The Role Of Pyroptosis In Hypoxic-ischemic Brain Damage In Newborn Rats

Research background and purpose:Hypoxic-ischemic brain damage(HIBD)refers to hypoxic-ischemic(HI)damage to the brain tissue caused by perinatal asphyxia,which is an acute death of newborns and chronic nervous system damage.The main reason.At present,mild hypothermia is a more effective treatment method for term infants with moderate to severe HIBD,but its treatment time window is narrow and the application is limited.New clinical strategies are still urgently needed.HIBD is usually characterized by neuronal cell death,and inflammation is one of the main causes of neuronal cell death after HIBD.Post-HIBD inflammation is driven by pro-inflammatory cytokines(such as: IL-18 and IL-1β)and can aggravate the severity of brain damage.Studies have shown that activation of pro-inflammatory cytokines is regulated by activation of NLRP3 inflammatory bodies in a variety of brain injury diseases.As a new method of programmed cell death accompanied by inflammatory response,the classic pathway of cell death depends on the activation of Caspase-1,and its role in HIBD is not very clear.In the process of cell scorch,the activation of NLRP3 inflammatory bodies is necessary for the activation of Caspase-1,which will eventually lead to the activation and secretion of proinflammatory cytokines IL-18 and IL-1β,increase the inflammatory response,and eventually cause cell damage.Caspase-1 specific inhibitor VX-765 can penetrate the blood-brain barrier and can effectively inhibit the pro-inflammatory process mediated by Caspase-1 activation.In this study,the HIBD neonatal rat model was used to detect the expression of scorch cell death related indicators,and to investigate whether NLRP3 inflammatory body-mediated scorch cell death is involved in the pathogenesis of HIBD.VX-765 inhibits the key link of scorch cell death Caspase-1.Whether activation can reduce the severity of hypoxic-ischemic brain injury,thereby exerting neuroprotective effects and improving learning and memory functions,aims to provide a theoretical basis for neonatal HIBD treatment.Materials and Methods:1.Seven-day-old newborn SD rats were randomly divided into a sham operation group and a model(HIBD)group,and a modified rice method was used to establish amodel of HIBD newborn rats.Brain tissues were taken at 24 h,48h,and 72 h after surgery.Western blot was used to detect the changes in NLRP3,ASC,pro-Caspase-1,and Caspase-1 protein expression in the left hippocampus and the peak time of expression.At 72 hours after operation,HE staining and Nissl staining were used to observe the pathological changes and neuronal damage in the left hippocampal DG region;immunohistochemical staining was used to detect the expression of NLRP3,ASC,and Caspase-1 proteins in the left hippocampal DG region;Western blot The expression of NLRP3 inflammatory corpuscle-mediated cell death related molecules(NLRP3,ASC,Caspase-1,GSDMD,IL-1β,IL-18)and mRNA expression in the left hippocampus were detected by RT-PCR and RT-PCR.2.Seven-day-old newborn SD rats were randomly divided into a sham operation group,a model(HIBD)group,and a Caspase-1 inhibitor VX-765 treated HIBD(VX-765)group.The VX-765 group was injected intraperitoneally with VX-765solution(50 mg / kg)at 0,24,and 48 hours after surgery.The Sham group and the HIBD group were injected with the same amount of normal saline.At 72 hours after the operation,Western blot and Rt-PCR were used to detect the expression of cytosolic death-related proteins and mRNA(Caspase-1,GSDMD,IL-1β,IL-18)in the left hippocampus of rats;Changes of inflammatory cytokines(IL-1β,IL-18)levels in hippocampal tissue;HE staining and immunohistochemical staining were used to observe the pathological changes and neuronal damage of brain tissue in the left hippocampal CA1 region of rats;Tissue infarct area;Morris water maze and other behavioral experiments to assess learning and memory function in rats.Results:1.Western blot results showed that compared with the Sham group,the expression levels of NLRP3 protein increased at 24 and 48 hours in the HIBD group;the expression of ASC and Caspase-1 protein increased at 72 hours after the operation;pro-Caspase-1 protein expression level did not change significantly.The peak expressions of NLRP3,ASC,and Caspase-1 proteins in the left hippocampus of HIBD newborn rats were at 24 h,72h,and 72 h,respectively.2.The HE staining results showed that compared with the Sham group,the number of cell layers in the hippocampal DG area on the left side of the HIBD group wasrelatively reduced and the cell arrangement was relatively irregular.3.Nissl staining results showed that the number of neurons in the left hippocampal DG region in the HIBD group was greater than that in the Sham group.4.Immunohistochemical staining results showed that the expression of NLRP3,ASC,and Caspase-1 in the hippocampal DG region of the left side of the HIBD group was enhanced.5.Western blot and RT-PCR results showed that dysfunction-related proteins and mRNA(NLRP3,ASC,Pro-Caspase-1,Caspase-1,GSDMD,IL-1 β,IL-18)in the hippocampus of the left side of the HIBD group The expression level increased,and the difference was statistically significant(P <0.05).6.After VX-765 intervention,Western blot and RT-PCR results showed that compared with the Sham group,HIBD-associated proteins and mRNAs(Caspase-1,GSDMD,IL-1β,IL-18)expression level was increased,and the difference was statistically significant(P <0.05).Compared with the HIBD group,the expression levels of coke death-related proteins and mRNA in the left hippocampal tissue cells of the newborn rats in the VX-765 group were significantly reduced.It was statistically significant(P <0.05).7.After VX-765 intervention,the ELISA test results showed that compared with the Sham group,the levels of inflammatory cytokines(IL-1 β,IL-18)in the left hippocampus of newborn rats after HIBD increased significantly(P <0.05);Compared with the HIBD group,the levels of inflammatory cytokines(IL-1β,IL-18)in the left hippocampus of newborn rats in the VX-765 group were significantly reduced(P<0.05).8.After VX-765 intervention,the immunohistochemical results showed that compared with the Sham group,neurons in the left hippocampal CA1 region of newborn rats were significantly lost and the number of NeuN + cells was significantly reduced.Neuron loss in the hippocampal CA1 region of newborn rats in group 765 was reduced,and the number of NeuN + cells was reduced.9.After VX-765 intervention,the results of TTC staining showed that compared with the Sham group,the infarct area of the left cerebral hemisphere in rats increasedsignificantly after HIBD;but compared with the HIBD group,the left hemisphere of the VX-765 group rats The infarct size did not decrease significantly.10.After VX-765 intervention,the results of Morris water maze and other behavioral experiments showed that compared with the Sham group,the escape latency of the rats after HIBD was prolonged and the number of puncture steps was reduced;compared with the HIBD group,the rats of the VX-765 group The escape latency was shortened and the number of crossings was not significant.conclusions:NLRP3 inflammatory corpuscle-mediated pyroptosis has been involved in the pathogenesis of neonatal rats with HIBD;VX-765 inhibits the activation of Caspase-1,which effectively inhibits the occurrence of cellular death in the hippocampus of injured rats after HIBD and reduces The inflammatory response and neuron damage in the hippocampus of neonatal rats after HIBD were not reduced,but the area of cerebral hemisphere infarction on the injured side of HIBD rats could not be significantly reduced,and learning and memory functions were improved.