward trend in the first week,and decreased significantly in the second and third weeks（P<0.05）.In the new object recognition experiment,the discrimination index of CO group rats was significantly lower than that of Control group rats in the first,second and third weeks,and the difference between the two groups at three time points was statistically significant（P<0.05）.The results of Y maze experiment were consistent with the results of new object recognition.The time of entering the new opposite arm in CO group was significantly lower than that in Control group（P<0.05）.（2）The pathological results showed that compared with the Control group,the staining results of corpus callosum myelin in the CO group at the first,second and third weeks after CO poisoning showed that the myelin staining in the CO group was uneven,sparse,the staining of corpus callosum became lighter,and the myelin sheath was broken and lost.Through the behavioral results and pathological myelin staining of Control group and CO group,it was found that the clinical and imaging characteristics of CO group rats were similar to DEACMP,indicating that DEACMP model rats were successfully prepared.（3）Western blot results showed that compared with the Control group,the expression levels of RhoA,P-P38 in hippocampus,RhoA,ROCK2,P-P38 and TNF-α in frontal lobe and RhoA,ROCK2,P-P38,IL-6 and TNF-α in striatum were significantly increased in CO group at the first week after CO poisoning.In the second week,the expression levels of RhoA,ROCK2,P-P38,IL-6 and TNF-α in hippocampus,RhoA,ROCK2 and TNF-α in frontal lobe and RhoA,ROCK2,P-P38,IL-6 and TNF-α in striatum were significantly increased in CO group.At the 3rd week,the expression levels of RhoA,ROCK2,P-P38,IL-6 and TNF-α in hippocampus,RhoA,ROCK2,P-P38,IL-6 and TNF-α in frontal lobe and RhoA,ROCK2,P-P38 and TNF-α in striatum were significantly increased in CO group（P<0.05）.（4）The immunofluorescence results showed that compared with the Control group,the immunofluorescence staining results of hippocampal CA1 area in the CO group showed that the average fluorescence intensity of IL-6 and TNF-α increased significantly in the first week,second week and third week after CO poisoning.The results of Western blot and immunofluorescence in Control group and CO group showed that RhoA,ROCK2,P-P38,IL-6 and TNF-α in CO group the protein expression increased or significantly increased at different time nodes,indicating that DEACMP model rats are related to immune imbalance and inflammatory activation mediated by Rho/ROCK signaling pathway.2.Effects of rock inhibitor H1152 on behavior,pathology and protein molecules in DEACMP model rats（1）The behavioral results showed that in the open field experiment,compared with the CO group,the total distance of CO+H1152 group increased in the first week,second week and third week after inhibitor intervention（P<0.05）.At the same time,in the runner fatigue experiment,compared with the CO group,the climbing ability of the CO+H1152 group was significantly enhanced in the second week（P<0.05）.In the new object recognition experiment,the discrimination index of CO+H1152 group was significantly higher than that of CO group in the first and second weeks,and the difference between the two groups was statistically significant（P<0.05）.There was significant difference in the time of entering the maze in the three weeks（P<0.05）.（2）The pathological results showed that compared with CO group,the phenomena of myelin breakage and loss in CO+H1152 group were improved in the first,second and third weeks after administration of inhibitor H1152.（3）Western blot results showed that in the first week after administration of inhibitor H1152,compared with CO group,the expression levels of RhoA,P-P38,TNF-α in hippocampus,TNF-α in frontal lobe and RhoA,P-P38,IL-6 and TNF-α in striatum were significantly reduced in CO+H1152 group.In the second week,the expression of RhoA,ROCK2 and TNF-α in hippocampus,Rho A,ROCK2,P-P38,IL-6 and TNF-α in frontal lobe and RhoA,ROCK2,P-P38,IL-6 and TNF-α in striatum decreased significantly in CO+H1152 group.At the 3rd week,the expression of RhoA,TNF-α in frontal lobe and P-P38 protein in striatum decreased significantly in CO+H1152 group（P<0.05）.In summary,the results of various indicators suggest that ROCK inhibitors interfere with the Rho/ROCK pathway to improve the behavioral phenotype of DEACMP model rats and reduce neuropathological damage.（4）Immunofluorescence results showed that compared with CO group,the immunofluorescence staining results of hippocampal CA1 area in CO+H1152 group showed that the average fluorescence intensity of IL-6 decreased significantly in the first week after the administration of inhibitor,with statistical difference.The average fluorescence intensity of IL-6 decreased in the second and third weeks.The average fluorescence intensity of TNF-α decreased significantly at week 1,week 2 and week 3（P<0.05）.Conclusions1.The pathogenesis of DEACMP is related to the immune imbalance and inflammatory activation caused by Rho/ROCK signaling pathway.2.ROCK inhibitor H1152 can reduce the degree of nerve injury in DEACMP model rats by interfering with Rho/ROCK pathway. |
PDF Full Text Request