| Part 1 Effects of Remimazolam on Cerebral Ischemia Reperfusion Injury of RatsObjective Stroke is the second leading cause of death in humans,and the number of deaths is increasing year by year,and the proportion of stroke patients undergoing surgery is also increasing year by year.Perioperative brain protection of stroke patients has always been the focus of anesthesiologists.Remimazolam is a novel intravenous anesthetic.In this study,the effects of remimazolam on cerebral ischemia-reperfusion injury of rats were observed by establishing the rat model of cerebral ischemia-reperfusion injury.Methods Male SD rats aged 7 to 8 weeks were selected to establish the ischemia reperfusion injury model of middle cerebral artery occlusion.They were divided into Sham operation group(Sham group),middle cerebral artery occlusion group(MCAO group),low-dose remimazolam treatment group(MCAO+RL group),medium-dose remimazolam treatment group(MCAO+R_M group)and high-dose remimazolam treatment group(MCAO+R_H group).In Sham group,the right common carotid artery,external carotid artery and internal carotid artery bifurcation were separated,and no intraluminal filament was inserted.In MCAO group,the right middle cerebral artery of rats was embolized and ischemic for 2 h,and the normal saline was immediately pumped through the tail vein after reperfusion.Rats in MCAO+RL group,MCAO+R_M group and MCAO+R_H group were pumped with remimazolam 5 mg/kg,10 mg/kg or 20mg/kg immediately after reperfusion,respectively.Longa nerve defect score was performed 24 h after reperfusion,TTC staining was used to calculate cerebral infarction volume,and transmission electron microscopy was used to observe the ultrastructure of cortical neurons in each group.The expression of NLRP3,ASC,Caspase-1 P20,GSDMD,IL-1β,and IL-18 were detected by immunohistochemistry,RT-PCR,Western blot and ELISA.Results 1.Behavior and morphology: the neurological defect scores of MCAO group,MCAO+RL group,MCAO+R_M group and MCAO+R_H group were significantly higher than those of Sham group after 2 h ischemia(P<0.05),and the scores of MCAO+RL group and MCAO+R_M group were significantly lower than those of MCAO group after 24 h reperfusion(P<0.05).Remimazolam significantly reduced the area of cerebral infarction,especially in MCAO+R_M group(P<0.05).The cell membrane of MCAO group and MCAO+R_M group was damaged,and the damage degree of MCAO+R_M group was lower than that of MCAO group.2.Immunohistochemical results:Compared with Sham group,the expressions of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 were increased in MCAO group(P<0.05).Compared with MCAO group,the expressions of NLRP3,ASC,Caspase-1,GSDMD and IL-18 in MCAO+RL group were decreased(P<0.05),and the expressions of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 in MCAO+R_M group were decreased(P<0.05).The expressions of NLRP3,ASC,GSDMD,IL-1β and IL-18 in MCAO+R_H group were decreased(P<0.05).Compared with MCAO+RL group,the expression of ASC,GSDMD,IL-1β and IL-18 in MCAO+R_M group was decreased(P<0.05),and the expression of ASC in MCAO+R_H group was decreased(P<0.05).Compared with MCAO+R_M group,the expressions of ASC,Caspase-1,GSDMD,IL-1β and IL-18 were increased in MCAO+R_H group(P<0.05).3.RT-PCR results: The NLRP3 mRNA expression in MCAO group and MCAO+R_H group was higher than that in Sham group(P<0.05),and the NLRP3 mRNA expression in MCAO+R_M group was lower than that in MCAO group(P<0.05).The ASC mRNA expression in MCAO group,MCAO+RL group,MCAO+R_M group and MCAO+R_H group was higher than that in Sham group(P<0.05),and the ASC mRNA expression in MCAO+R_M group was lower than that in MCAO group(P<0.05).The Caspase-1 mRNA expression in MCAO group,MCAO+RL group and MCAO+R_M group was higher than that in Sham group(P<0.05),and Caspase-1 mRNA expression in MCAO+R_M group was lower than that in MCAO group(P<0.05).GSDMD mRNA expression in MCAO group,MCAO+RL group,MCAO+R_M group and MCAO+R_H group was higher than that in Sham group(P <0.05).The GSDMD mRNA expression in MCAO+R_M group was lower than that in MCAO group(P<0.05).4.Western