The Protective Effect Of Hypoxic Preconditioning On Cerebral Ischemia-reperfusion Injury By Regulating NLRP3 Inflammasome-mediated Pyroptosis

According to global burden of disease study in 2019,stroke is not only the first cause of death in Chinese residents,but also an important cause of disability.Stroke can be divided into ischemic stroke and hemorrhagic stroke,of which ischemic stroke is the most common.From 2010 to 2019,the incidence rate of ischemic stroke in China has been rising continuously,and the incidence rate of ischemic stroke in 2019 has risen to 145/10 million.At present,the most effective way to treat patients with acute ischemic stroke is endovascular Thrombectomy or using thrombolytic medicines as soon as possible,so as to recanalize blood vessels and restore the blood circulation of brain tissue.However,with the recanalization of blood vessels,a large number of reactive oxygen species(ROS)were generated in the brain tissues of some patients,leading to secondary injury in the ischemic area,namely cerebral ischemia-reperfusion injury.Cerebral ischemia-reperfusion injury is a complex process involving many factors.The innate immune response of inflammation after ischemia plays an important role in the progression of cerebral ischemia-reperfusion injury.As an important part of innate immunity,pyroptosis mediated by inflammatory bodies composed of intracellular innate immune receptor NLRP3(NACHT,LRR and PYD domains-containing protein 3)has attracted more and more attention in the pathophysiological process of cerebral ischemia-reperfusion injury.Hypoxic/ischemic preconditioning is an effective mechanism of endogenous protection by mobilizing tissues or cells.HPC refers to a sublethal hypoxic/ischemic stimulation to tissues or cells,which can make tissues or cells tolerate the subsequent lethal hypoxic/ischemic stimulation.Professor Guowei Lv proposed that the tissue and cellular mechanism of hypoxia tolerance may be the change of gene expression when cells face the choice of life and death under extreme conditions,which is specifically manifested as up-regulation of "good genes"with brain protection and down-regulation of "bad genes" with brain injury.This experiment aims to study whether hypoxic preconditioning can protect against ischemia-reperfusion injury by regulating cell pyroptosis pathway mediated by NLRP3 inflammatory bodies in vivo and in vitro.OBJECTIVE:1.To establish the model of oxygen-glucose deprivation(OGD)/Reoxygenation of Mousederived microglia BV2 cells,and to observe the expression changes of NLRP3 during this process.2.To study the effects of cerebral ischemia-reperfusion injury in mice after hypoxic preconditioning and the expression of NLRP3 and its downstream molecules in the brain during this process.METHODS:PART 1:Effects of hypoxic preconditioning on NLRP3 expression in BV2 cells after oxygen-glucose deprivation/reoxygenation(OGD/R)injury.1.Establishment of OGD/R model of BV2 cellsIn this experiment,Mouse-derived microglia BV2 cells were selected as the research object.BV2 cells were randomly divided into 4 groups,and OGD/R model was determined according to the cell viability.2.Establishment of OGD/R after HPC model of BV2 cellsIn this experiment,BV2 cells were randomly divided into 4 groups according to the hypoxia exposure treatment of mouse hypoxic preconditioning model.HPC+OGD/R model was determined by cell viability.3.Effects of HPC on the expression of NLRP3 in OGD/R treated BV2 cells by Western blotIn this experiment,Western blot analysis was conducted to detect NLRP3 expression changes in OGD/R and HPC+OGD/R models of BV2 cell.PART 2:Effects of hypoxic preconditioning on NLRP3 inflammasome mediated pyroptosis in ischemic-reperfusion injury mice brain tissue.1.Establishment of cerebral ischemia-reperfusion model after hypoxic preconditioning in mice.1.1 Replication of hypoxic preconditioning model in miceIn this study,the hypoxic preconditioning model of mice was replicated according to literature reports,and the hypoxic tolerance limit time of the 2nd,3rd and 4th times of hypoxic exposure was used to judge whether the model was successfully replicated.1.2 Establishment of cerebral ischemia/reperfusion model after hypoxic preconditioning in mice.In this study,Western blot was used to detect the expression of NLRP3 during different duration of reperfusion in mice brain after hypoxic preconditioning,so as to determine the appropriate duration for reperfusion after hypoxic preconditioning in mice brain ischemia/reperfusion model.2.Effects of hypoxic preconditioning on NLRP3 induced pyroptosis in mice ischemiareperfusion brain tissue.2.1 Neurological score2.2 TTC staining of mice brain tissue and calculation of relative infarct area2.3 HE staining of mice brain tissue2.4 TUNEL apoptosis detection in mice brain tissue2.5 Western blot was used to detect the expression of NLRP3,Caspase-1 and GSDMD in mice brain tissues.2.6 The expression of NLRP3 was detected by immunofluorescence in mice brain tissues.2.7 The contents of IL-8,IL-6 and IL-1β in mice brain tissues were detected by ELISA.RESULTS:PART 1:Effects of hypoxic preconditioning on NLRP3 expression in BV2 cells after oxygen-glucose deprivation/reoxygenation(OGD/R)injury.1.Establishment of OGD/R model of BV2 cellsIn OGD/R model,BV2 cells were treated with oxygen-glucose deprivation for 1,2,3,or 4 h,and were all reoxygenated with glucose and reoxygenation for 2 h.Respectively,the cell vitality of BV2 cells decreased gradually.When the time duration of OGD was 3 h,the cell vitality of BV2 cells decreased to the lowest.Therefore,OGD 3 h/R 2 h was used as BV2 cell