Effect Of 5-BDBD,a Selective Blocker Of P2X4,on The Activation Of NLRP3 Inflammasome In Microglia

Aim Due to the ageing of the population,the morbidity and disability of neurodegenerative diseases are increasing,which has a considerable impact on the social economy.A growing number of evidence supports the notion that the neurodegenerative,such as Alzheimer’s disease(AD),Parkinson’s disease(PD)and Amyotrophic Lateral Sclerosis(ALS)does not only involve the neuronal compartment but also imply a strong interaction with the immunological cells in the Central Nervous System(CNS),mainly the microglia.This paper mainly discusses the effect of 5-BDBD,a specific blocker of P2X4,on the activation of NLRP3 inflammasome in microglia.Methods Microglia in exponential phase were used for the experiment.A model of NLRP3 inflammasome activation in microglial cells was established by using lipopolysaccharide(LPS)+ triphosphate(ATP)bisignal interference.There are 8 groups according to different intervention methods: control group,ATP group,LPS group,LPS+ATP group,5-BDBD group,ATP+5-BDBD group,LPS+5-BDBD group,LPS+ATP+5-BDBD group.The influence of LPS and ATP on the vitality of BV2 cells was detected by CCK-8 method,and according to this result,the appropriate drug concentration was selected for subsequent experiments.ResultsThe result of CCK-8 showed that the concentration of 1μg/mL LPS and 0-1mmol/L ATP did not affect the vitality of BV2(P>0.05),but the ATP concentration of more than 2mmol/L had an effect on the survival rate of BV2.The BV2 cells of ATP concentration groups we selected,2mmol/L,3mmol/L,and 5mmol/L were all less active than the control group,the differences were statistically significant(P<0.05,P<0.001,P<0.001).Although the ATP concentration of 2mmol/L had a damaging effect on the cells,it affected the vitality of the cells(79.52 ± 3.873%),but through our follow-up experiments and reading a lot of literature,it is proved that the ATP concentration below 2mmol/L plus LPS can’t activate the downstream pathway well,but the 2 mmol/L ATP plus 1μg/m L LPS had good compatibility,and he downstream pathway can also be activated very well.Therefore,we selected the 1μg/m L LPS and 2mmol/L ATP as the drug concentration of the follow-up experiment.The results of Real time PCR showed that the NLRP3 mRNA levels of LPS group and LPS+ATP group were significantly increased compared with the control group,the difference was statistically significant(P<0.001,P<0.001),what’s more compared with LPS group,the level of NLRP3 mRNA of LPS+ATP group was increased,and the difference was statistically significant(P<0.05).Compared with the control group,the expression of P2X4 mRNA was significantly increased after adding 2mmol/L ATP to treat BV2 cell 3 h,and the difference was statistically significant(P<0.001).The western blot results showed that,compared with the control group,the expression of P2X4 protein in BV2 was increased significantly after adding ATP(2 mmol/L)treatment for 3 h,and the difference was statistically significant(P<0.001).Compared with the control group,the BV2 cells after LPS + ATP dual signal processing,their NLRP3 protein level increased,and the difference was statistically significant(P<0.001),but treated with LPS or ATP alone,the BV2 cells NLRP3 protein levels group has no statistical significance compared with the control(P > 0.05).Compared to control group,caspase-1(P10)changes in protein levels of LPS group and ATP group have no statistical significance(P > 0.05),but LPS + ATP group of caspase-1(P10)protein expression level increased significantly,the difference was statistically significant(P<0.001),however,after adding P2X4 selective blocker 5-BDBD,the results indicated that the expression level of caspase-1(P10)protein in LPS+ATP+ 5-BDBD group was significantly decreased compared with LPS+ATP group,and the difference was statistically significant(P<0.001).ELISA was used to detect the content of IL-1β of each group,and the results indicated that there was no statistically significant change in the content of IL-1β in the LPS group and ATP group compared with the control group(P>0.05),after the LPS+ATP dual signal processing BV2 cells,the content of IL-1β in the supernatant was significantly increased,and the difference was statistically significant(P<0.01).However,after adding P2X4 specific blocker 5-BDBD,the results indicated that compared with LPS+ATP group,the content of IL-1β was significantly decreased in LPS+ATP+5-BDBD group,and the difference was statistically significant(P<0.05).Conclusion P2X4 specific blocker 5-BDBD can inhibit the inflammatory effect of NLRP3 inflammatory in BV2 cells processing with LPS+ATP dual signal,these include the activation of caspase-1(P10)and the production of IL-1β,which will provide new ideas for the anti-inflammatory treatment of neurodegenerative diseases.