Blot results: Compared with Sham group,the protein expressions of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 were increased in MCAO group(P<0.05).Compared with MCAO group,the protein expressions of ASC,IL-1βand IL-18 in MCAO+RL group were decreased(P<0.05),and the protein expressions of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 in MCAO+R_M group were decreased(P<0.05).The protein expressions of Caspase-1 and IL-1β in MCAO+R_H group were decreased(P<0.05);Compared with MCAO+RL group,the protein expressions of NLRP3,ASC,Caspase-1,GSDMD and IL-1β in MCAO+R_M group were decreased(P<0.05),while the protein expressions of Caspase-1 in MCAO+R_H group were decreased(P<0.05),and the protein expressions of ASC and IL-18 were increased(P<0.05).Compared with MCAO+R_M group,the protein expressions of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 were increased in MCAO+R_H group(P<0.05).5.ELISA results: The concentration of IL-1β in MCAO group,MCAO+RL group,MCAO+R_M group and MCAO+R_H group was higher than that in Sham group(P<0.05),and the concentration of IL-1β in MCAO+R_M group was lower than that in MCAO group,MCAO+RL group and MCAO+R_M group(P<0.05).Conclusions 1.Remimazolam can alleviate neuronal injury,cerebral infarction and inflammatory response caused by focal transient cerebral I/R injury of rats,reduced neurological deficit scores,and have ameliorative effect on brain I/R injury in rats;2.The ameliorative effect of remimazolam on cerebral I/R injury of rats was not dose-dependent,and 10 mg/kg had the best effect.Part 2 Effect of Remimazolam on Oxygen-Glucose Deprivation and Reoxygenation Injury in Primary Cortical NeuronsObjective To better understand the protective effect and mechanism of remimazolam on cortical neurons in rats,the present study investigated the effect of remimazolam on primary cortical neurons by establishing in vitro primary cultured cell model of oxygen-glucose deprivation reperfusion reperfusion injury.Methods Primary cortical neurons cultured for 7 days in vitro were divided into control group(C group),drug control group(C+R1 group,C+R10group,C+R20 group,C+R50 group),model group(OGD group)and treatment group(R1 group,R10 group,R20 group,R50 group)with different concentrations of remimazolam.Neurons in C group were cultured normally,and neurons in C+R1 group,C+R10 group,C+R20 group and C+R50 group were cultured for 12 h with 1 μM,10 μM,20 μM and 50 μM remimazolam,respectively,in the neuronal medium for 7 days.OGD group neurons were deprived of oxygen and glucose for 2 h and reoxygenated for 12 h;Neurons in R1,R10,R20 and R50 groups were deprived of oxygen and glucose for 2 h,and were incubated for 12 h with 1 μM,10 μM,20 μM and 50 μM remimazolam,respectively.Neu N immunofluorescence was used to identify the purity of cortical neurons,and cell morphology was observed under light microscope.CCK-8 and LDH were used to detect cell viability and cytotoxicity.Transmission electron microscopy was used to observe the ultrastructure of neurons,and the expressions of NLRP3,ASC,Caspase-1 and GSDMD were detected by RT-PCR and Western Blot.The concentration of IL-1β was determined by ELISA.Results With the increase of remimazolam concentration,normal cortical neurons showed dose-dependent impairment.When the remimazolam concentration was 50 μM,neurons were significantly damaged.CCK-8 results:Normal neuronal activity was not affected when the remimazolam concentration was less than 20 μM.The neuronal activity of OGD group was lower than that of C group and R10 group(P<0.05).LDH results: The release of LDH in OGD group,R1 group,R10 group,R20 group and R50 group was more than that in C group(P<0.05),the release of LDH in R10 group was less than that in OGD group(P<0.05),and the release of LDH in R20 and R50 groups was more than that in R1 and R10 group(P<0.05).Transmission electron microscopy(TEM)results: OGD group had multiple vacuoles,cell membrane blistering and formation of multiple pores,and R10 group had less neuron damage.RT-PCR results showed that the mRNA expressions of NLRP3,ASC,Caspase-1,and