| Neonatal hypoxic-ischemic encephalopathy(HIE)refers to hypoxic-ischemic brain damage due to partial/complete cerebral hypoxia and reduced/stopped cerebral blood flow caused by many factors in the perinatal period,and is an important factor in the increased probability of acute neonatal death and the increased incidence of chronic neurological damage.TRPM2 channels are widely expressed in the CNS and are involved in the physiopathology of many CNS diseases,including hypoxic-ischemic brain injury.Its activation by oxidative stress mediates Ca2+ endocytosis,which in turn induces the activation of NLRP3 inflammatory vesicles,and the loss of control of inflammatory response mediated by this process is a key mechanism of neuronal damage caused by ischemia and hypoxia.Therefore,we propose to investigate the role of TRPM2 channel/NLRP3 inflammatory vesicles in the pathogenesis of ischemic-hypoxic brain injury through clinical studies,model animal and cellular experiments.Part Ⅰ.Expression of TRPM2/NLRP3 in peripheral blood of newborns with HIE and its clinical significanceObjective:To initially investigate the expression of TRPM2,NLRP3 and Caspase-1 mRNA in the postnatal peripheral blood of HIE neonates,to compare the expression levels of TRPM2,NLRP3 and Caspase-1 mRNA in neonates with different severity of HIE,and to analyze the correlation between the expression levels and resuscitation scores of the three,and to evaluate the value of the three in the assessment of the severity of ischemia and hypoxia in HIE neonates.The correlation between the expression levels and resuscitation scores was analyzed to evaluate the value of the three in assessing the severity of hypoxia in HIE neonates.Methods:1.Thirty newborns with HIE admitted to the neonatal unit of the Children’s Hospital of Soochow University from 2021-01 to 2021-12,and 32 healthy full-term newborns in the same period were collected as the control group to collect relevant clinical data.2.The newborns in the HIE group were classified into mild HIE group,moderate HIE group,and severe HIE group according to the classification criteria of HIE severity.3.Both groups of neonates underwent aEEG examination within 24 h after birth,and were classified into aEEG normal group,aEEG mild abnormal group,aEEG moderate abnormal group,and aEEG severe abnormal group according to the aEEG background classification.4.Resuscitation scores were performed in neonates in the different severity HIE groups and in the different degree aEEG abnormality groups.5.The expression levels of TRPM2,NLRP3 and Caspase-1 mRNA in peripheral blood mononuclear cells were detected by qRT-PCR.6.The correlation between the levels of TRPM2,NLRP3,Caspase-1 mRNA and the severity of HIE and resuscitation score was analyzed.Results:1.The aEEG abnormality rate in the HIE group was significantly higher than that in the control neonates;the aEEG moderate abnormality rate and aEEG severe abnormality rate in the moderate HIE group were significantly higher than that in the mild HIE group;the aEEG severe abnormality rate in the severe HIE group was significantly higher than that in the mild HIE group and the moderate HIE group;the differences were statistically significant(p<0.05).2.The resuscitation scores of the three groups,mild HIE group,moderate HIE group and severe HIE group,showed a gradual increase,and the difference was statistically significant(p<0.05).3.The resuscitation scores of the three groups,aEEG mild abnormality group,aEEG moderate abnormality group,and aEEG severe abnormality group,showed a gradual increase,and the difference was statistically significant(p<0.05).4.The levels of TRPM2,NLRP3,and Caspase-1 mRNA in peripheral blood mononuclear cells of neonates in the HIE group were significantly higher than those of neonates in the control group,and increased with the severity of HIE,with statistically significant differences(p<0.05).5.The levels of TRPM2,NLRP3,and Caspase-1 mRNA expression in raw peripheral blood mononuclear cells of neonates with HIE were positively correlated with HIE severity and resuscitation score(p<0.05).Conclusion:1.The expression of TRPM2,NLRP3 and Caspase-1 mRNA levels in peripheral blood of neonates in the HIE group were significantly upregulated early in the course of HIE.2.The expression levels of TRPM2,NLRP3,and Caspase-1 mRNA were positively correlated with the severity of HIE and resuscitation score.Part Ⅱ Significance of TRPM2/NLRP3 expression in peripheral blood and brain tissues of neonatal rats with HIBDObjective:To further observe the changes in the expression of TRPM2/NLRP3 and related inflammatory factors in peripheral blood and brain tissue of neonatal rats with HIBD.METHODS:A HIBD model was constructed using 7-day-old neonatal SD rats randomly divided into sham-operated and HIBD groups by ligating the left common carotid artery using the modified Rice-vannucci method followed by 2.5 hours of hypoxia,and the following indices were observed.1.TTC staining method to detect the volume of cerebral infarction in neonatal rats.2.Histomorphological changes in the brain cortex of neonatal