The Role Of TIGAR In Neonatal Rats With Hypoxic-ischemic Brain Damage And Its Mechanism

Aim:To observe the expression of TIAGR during hypoxic-ischemic brain damage in newborn rats;the role of TIGAR in hypoxic-ischemic brain damage and its mechanism of pyropsis preliminary study.Methods:Hypoxic-ischemia brain damage(HIBD)model in newborn rats and in vitro cultured rat microglial cell line(HAPI)sugar deprivation and reperfusion(Oxygen and glucose deprivation/reoxygenation,OGD/R)model.Western blot was used to detect the expression of TIGAR and pyropsis-related proteins GSDMD-N,caspase-1,IL-1β in cerebral cortex tissues after HIBD and in rat microglia after OGD/R.After transfection of rat microglial cell lines with the constructed lentivirus that interferes with TIGAR,the immunofluorescence and Western blot methods were used to detect TIGAR infection rate and protein expression,CCK-8 and LDH kits were used to detect the effect of TIGAR on the viability and toxicity of rat microglial cell lines after OGD/R.Interfering TIGAR constructed by lentivirus was injected into the rat brain through the left striatum and the left ventricle,and the TIGAR infection rate and protein expression were detected by immunofluorescence,small animal fluorescence in vivo imaging technology and Western blot,the volume of cerebral infarction was used to evaluate the effect of TIGAR on hypoxic-ischemic brain injury.After transfecting rat microglial cell lines with the constructed lentivirus that interferes with TIGAR,Western blot was used to detect the effect of LV-sh_TIGAR on the content of pyropsis-related proteins in microglial cell lines of rats 24 hours after OGD/R.In LV-sh_TIGAR rat microglia cell line,after applying NADPH,DHE staining was used to detect the effects of NADPH on OGD/R induced intracellular ROS levels.NADPH/NADP+ and GSH/GSSG kits were used to detect the effects of NADPH on NADPH and GSH/GSSG levels in OGD/R induced cells.The effect of the content of pyropsis-related proteins caused by OGD/R at 24 hours was detected.CCK-8 and LDH kits were used to detect the effect of NADPH on cells viability and toxicity after OGD/R.Meanwhile,in LV-sh TIGAR newborn rats,the effects of NADPH on hypoxic-ischemic brain injury were detected by TTC staining,HE staining,electron microscopy,and immunohistochemical staining after applying NADPH.Result:After HIBD,the expression levels of TIGAR,GSDMD-N,caspase-1,and IL-1β in the cerebral cortex tissue increased and reached a peak at the time point of 24h;TIGAR,GSDMD-N,The expression of caspase-1 and IL-1β increased,and reached the peak at 24h.Interfering with TIGAR in rat microglial cell line effectively interferes with TIGAR and effectively reduces TIGAR expression.Interfering with TIGAR aggravates OGD/R induced damage to rat microglial cell lines.Interfering TIGAR constructed by lentivirus can effectively infect brain tissues and interfere with TIGAR expression.LV-sh_TIGAR can increase cerebral infarction volume in HIBD rats.Interfering with TIGAR can increase GSDMD-N,caspase-1,IL-1β protein levels in rat microglial cell lines 24 hours after OGD/R.In LV-sh_TIGAR rat microglia cell line,after applying NADPH:NADPH significantly inhibited the interference of TIGAR induced death of rat microglia cell line;interfering with TIGAR significantly promoted the reduction of intracellular NADPH,GSH/GSSG and enhancement of ROS levels induced by OGD/R,while NADPH blocked the effect of interfering with TIGAR;interfering with TIGAR reduced GSDMD-N and IL-1β protein levels in rat microglial cell lines 24 hours after OGD/R.At the same time,in LV-sh_TIGAR newborn rats,after applying NADPH:Interfering with TIGAR reduces the cerebral infarction volume of rats after HIBD,reduces pathological damage to cerebral cortex and damage to cerebral cortex cells,and reduces the expression of GSDMD-N in cerebral cortex.Conclusion:TIGAR has protective effects on hypoxic-ischemic brain damage in newborn rats.The mechanism may be related to increasing the endogenous antioxidant NADPH and reducing intracellular ROS levels and inhibit the activation of pyropsis.