| Background and Objective:Cerebral Stroke is a common clinical emergency,and its incidence is increasing year by year.The current pharmacological treatment for ischemic stroke is mainly timely thrombolysis and restoration of blood perfusion to the ischemic focus.However,as Ischemia-Reperfusion Injury(IRI)after restoration of blood flow often causes more severe brain tissue and nerve cell damage,which further becomes an important factor leading to poor prognosis and functional impairment of patients.The exact mechanism of cerebral ischemia-reperfusion injury is still unknown.The phenomenon of pyroptosis was first observed in Shigella fowler-infected macrophages.This cell death was first referred to as apoptosis because it shares certain features with apoptosis,such as DNA fragmentation,nuclear sequestration,and dependence on caspase pathway activation.In 2005,Susun L first defined caspase-1-dependent programmed cell death separately and officially named it as pyroptosis.Pyroptosis is a programmed cell death triggered by pathogenic microbial infection or other danger signals and accompanied by an inflammatory response,characterized by the occurrence of cell swelling,lysis and release of cytoplasmic contents,which in turn is associated with altered cell membrane permeability due to the aforementioned inflammatory response.pyroptosis has now been found in ischemia-reperfusion injury in organs such as the heart and kidney,but it is still inconclusive whether pyroptosis plays a role in tissue cell injury during cerebral ischemia-reperfusion.Propofol,a commonly used intravenous anesthetic drug,is widely used for induction and depth maintenance during general anesthesia,and also as a sedative required for regional anesthesia and for sedation of patients undergoing clinical examinations,for painless gastroscopy,painless bronchoscopy,and other examinations.In addition to its characteristics of rapid induction,smooth maintenance,and rapid awakening,propofol has also been found in clinical work to have many advantages outside the field of anesthetic sedation,i.e.,non-anesthetic efficacy.In recent years,studies on the non-anaesthetic efficacy of propofol have focused on its anti-inflammatory and antioxidant effects.Studies have shown that propofol has antioxidant effects,reduces intracellular calcium overload,inhibits apoptosis,reduces neutrophil and endothelial cell adhesion,regulates the balance of inflammatory cytokines,and directly improves impaired cellular energy metabolism.In terms of inflammation regulation,propofol can reduce the transfer of NF-k B into the nucleus by inhibiting TLR-4 expression in endotoxemia,thereby inhibiting CD14 gene expression and phosphorylation of Ik B,which in turn reduces cytokines such as IL-1?,TNF-a,and IL-18 in plasma and tissues.Current studies suggest that potential mechanisms for the protective effects of propofol include inhibition of activation of pro-inflammatory factors and counteracting oxidative stress.Regarding inflammation,some previous probes found that the degree of cortical damage and inflammatory response in rats,a model of brain injury,was positively correlated with the expression and activation of NLRP1 inflammatory vesicles in their cortex.At the same time,propofol can reduce the inflammatory response by down-regulating the expression and inhibiting the activation of NLRP1 inflammatory vesicles,alleviating its damage to cortical tissue.Therefore,it is generally accepted that propofol has antiinflammatory effects to reduce tissue and cellular damage from inflammatory responses occurring in the central nervous system and has become an attractive and popular candidate for the treatment of stroke.But does propofol's ability to counteract the local inflammation that occurs in the nervous system? Can it exert neuroprotective effects through this action? Whether this therapeutic effect can be achieved through the mechanism of reducing cellular pyroptosis by inhibiting the activation of inflammatory vesicles NLRP1-caspase-1 is unclear.Therefore,the aim of this study was to simulate the ischemia-reperfusion injury in tissues and cells through an animal model and a cellular assay of Oxygen and glucose deprivatiord Reperfusion process.By clarifying the activation of inflammatory response and the involvement of cellular pyroptosis in neural tissue and neuronal cells during this pathophysiological process,and exploring the underlying mechanisms.We also explored the specific protective mechanism of propofol on neuronal cells damaged by ischemia-reperfusion god by observing the protective effect of propofol on cerebral ischemia-reperfusion injury.Methods:1.Animal modeling of stroke and evaluation of brain injury: an animal model of cerebral ischemia-reperfusion injury in C57BL/6J mice was established using the middle cerebral artery occlusion method,and neurobehavioral changes were evaluated in mice after cerebral ischemia-reperfusion injury using neurobehavioral scores,and cerebral infarction was calculated by TTC staining Volume,wet weight/dry weight method was used to evaluate the degree of cerebral edema.2.Assessment of local inflammation in animal-modeled brain tissue in stroke:Realtime-PCR and Western Blot techniques were used to detect the gene and protein expression of NLRP1 inflammatory vesicles,cleaved caspase-1 and its downstream inflammatory factors cleaved IL-1? and cleaved IL-18 in brain tissue treated with simulated cerebral ischemia-reperfusion at different time points.expression changes at different time points.The role of caspase-1 in the process of ischemia-reperfusion injury of brain tissue and neuronal cells was investigated using caspase-1 inhibitor Ac-YVAD-CMK to inhibit caspase-1 activation.3.Assessment of pyroptosis death in animal-modeled brain tissue in stroke:Western Blot technique and immunofluorescence technique were used to detect the expression level and localization of GSDMD-N protein,an indicator protein for