Neuroprotection And Mechanisms Of Glibenclamide In A Mouse Model Of Radiation Brain Injury

BackgroundPartial or whole brain radiation therapy is an important adjuvant treatment for primary or metastatic tumors in the brain.Radiation-induced brain injury(RBI)is a common complication of radiotherapy for head and neck tumors.In addition to the obvious killing and suppressing effects on tumor cells,radiation therapy inevitably has a significant damaging effect on normal tissues adjacent to tumors in the radiation area.RBI causes degenerative changes in brain tissue,cognitive decline,and even severe dementia,which seriously affects the quality of life of patients undergoing radiation therapy and is the leading cause of death and disability.Its pathophysiological mechanisms still needs further research,and currently there is no effective preventive and therapeutic measures in clinical practice.Persistent neuro-inflammation induced by ionizing radiation is the main pathophysiological changes.In recent years,it has been found that the newly formed SUR1-TRPM4(sulfonylurea receptor 1-transient receptor potential M4)channel is induced in a variety of central nervous system diseases.This channel is closely related to the mechanism of cell swelling death,blood-brain barrier disruption,and brain edema.Glibenclamide,a specific inhibitor of this channel,can significantly inhibit neuron death,reduce blood-brain barrier disruption,and brain edema.At the same time,glibenclamide has been reported to exert anti-inflammatory effects in a variety of inflammation-related diseases.It can inhibit the activation of NLRP3 inflammasome and IL-1β,but the specific target and mechanism are unknown.To date,no studies have been conducted on the application of glibenclamide in RBI animal models.Whether glibenclamide can reduce the inflammatory response of radiation brain injury by inhibiting NLRP3 inflammasome and whether this effect through SUR1-TRPM4 needs further exploration.The purpose of this study was to evaluate the protective effect of glibenclamide on the pathological and functional impairment of the model by constructing a mouse model of RBI,and to explore the mechanism of action.Methods(1)Using Faxitron MultiRad 225 irradiator to construct a mouse whole brain radiation model and evaluating the pathological changes of the model.(2)Preliminary evaluation of the effect of glibenclamide on cognitive impairment in RBI model.(3)Observe the expression of NLRP3 inflammasome associated protein,SUR1,and TRPM4 in the RBI model.(4)Using Trpm4 knockout mice to explore the anti-inflammatory and neuro-protective mechanisms of glibenclamide in the RBI model.(5)Using microglia cell line radiation model to explore the anti-inflammatory mechanisms of glibenclamide.Results(1)Using the Faxitron MultiRad 225 irradiator,perform 30Gy(3Gy/min)whole brain radiation modeling on mice.It was found that the mice had a poor response from 9 to 14 days after modeling,and then gradually improved.No mouse death was observed in 2 months after modeling.At different time points within 9 days after modeling,no brain edema was detected by brain water content detection.Evans blue extraction experiment showed mild blood-brain barrier disruption 3 to 5 days after irradiation.MR(T2WI,ADC)within 1 week after radiation showed no obvious cytotoxic and vasogenic edema of brain tissue.The brain atrophy was gradually aggravated which was indicated by lateral ventricle enlargement in MR 2 months after radiation.Immunofluorescence experiments at 2 months after radiation showed astrocytic(obvious in the hippocampus)and microglial activation,and inhibition of immature neurons in the hippocampal DG zone,suggesting chronic neuro-inflammation and neuro-regeneration impairment.(2)Morriz water maze experiment showed that glibenclamide can reduce memory and cognitive impairment in 2 months after radiation modeling.(3)Western blotting experiments showed that the up-regulation of NLRP3 inflammatory body-associated proteins(NLRP3,Caspase-1,IL-1β),SUR1,and TRPM4 after radiation modeling,and NLRP3 inflammatory body activation(Caspase-1 P10,IL-1 β P17).Immunofluorescence colocalization showed activated microglial cells express SUR1 and TRPM4.(4)Wild type and Trpm4-/-mice were used for whole-brain radiation modeling,and glibenclamide and vehicle control intervention were applied to the two genotype mice,respectively.Glibenclamide and Trpm4-/-can inhibit NLRP3 inflammasome activation,astrocytic and microglial activation,restore neural regeneration,reduce lateral ventricle enlargement and cognitive impairment after radiation modeling.There was no difference between the two groups of Trpm4-/-mice with glibenclamide and vehicle control intervention.(5)BV2 cells radiation model showed that SUR1,TRPM4 and NLRP3 up-regulated after radiation,and activation of NLRP3 inflammasome 24h after radiation.Conclusion(1)The experimental method of using Faxitron MultiRad 225 irradiator to construct a whole-brain radiation model in mice is feasible,which can simulate the pathophysiology and cognitive impairment of the chronic inflammatory response of radiation brain injury.(2)Glibenclamide inhibits the activation of NLRP3 inflammatory bodies,reduces pathological changes and functional impairment,and exerts anti-inflammatory and brain protective effects by blocking SUR1-TRPM4 in a mouse whole brain radiation model.