Edaravone Dexborneol Inhibits NLRP3 Inflammasome And Induces Autophagy To Alleviate Cerebral Ischemia-Reperfusion Injury In Rats

Background:Stroke is a common cerebrovascular disease caused by cerebral vascular rupture or thromboembolism,which is divided into hemorrhagic stroke and ischemic stroke,of which ischemic stroke accounts for about 80% of stroke events.Ischemic stroke includes two related pathological processes: ischemic stroke injury and ischemic stroke reperfusion(I/R)injury.With the normalization of intravenous thrombolysis and intravascular thrombectomy,the I/R injury after occlusive vessel recanalization has become a research hotspot.At present,the treatment of I/R injury is very limited,and its therapeutic effect is not satisfactory.Therefore,it is of great value to clarify the exact pathological mechanism of I/R injury and to explore a new neuroprotective strategy after I/R injury.NOD-like receptor protein 3(NLRP3)inflammasome is a multiprotein complex in immune cells,which is mainly involved in neuronal inflammation-mediated cell injury after I/R.Studies have shown that inhibition of NLRP3 inflammasome can induce autophagy enhancement and reduce nerve inflammatory injury.Edaravone dexborneol(C.EDA)is a commonly used drug in the treatment of acute ischemic stroke,which has been proved to have anti-inflammatory and antioxidant cytoprotective effects.However,it is not clear whether C.EDA can effectively reduce the damage of I/R.Objective:The purpose of this study was to explore the protective effect of C.EDA on NLRP3 inflammasome and autophagy through in vivo experiments,so as to provide experimental basis for the treatment of I/R injury.Methods:1.Model construction and confirmation: thread occlusion method was used to establish the rat I/R injury model after ischemic stroke for 90 min and reperfusion for 72 h.And the laser speckle system was used to monitor the changes of cerebral cortical blood flow at 10 min before ischemic stroke,10 min during ischemic stroke and 10 min after reperfusion to ensure the successful establishment of rat I/R model.2.Experimental grouping and intervention: 65 male Sprague Dawley rats weighing250g-280 g were divided into the following three groups: sham operation group(Sham),I/R group and C.EDA treatment group.C.EDA treatment of 3 mg/kg was given immediately after reperfusion,and then was given every 12 h to 72 h.3.Neuroprotection: Zea longa score was used to evaluate the degree of neurological impairment 72 hours after I/R injury.TTC staining was used to detect the cerebral infarct volume after I/R injury.Evans blue dye permeability was used to evaluate the integrity of blood-brain barrier after I/R injury.4.Molecular experiment: Western blot was used to detect the expression of Nuclear factor-kappa B(NF-κB),Phosphorylated nuclear factor-kappa B(p-NF-κB),Domain-like-receptor family pyrin domain-containing 3(NLRP3),Cysteinyl aspartate specific proteinase 1(Caspase-1),interleukin-1β(IL-1β),TIR domain containing adaptor inducing interferon β(TRIF),Inducible nitric oxide synthase(i NOS),Arginase-1(Arg-1)in the cerebral cortex of rats in Sham,I/R and C.EDA groups.The expressions of NF-κB,NLRP3,microtubule associated protein la light chain 3(LC3B),iba-1,i NOS and Arg-1 in the cerebral cortex of rats with ischemia-reperfusion were detected by immunofluorescence method.Ressults:1.During the establishment of I/R model in rats,the blood flow of the injured cerebral cortex decreased significantly within 10 min of ischemia(39.69±7.41%),and increased significantly after reperfusion(73.03±6.35%).Compared with Sham group,the infarct volume,Zea longa score and blood-brain barrier permeability in I/R group were significantly increased(P<0.001).The infarct volume,Zea longa score and blood-brain barrier permeability of rats in C.EDA group were significantly lower than those in I/R group(P<0.05).2.Compared with Sham group,the levels of NLRP3,Caspase-1 and IL-1β in cerebral ischemia reperfusion group were significantly increased in I/R group(P<0.01).The levels of NLRP3,Caspase-1 and IL-1β in the cortex of C.EDA group were significantly lower than those in I/R group(P<0.05).3.Compared with the Sham group,the expression of iba-1,i NOS,Arg-1 and LC3 B in the cerebral ischemia reperfusion side of the I/R group increased(P<0.05).The expression of iba-1 and i NOS was significantly decreased,while the expression of Arg-1 and LC3 B was significantly increased in the cortex of C.EDA group compared with that of I/R group(P<0.05).4.Compared with Sham group,the expression of NF-κB,p-NF-κB was significantly increased and the expression of TRIF protein was significantly decreased in I/R group(P<0.05).The expression of NF-κB,p-NF-κB was significantly decreased and the expression of TRIF protein was increased in the cortex of C.EDA group compared with that of I/R group(P<0.05).Conclusion:Edaravone dexborneol can reduce ischemic stroke-reperfusion injury by inhibiting NLRP3 inflammasome and inducing autophagy,and its mechanism may be related to down-regulating the expression of NF-κB signal pathway protein.