GSDMD in OGD group,R1,R20,and R50 groups were higher than those in C group(P<0.05).The mRNA expressions of NLRP3,ASC,Caspase-1,and GSDMD in R10 group were lower than those in OGD group(P<0.05).Western blot results showed that the protein expressions of NLRP3,GSDMD,ASC,and Caspase-1 in OGD,R1,and R50 groups were higher than those in C group(P<0.05).Compared with OGD group,the protein expressions of NLRP3,GSDMD,ASC,and Caspase-1 in R10 group were decreased(P<0.05),and the protein expressions of GSDMD,ASC,and Caspase-1 in R20 group were decreased(P<0.05).Compared with R1 group,Caspase-1 protein expression in R10 and R20 groups was decreased(P<0.05);Compared with R10 group,Caspase-1 protein expression was increased in R50 group(P<0.05).ELISA results: The concentration of IL-1β in OGD,R1,R20,and R50 groups was higher than that in C group(P<0.05),and the concentration of IL-1β in R10 group was lower than that in OGD group(P<0.05).Conclusions 1.In a certain range of concentrations,remimazolam can reduce OGD/R injury of primary cortical neurons cultured in vitro,which may be related to inhibition of NLRP3/ASC/Caspase-1 inflammasome activation,alleviate relative neuronal pyroptosis,and reduction of the inflammatory cascade induced by it.2.Remimazolam has dose-dependent toxicity on cortical neurons,and has toxic effect on cortical neurons when the concentration exceeds 20 μM.Part 3 Effect of NF-κB/NLRP3 Inflammasome Dependent Pyroptosis on Remimazolam Ameliorating Cerebral Ischemia-Reperfusion Injury in RatsObjective The essence of cerebral ischemia-reperfusion injury is the dysfunction and death of brain tissue and cells,but the exact mechanism is still unclear.The intense inflammatory cascade caused by neuronal ischemia and hypoxia after stroke and reperfusion injury after vascular revascularization deserves attention.NF-κB is a key promoter of inflammatory response,and activation of NLRP3 inflammasome is an important signal of progressive amplification of inflammation.In this study,we investigated the role of NF-κB/NLRP3 inflammasome signaling pathway in the improvement of ischemic reperfusion injury of cortical neurons by remimazolam at both animal and cellular levels.Methods SD rats were randomly divided into Sham group,MCAO group,DMSO group,remimazolam treatment group(Re group),NF-κB inhibitor JSH-23 group(JSH group)and NLRP3 inhibitor MCC950 group(MCC group).Rats in the DMSO group were intraperitoneally injected with the same amount of solvent before modeling,rats in the Re group were immediately reperfusion and pumped into the tail vein with 10 mg/kg remimazolam,rats in the JSH-23 group were intraperitoneally injected with 10 mg/kg JSH-23 2 hours before modeling,and rats in the MCC950 group were intraperitoneally injected with 5 mg/kg MCC950 2 hours before modeling.Cell experimental grouping: Primary cortical neurons cultured for 7 days in vitro were divided into C group,C+R10 group,OGD group,DMSO group,R10 group,JSH group and MCC group.Neurons in C group were cultured normally,and neurons in C+R10 group were cultured with 10 μM remimazolam for 12 h.OGD group neurons were deprived of oxygen and glucose for 2 h and reoxygenated for 12 h;In DMSO group,the neurons were immediately added with the same concentration of DMSO solvent and incubated for 12 h.In R10 group,10 μM remimazolam was added to the medium and incubated for 12 h.In the JSH group,10 μM JSH-23 was added and incubated for 2 h during oxygen-glucose deprivation,and JSH-23 was incubated for 12 h after reoxygenation.In MCC group,10 μM MCC950 was added and incubated for 2 h during oxygen-glucose deprivation,and MCC950 was incubated for 12 h during reoxygenation.The expressions of NF-κB,NLRP3,ASC,Caspase-1,and GSDMD were detected by immunofluorescence,RT-PCR and Western Blot,and the concentration of IL-1β was detected by ELISA.Results 1.Cell immunofluorescence results: Compared with C group,the fluorescence intensity of NF-κB p65,NLRP3,Caspase-1,and GSDMD in OGD group,DMSO group and R10 group was increased(P<0.05),and the fluorescence intensity of NF-κB p65 in MCC group