pyroptosis pathway activation,in tissues to clarify the presence of pyroptosis in neural tissues,and the use of caspase-1 inhibitor Ac-YVAD-CMK to inhibit caspase-1activation to explore the key role of caspase-1 in the focal death of ischemiareperfused neuronal cells.X4.Cell modeling and neuronal cell injury assessment in stroke: Oxygen and glucose deprivatiord/Reperfusion modeling of ischemia-reperfusion injury neuronal cells,CCK-8 method and lactate dehydrogenase(LDH)content assay to assess hypoxia-reperfusion.The survival rate of primary neuronal cells in mice after hypoxia-reoxygenation treatment was assessed by CCK-8 and lactate dehydrogenase(LDH)content measurements.5.Cytomodelling and neuronal cell inflammation assessment in stroke: detection of NLRP1 inflammatory vesicles,cleaved caspase-1 and its downstream inflammatory factors cleaved IL-1?,cleaved IL-18 genes in OGD/R neuronal cells by Realtime-PCR,Western Blot technique,ELISA and protein expression changes at different time points.Inhibition of caspase-1 activation using caspase-1 inhibitor AcYVAD-CMK to investigate the role of caspase-1 in the OGD/R process in neuronal cells.6.Cellular modeling in stroke and assessment of neuronal cell pyroptosis:Western Blot technique was used to detect the expression level of GSDMD-N protein,an indicator protein for pyroptosis pathway activation,to clarify the presence of pyroptosis in neuronal cells.caspase-1 inhibitor Ac-YVAD-CMK was used to inhibit caspase-1 activation and to explore the the critical role of caspase-1 in OGD/R neuronal cells.7.Assessment of the neuroprotective effects of propofol in stroke-molded animals: neuroprotective effects on mice with cerebral ischemia-reperfusion injury after intervention with propofol were examined by neurobehavioral scoring,water maze test,cerebral infarct volume measurement,and brain edema degree measurement.8.Assessment of the inflammatory response and pyroptosis effect of propofol on neural tissue in stroke-molded animals: the effect of propofol pretreatment on NLRP1 inflammatory vesicles,cleaved caspase-1 and its downstream inflammatory factors cleaved IL-1 ?,cleaved IL-18 and GSDMD-N in brain tissue of IRI mice was examined using Western Blot technique.9.Exploration of the protective effect and mechanism of propofol on stroke modelling cells: silencing NLRP1 gene,observing the protective effect of propofol on hypoxic-reoxygenated neuronal cells,silencing caspase-1 gene under the effect of propofol intervention and OGD/R,observing the focal death of neuronal cells,and exploring the mechanism of the effect of propofol on hypoxic-reoxygenated neuronal cells.Results:1.The results of in vivo experiments showed that mice with reduced neurobehavioral scores,brain tissue infarction and severe brain edema after cerebral ischemia-reperfusion,and improved neurobehavioral scores,reduced infarct size and brain edema after using caspase-1 inhibitor Ac-YVAD-CMK.2.A dramatic inflammatory response occurs after cerebral ischemia-reperfusion in mice,and NLRP1,cleaved caspase-1,a key factor in the pyroptosisdeath pathway,and its downstream inflammatory factors cleaved IL-1? and cleaved IL-18 are elevated early in reperfusion and continue to rise over time,slowly decreasing after reaching a peak.The inflammatory response was attenuated with the caspase-1inhibitor Ac-YVAD-CMK.3.Neuronal cell pyroptosis occurred early in the reperfused mouse brain tissue and increased gradually over time,decreasing slowly after reaching the peak.4.The results of cellular experiments showed that neuronal cell death rate increased and LDH content increased after OGD/R,and that neuronal cell death rate decreased and LDH content decreased after using caspase-1 inhibitor Ac-YVADCMK.5.NLRP1,cleaved caspase-1 and its downstream inflammatory factors cleaved IL-1? and cleaved IL-18 were elevated in neuronal cells after OGD/R at the early stage of reoxygenation and continued to increase over time,slowly decreasing after reaching a peak.Use of caspase-1 inhibitor Ac-YVAD-CMK attenuates the inflammatory response.6.GSDMD-N protein in post-OGD/R neuronal cells is elevated early in reoxygenation,continues to rise over time,and slowly decreases after reaching a peak.The use of caspase-1 inhibitor Ac-YVAD-CMK attenuated neuronal cell pyroptosis.7.Animal experiments confirmed that the use of propofol improved neurobehavioral scores,learning,and memory impairment after cerebral ischemiareperfusion in mice,and reduced brain edema after cerebral ischemia-reperfusion injury in mice.8.Assessment of the effects of propofol on neurological tissue inflammatory response and pyroptosis in stroke-molded animals: detection of the effects of propofol pretreatment on NLRP1 inflammatory vesicles,cleaved caspase-1 and its downstream inflammatory factors cleaved IL-1?,cleaved IL-18 and GSDMD-N in brain tissue of IRI mice using Western Blot technique.9.Exploration of the protective effect and mechanism of propofol on stroke modelling cells: silencing NLRP1 gene,observing the protective effect of propofol on hypoxic-reoxygenated neuronal cells,silencing caspase-1 gene in the presence of propofol intervention and OGD/R,observing the pyroptosis of neuronal cells,and exploring the mechanism of the effect of propofol on hypoxic-reoxygenated neuronal cells.Conclusions:1.Cerebral ischemia-reperfusion injury can cause neurological dysfunction and neuronal inflammatory response in mice.Its process revealed brain tissue NLRP1 inflammatory vesicles with caspase-1 activation-mediated neuronal pyroptosis.2.Propofol attenuated ischemia-reperfusion injury in brain tissue of MCAO mice and attenuated neuronal inflammatory response after OGD/R.3.Propofol can inhibit cellular pyroptosis during neuronal hypoxiareoxygenation by inhibiting the NLRP1-caspase-1 pathway.It is associated with its ability to alleviate neural injury and reduce neuronal mortality after OGD/R. |
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