The Role Of TRPM2 In Mediating Neuronal And Microglial Death Induced By DDVP Or H₂O₂

Background TRPM2 is a non-selective cation channel that can be activated by H₂O₂,causing death of related neurons and microglia.Numerous studies have shown that TRPM2 is an ion channel associated with oxidative stress,which combines oxidative stress with channel to facilitate the discovery of its potential targeting,thus providing a better treatment for diseases associated with oxidative stress Theoretical basis.Organophosphorus pesticides are widely used in agriculture.Ingestion or suicide in daily life can cause acute poisoning.At present,studies on the toxicity of organophosphorus pesticides mainly focus on the function of cholinergic neurons,but not on bile There are few studies on alkaline neurons and microglia.This project aims to study its effect on cell death in neurons and microglia,and is helpful for a understanding of the mechanism of the organophosphates.providing a new basis for clinical treatment of organophosphate poisoning.Part Ⅰ Effect of TRPM2 overexpression on SH-SY5 Y cell death induced by H₂O₂ and DDVP Objective: To investigate the effect of low concentrations of H₂O₂ on TRPM2 ion channels in wild-type and TRPM2 overexpressing SH-SY5 Y cell lines at different cell densities.The role of TRPM2 in different concentrations of H₂O₂ and DDVP-induced SH-SY5 Y cell death.Methods: Cells were seeded at different densities and treated with different concentrations of H₂O₂ and DDVP for 24 h.Cell viability was measured using CCK-8 kits.Results:（1）After treatment with low concentrations of H₂O₂（0-100μM）,the survival rate of SH-SY5 Y and its overexpressing cell lines decreased and was concentration-dependent,but different cell densities did not cause a large change in cell viability;（2）At low concentrations of H₂O₂（30,100μM）,the survival rate of SH-SY5 Y overexpressing cell lines was not significantly lower than that of wild group cells（SH-SY5 Y cells）,and the statistical difference was not significant（P > 0.05）.（3）When the concentration of H₂O₂ was 300μM,400μM,500μM,the cell viability of the wild group（WT）was significantly higher than that of the overexpression group（OE）,and there was statistical difference（###P <0.001,##P < 0.01,#P < 0.05）（4）When the concentration of DDVP was 100μM,the cell survival rate of the wild group（WT）was higher than that of the overexpression group（OE）,and there was a statistical difference（#P < 0.05）.Conclusion: Different cell densities had no significant effect on the death of SH-SY5 Y cells and their overexpressing cell lines induced by low concentrations of H₂O₂;at a certain concentration,TRPM2 was induced by H₂O₂ or DDVP in SH-SY5 Y cells and its overexpressing cell lines.Playing a role in death。Part Ⅱ Effects of TRPM2-knockout on SH-SY5 Y cell death induced by H₂O₂ and DDVP Objective: Using the constructed TRPM2 knockout cell line and wild-type SH-SY5 Y cells to investigate the role of TRPM2 in the death of different concentrations of H₂O₂ and DDVP-induced knockout cell lines Methods: H₂O₂-and DDVP-induced cell death was detected by propidium iodide（PI）/HOECHEST staining,and cell viability was detected using CCK-8 kits.Results:（1）After treatment with different concentrations of H₂O₂,the mortality of the cells was concentration-dependent,and the cell survival rate of the knockout group was higher than the wild-type group,but there was no statistical difference（P > 0.05）.（2）After treatment with different concentrations of H₂O₂,the mortality of the cells was oncentration-dependent.When the concentration of H₂O₂ was 100μM,the death rate of the knockout group（KO）cells was lower than that of the wild type group（KO）.Statistically significant（#P <0.05）（3）After treatment with different concentrations of DDVP,the mortality of the cells was concentration-dependent;at the DDVP concentration of 100μM,the survival rate of the knockout group（KO）cells was higher than the wild type group（WT）,and Statistically significant（#P < 0.05）;（4）Among the deaths of SH-SY5 Y cells and knockout cell lines induced by DDVP,the knockout group（KO）cells had a lower mortality rate than the wild type group（KO）when the concentration of DDVP was 100μM,and Statistically significant（#P <0.05）;Conclusion: TRPM2 plays a minor role in the death of SH-SY5 Y cell line caused by H₂O₂;TRPM2 plays a role in the death of DDVP-induced SH-SY5 Y cell line at a concentration of 100μM.Part Ⅲ TRPM2 mediates the role of DDVP in the death of RM microglia Objective: To explore the role of TRPM2 in DDVP-induced RM cell death.Methods: The survival rate and mortality of microglia were detected by CCK-8 method and PI staining respectively.The ROS level in microglia was detected by DCFH-DA,the mitochondrial changes in cells were detected by Mito Tracker fluorescent probe,and Lyso Tracker Probe detects lysosomal changes in cells.Methods:（1）With the increase of DDVP concentration,the survival rate of microglia decreased,showing concentration dependence.The concentration of DDVP at 300μM,the survival rate of microglia decreased significantly and was statistically significant（n=3,bar=standard error,*P <0.05）.（2）DDVP was added to detect intracellular ROS,mitochondria and lysosomes,and the morphology of the cells was found to change,indicating that DDVP has an effect on the induction of microglial death.DDVP increased ROS,and mitochondrial and lysosomal morphological structures and fluorescence density changed.（3）Using PARP and TRPM2 inhibitors,the concentration of DDVP was 300μM,and the survival rate of microglia decreased after using PJ34,DPQ,and 2-APB inhibitors.There was no statistical difference in using PJ34,using DPQ,2-APB has statistical difference（*P <0.05）Conclusion: DDVP can induce RM cell death and TRPM2 play a role in it.