model to simulate cerebral ischemia-reperfusion in vitro.2.Establishment of OGD/R after HPC model of BV2 cellsBV2 cells prepared for OGD/R were divided into 4 different hypoxic exposure groups.Among them,the cells viability of BV2 that underwent OGD/R after 1%O2 for 2 h and reoxygenation for 2 h was the highest.Therefore,the above hypoxic exposure condition was used as the HPC condition for BV2 cells.Based on the above results,HPC+OGD/R model of BV2 cells was established.3.Effects of HPC on the expression of NLRP3 in OGD/R treated BV2 cells by Western blot;Western blot analysis showed that NLRP3 expression in HPC+OGD/R group and OGD/R group was significantly increased,while NLRP3 expression in HPC+OGD/R group was significantly lower than OGD/R group,but NLRP3 expression in both above groups was higher than that in N group and HPC group.PART 2:Effects of hypoxic preconditioning on NLRP3 mediated pyroptosis in ischemicreperfusion injury mice brain tissue1.Establishment of cerebral ischemia-reperfusion model after hypoxic preconditioning in mice1.1 Replication of hypoxic preconditioning model in miceAccording to the methods reported in the literature,we exposed mice to hypoxia.The results showed that the tolerance limits of the first,second,third and fourth hypoxic exposure were 22.53 ± 1.50 min,45.27 ± 5.51 min,78.13 ± 10.52 min and 113.53 ± 14.97 min,respectively,which were close to 2,4 and 6 times of the tolerance time of the first hypoxic exposure.The results were consistent with the literature reported,suggesting that the mouse hypoxic preconditioning model was successfully replicated.1.2 Establishment of cerebral ischemia/reperfusion model after hypoxic preconditioning in mice.We treated the mice with hypoxic preconditioning for 60 min,then gave the middle cerebral artery occlusion to simulate cerebral ischemia for 60 min,and last treated mice with reperfusion for 12,24 or 36 h respectively.After Western blot analysis,the NLRP3 expression in the brain tissues of mice reperfusion for 12 h was the lowest.Therefore,60 min ischemia/12 h reperfusion was selected as the model processing method of OGD/R in subsequent experiments.2.Effects of hypoxic preconditioning on NLRP3-mediated pyroptosis in mice ischemiareperfusion brain tissue.2.1 Neurological scoreThe mice with neurological score greater than 3 in the I/R group were included in the experiment,and the inclusion rate was about 80%.The neurological scores of HPC+I/R group and I/R group were significantly higher than those of control group and HPC group,but the neurological scores of HPC+I/R group were significantly lower than those of I/R group.These results suggest that hypoxic preconditioning can alleviate the neurological injury induced by ischemia-reperfusion in mice.2.2 TTC staining of mice brain tissue and calculation of relative infarct areaThe pictures of TTC staining of mouse brain tissue showed that the relave infarct area of HPC+I/R group and I/R group was significantly larger than that of control group and HPC group,but the relave infarct area of HPC+I/R group was smaller than that of I/R group,which was further validated by calculation of relative infarct area.2.3 HE staining of mice brain tissueBy HE staining of mouse brain tissue,we found that the brain tissue structure,especially hippocampus of control group and HPC group were complete and arranged clearly,without tissue damage and inflammatory cell infiltration,while tissue damage occurred in HPC+I/R group and I/R group,but the degree of tissue damage in HPC+I/R group was significantly less than that in I/R group.2.4 TUNEL apoptosis detection in mice brain tissueAccording to the results of TUNEL apoptosis detection,the number of apoptotic cells in HPC+I/R group and I/R group increased significantly compared with control group and HPC group,but the number of apoptotic cells in HPC+I/R group decreased significantly compared with I/R group.2.5 The expression of NLRP3,Caspase-1 and GSDMD in mice brain tissues by Western blotWe found that the expressions of NLRP3,Caspase-1 and GSDMD in the HPC+I/R group and I/R group were significantly higher than those in control group and HPC group,but the expressions of all the above proteins in HPC+I/R group was significantly lower than that in I/R group.2.6 The expression of NLRP3 in mice brain tissues by immunofluorescenceThe results of immunofluorescence detection of NLRP3 expression in mice brain tissue in each experimental groups were the same as those of Western blot.that is,the expression of NLRP3 in HPC+I/R group and I/R group was significantly higher than that in control group and HPC group,but that in HPC+I/R group was significantly lower than that in I/R group.2.7 The contents of IL-8,IL-6 and IL-1β in mice brain tissues by ELISABy ELISA detection,it was found that compared with control group and HPC groups,the contents of IL-1β,IL-6 and IL-8 in I/R group and HPC+I/R group were significantly increased.However,HPC+I/R group was significantly lower than I/R group.CONCLUSION:Hypoxia preconditioning has certain protective and ameliorative effects on cerebral ischemia-reperfusion injury in mice,which may be related to decrease NLRP3 expression and alleviate NLRP3 inflammasome mediated pyroptosis during cerebral ischemia-reperfusion injury in mice.This finding proposes the neuroprotective mechanism that hypoxic preconditioning plays a neuroprotective role in cerebral ischemia-reperfusion injury through NLRP3 mediated pyroptosis for the first time,which not only enriches the theoretical mechanism of hypoxic preconditioning neuroprotection,but also provides a theoretical basis for its clinical application.