rats were observed by HE staining.3.The expression of TRPM2,NLRP3 and Caspase-1 mRNA levels in peripheral blood of neonatal rats was detected by qRT-PCR.4.Western Blot method was used to detect the expression of TRPM2,NLRP3,CLCaspase-1,IL-18 and IL-1β protein levels in cerebral cortex at different time points.Results:1.TTC staining:neonatal rats in the Sham group had no abnormal brain tissue morphology,bilateral symmetry,no swelling and no local infarction;rats in the HIBD group had severe bilateral brain tissue swelling,and the ipsilateral left side of the ligated brain had obvious local infarction with edema foci.Comparing the brain tissue damage in the two groups,the differences were statistically significant(p<0.05).2.HE staining:neurons and glial cells in the cortex of the Sham group had normal morphology,clear nuclei,and no cell edema;the interstitium had no hemorrhage,necrosis,or edema;the cells in the cortex of the HIBD group were swollen and prominent,and some of the nuclei were fixed;the interstitial tissue was loose and edematous,and the extracapillary space was widened.The ratio of damaged cells/total cells in the high magnification field was significantly higher than that of the Sham24 h group,and the difference was statistically significant(p<0.05).3.The expression of TRPM2,NLRP3 and Caspase-1 mRNA in peripheral blood of neonatal rats at the early stage of HIBD(24h)was significantly upregulated by qRT-PCR compared with the control group,and the difference was statistically significant(p<0.05).4.The expression of TRPM2,NLRP3,CL-Caspase-1,IL-18,and IL-1β protein levels in the cerebral cortex of neonatal rats at different time points during the early stages of HIBD were significantly upregulated by Western Blot compared with the control group(p<0.05).Conclusion:The expression of TRPM2,NLRP3,and Caspase-1 mRNA in peripheral blood was significantly upregulated in the early post-HIBD period,and the expression of TRPM2,NLRP3,CL-Caspase-1,IL-18,and IL-1β protein levels were significantly upregulated in cerebral cortex tissues,suggesting that TRPM2 is involved in the pathophysiological process of HIBD,and interfering with TRPM2 expression may be a potential target for clinical treatment of HIBD.Part Ⅲ TRPM2 is involved in hypoglycemic hypoxia-induced apoptosis and NLRP3 inflammatory vesicle activation in neuronal cellsObjective:To observe the effect of interference with TRPM2 expression in PC 12 cells on apoptosis,NLRP3,Caspase-1,CXCL2,IL-1β and other molecules expression,and to explore in depth the mechanism of the role of TRPM2/NLRP3 activation in inflammatory injury and apoptosis in HIBD neuronal cells.Methods:1.Rat PC 12 cell line was established as a model of sugar-oxygen deprivation(OGD).2.Using TRPM2 to interfere with PC 12 cells and establish a OGD model.3.Experimental groups:1)normal control group(Control);2)interfering control group(sh-NC);3)interfering TRPM2 group(TRPM2-shRNA);4)OGD model(OGD);5)interfering control group+OGD model(sh-NC+OGD);6)interfering TRPM2 group+OGD model(TRPM2-shRNA+OGD).4.The expression of apoptosis,mitochondrial membrane potential,ROS,Ca2+concentration and IL-1β were detected in each group by flowmetry at 24h after model establishment.5.The expression of NLRP3,Caspase-1 and CXCL2 mRNA in each group was detected by qRT-PCR method at 24h after model establishment.6.The expression of NLRP3,CL-Caspase-1 and CXCL2 proteins in each group was detected by WB method at 24h after model establishment.Results:1.In the control group,PC 12 neuronal cells were elongated or polygonal in shape,and some of the cells had sympathetic neuron-like neural protrusions;in the OGD group,PC 12 neuronal cells were rounded and gradually lost their original morphology,and the nuclei were solidified,generating many granular spots,and the nuclei of the more apoptotic cells were broken into fragments and disintegrated.2.At 24 h after OGD model establishment,apoptosis increased,mitochondrial membrane potential increased,ROS,Ca2+ concentration,and IL-1β expression increased in OGD group and sh-NC+OGD group,and apoptosis decreased,mitochondrial membrane potential decreased,ROS,Ca2+ concentration,and IL-1β expression decreased in TRPM2shRNA+OGD group.3.At 24 h after OGD model establishment,CXCL2,NLRP3,Caspase-1 mRNA expression was elevated in OGD group and sh-NC+OGD group,and CXCL2,NLRP3,Caspase-1 mRNA expression was decreased in TRPM2-shRNA+OGD group.4.At 24 h after OGD model establishment,CXCL2,NLRP3,CL-Caspase-1 protein expression was elevated in OGD group and sh-NC+OGD group,and CXCL2,NLRP3,CLCaspase-1 protein expression was decreased in TRPM2-shRNA+OGD group.Conclusion:OGD produces ROS,activates Ca2+pathway,which causes apoptosis in PC 12 cells,and activates NLRP3 inflammatory complex pathway and CXCL2 expression;interfering with TRPM2 expression results in the inhibition of Ca2+ channel and the reduction of ROS production,and inhibits NLRP3 inflammatory complex pathway and CXCL2 expression,which in turn reduces PC 12 cell apoptosis and protect PC 12 neuronal cells. |
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