Study On The Mechanism Of NaHS In The Treatment Of Stroke By Reducing NLRP3/Caspase-1/GSDMD

BackgroundThe high mortality and disability rate of ischemic stroke,the continuous progress of reperfusion injury after thrombolytic therapy and the difficulty of repair of ischemic injury seriously affect the quality of life of stroke patients.Exploring the occurrence and development mechanism of post-stroke neuronal inflammation and cell death is still the main target of studying new drugs for the treatment of stroke.Inflammatory mediators released from the injured area after cerebral ischemia promote inflammation,edema,apoptosis,pyroptosis and necrosis.Pyroptosis is an inflammatory caspase 1,4 and 11activation that completes the N-terminal shear of GSDMD to form pores,and finally releases proinflammatory factor IL-1β,cell death mode of IL-18.Among them,the discovery of GSDMD upgrades people’s understanding of pyroptosis to a new level,and Gasdermin-dependent protein is also a necessary molecular feature in the process of pyroptosis.Neuronal pyroptosis after ischemia-reperfusion is closely related to nervous system inflammation.Interpreting the mechanism of neuronal inflammation and pyroptosis after stroke and finding drugs that can intervene this process will become a new direction of stroke research.The high lipid solubility and rapid permeability of NaHS through cell membrane enable it to produce neuromodulatory effect within seconds.The effect of NaHS in animal models has been widely explored.More and more evidences show that NaHS or its donor compound sodium hydrosulfide（NaHS）has protective effects on inflammation and nerve cells injury caused by nervous system diseases.Physiological and pathological effects of NaHS are gradually recognized.The research of NaHS in nervous system diseases has made new progress in recent years.In stroke,NaHS can significantly alleviate pathology of ischemia-reperfusion rats,reduce the volume of cerebral infarction,improve prognosis of post-stroke animals and cerebral edema.It can reduce the serum lipid peroxidation product malondialdehyde（MDA）and the activity of lactate dehydrogenase（LDH）in rats with focal cerebral ischemia-reperfusion injury.The infarct area decreased by 50%in cerebral ischemia rats after 2 days of exposure to NaHS（80 ppm H₂S in 19.5%O~2）.However,the protective mechanism of NaHS in stroke is not clear.This experiment aims to explore the mechanism of H₂S in reducing the infarct area after stroke.Objective1.To explore the activation of microglia and the survival rate of neurons in the injured cortex after cerebral ischemia-reperfusion in rats,and whether hydrogen sulfide can increase the survival rate of neurons by inhibiting the activation of microglia and improve stroke.2.To explore whether inflammation and pyroptosis occur in the injured cortex after cerebral ischemia-reperfusion in rats and type of nerve cells in which pyroptosis occurs;whether hydrogen sulfide can take cerebral protective effect by inhibiting the pyroptosis of injured cortical cells after cerebral ischemia-reperfusion.Methods1.280～350g SD rats were taken to establish the middle cerebral artery embolization model of SD rats（suture method）.The experiment was divided into four groups.In the first group,the right middle artery was separated than intraperitoneal injection of sodium chloride within 15 minutes for sham operation without suture;In the second group,the right middle cerebral artery was embolized than intraperitoneal injection of sodium chloride within 15 minutes,ischemia for 2 hours and reperfusion for24 hours;In the third group,the right middle cerebral artery was embolized than intraperitoneal injection of sodium hydrosulfide within 15 minutes,ischemia for 2 hours and reperfusion for 24 hours;In the fourth group,the right side was separated for sham operation without thread plug,than intraperitoneal injection of sodium hydrosulfide within 15 minutes2.After completing the experiment,the brain of each group was taken for TTC staining to detect the area of brain obstruction in each group;m NSS detect neurological function score.3.After each group completed the experiment,the injured cerebral cortex was taken and the proinflammatory factor IL-1β,IL-18 was detected by ELISA.The expression of NLRP3,Caspase-1,GSDMD was detected by Western blot.4.After completing the experiment,the brains of each group were taken for frozen sections,the activation of microglia was detected by immunofluorescence,and the location of pyroptosis of nerve cells on the injured side was located.Results1.NaHS decreased the area of obstruction on the injured side of ischemia-reperfusion rats and increased the neurological function score.2.NaHS reduces the expression of IL-1β,IL-18,NLRP3,Caspase-1 and GSDMD protein improved the I/R brain injury in rats.3.After ischemia-reperfusion,cortical cell pyroptosis mainly occurred in the ischemic area,and the number of neuronal pyroptosis was higher than that of microglia.Microglia pyroptosis mainly occurred in the ischemic junction area.Conclusion1.NaHS can reduce the activation of microglia and increase the survival rate of neurons.2.NaHS can reduce the expression of NLRP3,Caspase-1 and GSDMD,reduce the pyroptosis of nerve cells and improve the ischemia-reperfusion injury.