Efficacy And Mechanism Of Fasudil Hydrochloride Combined With Hyperbaric Oxygen In The Treatment Of Brain Injury Caused By Carbon Monoxide Poisoning

Objective: To investigate the molecular mechanism and the treatment effect of hydrochloride Fasudil combined with Hyperbaric Oxygen on acute brain injury induced by carbon monoxide(CO)poisoning based on Ras homologous gene(Rho)-Rho-associated-kinases(Rock)signaling pathway.Methods: According to random number table method,a total of 135(7 to 8 weeks)healthy male SD rats were divided into three groups: a normal control group(NC group,n=45),a hyperbaric oxygen therapy group(HBO group,n=45)and a Fasudil combined with HBO treatment group(Fasudil+HBO group,n=45).Rat models of acute severe CO poisoning were established in the HBO group and Fasudil+HBO treatment group by inhalation method in a hyperbaric oxygen chamber.All rats received hyperbaric oxygen therapy until killed.Rats in the Farsudil+HBO group were intraperitoneally injected with hydrochloride Farsudil for intervention [15 mg/(kg·d),once a day for 2 weeks],while those in the HBO group and NC group received the same volume of normal saline.The neurobehavioral scores of rats in each group were evaluated by Morris water maze test and shuttle box test at 1 day,7 days,1 month and2 months after modeling.The histopathological changes were observed in rat brain tissue by HE staining.The ultrastructures of brain tissues were observed by transmissionelectron microscopy at 7 days after modeling.Immunohistochemical staining and immunofluorescence staining were used to detect the staining intensity of neurite outgrowth inhibitor(Nogo),oligodendrocyte myelin glycoprotein(Omgp)and Rock positive cells in the brain tissue of rats in each group.The protein expressions of Nogo,OMgp and Rock in brain tissues were detected by Western blotting.Results: Compared with the NC group,the escape latency was significantly prolonged,the T1/T total was obviously decreased,and the escape time and active escape time were significantly prolonged on the first day after CO poisoning in the HBO group,which was more significant in the later stage of poisoning(7 days to 2months),and the difference was statistically significant(P< 0.05).Compared with the HBO group,the escape latency was significantly shortened,the T1/T total was gradually increased,and the escape time and active escape latency were significantly shortened after hydrochloride Fasudil treatment(>7 days),and the difference was statistically significant(P< 0.05).After HE staining,the structure of nerve cells in the NC group was intact,the nucleus was blue,the cytoplasm was purplish red,the outline was clear,neatly arranged and stained evenly.Compared with the NC group,the arrangement of nerve cells in the HBO group was disordered,the nucleus was pyknotic and the staining was deeper.In the Fasudil+HBO group,normal cells and abnormal cells were intertwined and coexisted.In the HBO group,the ultrastructures of brain tissues and blood-brain barrier were obviously damaged,and the changes in nucleus,mitochondria and synaptic structure were significant,while treatment of hydrochloride Fasudil could effectively maintain the integrity of ultrastructures and functions of brain tissues,and reduce brain edema.On 1 day,7 days,1 month and 2months after modeling,the staining intensity of Nogo,OMgp and Rock positive cells and protein expression levels of Nogo,OMgp and Rock in the HBO group were significantly higher than those in the NC group at the same time point(P<0.05);the staining intensity of Nogo,OMgp and Rock positive cells and protein expression levels of Nogo,OMgp and Rock in the Fasudil+HBO group were significantly lower than those in the HBO group at the same time point(P<0.05).Conclusion: CO poisoning can cause cognitive impairment,and hydrochloride Fasudil treatment can significantly improve the cognitive function of rats.The activation of Nogo-OMgp/Rho-Rock signaling pathway related molecules(Nogo,OMgp and Rock)is closely related to the acute brain injury caused by CO poisoning;hydrochloride Fasudil can effectively down-regulate the protein expressions of Nogo,OMgp and Rock,therefore obviously alleviate brain injury after CO poisoning.