was increased(P<0.05).Compared with OGD group,the fluorescence intensity of NF-κB p65,NLRP3,Caspase-1,and GSDMD in R10 and JSH groups was decreased(P<0.05).The fluorescence intensity of NLRP3,Caspase-1,and GSDMD decreased in MCC group(P<0.05).Compared with R10 group,the fluorescence intensity of NF-κB p65,NLRP3 and Caspase-1,in JSH group was decreased(P<0.05),the fluorescence intensity of NF-κB p65 in MCC group was increased(P<0.05),and the fluorescence intensity of NLRP3,Caspase-1,and GSDMD was decreased(P<0.05).Compared with JSH group,the fluorescence intensity of NF-κB p65 in MCC group was increased(P<0.05),and the fluorescence intensity of GSDMD was decreased(P<0.05).2.RT-PCR results of cell experiments: Compared with C group,mRNA expressions of NF-κB p65,NLRP3,ASC,Caspase-1,and GSDMD in OGD group and DMSO group were increased(P<0.05),mRNA expressions of ASC and GSDMD in R10 group were increased(P<0.05).The mRNA expression of NF-κB p65 in MCC group was increased(P<0.05).Compared with OGD group,the mRNA expressions of NF-κB p65,NLRP3,ASC,Caspase-1,and GSDMD in R10 group and JSH group were decreased(P<0.05),while the mRNA expressions of NLRP3,ASC,Caspase-1,and GSDMD in MCC group were decreased(P<0.05).Compared with R10 group,the mRNA expression of NF-κB p65,ASC and GSDMD in JSH group was decreased(P<0.05),and the mRNA expression of NF-κB p65 in MCC group was increased(P<0.05).NLRP3,ASC,Caspase-1,GSDMD mRNA expression decreased(P<0.05);Compared with JSH group,the mRNA expression of NF-κB p65 in MCC group was increased(P<0.05),and the mRNA expression of NLRP3 was decreased(P<0.05).3.Western blot results of cell experiments: Compared with C group,the protein expression of NF-κB p65,NLRP3,ASC,and Caspase-1 p20 in OGD group,DMSO group,and R10 group was increased(P<0.05),and the protein expression of Caspase-1 p20 in JSH group was increased(P<0.05).The expression of NF-κB p65 protein was increased(P<0.05)and NLRP3 protein was decreased(P<0.05)in MCC group.The protein expression of NF-κB p65,NLRP3,ASC,and Caspase-1 p20 in R10 group,JSH group,and MCC group was lower than that in OGD group(P<0.05).Compared with R10 group,the protein expressions of NF-κB p65,NLRP3 and ASC,in JSH group were decreased(P<0.05),and the protein expressions of NLRP3,ASC,and Caspase-1 p20 in MCC group were decreased(P<0.05).Compared with JSH group,the protein expression of NF-κB p65 in MCC group was increased(P<0.05),and the protein expression of NLRP3 and Caspase-1 p20 was decreased(P<0.05).4.Western blot results of rats: Compared with Sham group,the protein expression of NF-κB p65,NLRP3,and ASC in MCAO group and DMSO group was increased(P<0.05).The protein expression of NF-κB p65 was increased in MCC group(P<0.05).Compared with MCAO group,the protein expressions of NF-κB p65,NLRP3,and ASC in Re group and JSH group were decreased(P<0.05),and the protein expressions of NLRP3 and ASC in MCC group were decreased(P<0.05).The protein expression of NLRP3 and ASC in MCC group was lower than that in Re group(P<0.05).Compared with JSH group,the protein expression of NF-κB p65 in MCC group was increased(P<0.05),and the protein expression of ASC was decreased(P<0.05).5.ELISA results of rats showed that IL-1β concentration in MCAO group and Re group was higher than that in Sham group(P<0.05);IL-1β concentration in Re group,JSH group,and MCC group was lower than that in MCAO group(P<0.05);IL-1β concentration in JSH group and MCC group was lower than that in Re group(P<0.05).6.ELISA results of cell experiments showed that the concentrations of IL-1β in OGD,DMSO and R10 groups were higher than those in C group(P<0.05),and the concentrations of IL-1β in R10,JSH,and MCC groups were lower than those in OGD group(P<0.05).The IL-1β concentration in JSH group and MCC group was lower than that in R10 group(P<0.05).Conclusions Remimazolam plays ameliorative effects by inhibiting NF-κB mediated activation of NLRP3/ASC/Caspase-1 inflammasome and decreasing the degree of inflammatory cascade on cerebral ischemia reperfusion injury. |
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