Effect Of PP2X4R Gene On Inflammatory Injury Of Microglia Mediated By P2X4/NLRP3 Inflammatory Corpuscle Pathway

Objective Neurodegenerative diseases are the current research hotspots of the central nervous system.Recent studies have focused on the effects of immune responses and inflammatory mechanisms on their development,especially the cellular immunity and humoral immunity mediated inflammatory responses.The mechanism not only expressed in the neuronal cells,but also in microglia of the central system.This article explores the effect of lentivirus（LV-P2rx4）silencing of the P2X4 gene on P2X4/NLRP3 inflammatory avenue pathway-mediated activation of microglial inflammatory injury.Methods The infection pre-experiment was first performed to determine the optimal infection conditions for lentivirus-infected BV2 microglia.After that,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.BV2 microglia cells were randomly divided into6 groups according to different intervention methods:blank control group,activation model group,target gene negative control group,target gene negative control group+activation model group,empty virus negative control group,empty virus negative control group+activation model group.After the above-mentioned cells were treated,ribonucleic acid（RNA）and protein were extracted from each well,and the relative expression of NLRP3 and TNF-αmRNA and protein in the extracted RNA and protein were detected by Real time PCR and Western blots,respectively.Results1.The results of pre-experimental on lentivirus（lv-p2rx4）infection of BV2 microglia cells under electron microscope fluorescence showed that the fluorescence abundance of the conventional medium group with 50μg·mL^-11 Polybrene was higher than that of the control group,the conventional medium group,ENi.S group,ENi.S+Polybrene group,and the fluorescence abundance of the lentivirus with a density of 6×10⁷TU/mL was higher than that of the lentivirus with a density of 4×10⁷TU/mL and 8×10⁷TU/mL.Thus,the lentivirus density was determined to be 6×10⁷TU/mL,and the infection efficiency was the best in the conventional medium group with 50μg·mL^-11 Polybrene added.2.Real time PCR results showed that the expression of NLRP3 and TNF-αmRNA was significantly increased in the BV2 microglia group after LPS and ATP double signal treatment,indicating that the inflammatory model was successfully established.The mRNA expression levels of NLRP3 and TNF-αin the target gene negative control+activation model group were significantly lower than those in the activation model group and the empty virus negative control+activation model group,the difference was statistically significant(P_NLRP3<0.001,F_NLRP3=116.6;P_TNF-α<0.001,F_TNF-α=195.4).3.The results of Western blot showed that the expression of NLRP3 and TNF-αprotein in LPS and ATP groups was significantly higher than that in the un-added group,which was consistent with the Real time PCR results.It was verified that the microglia activation model was successfully established.Compared with the activation model group and the empty virus negative control group,the protein expression in the target gene negative control+activation model group was significantly reduced,and the difference was statistically significant(P_NLRP3<0.001,F_NLRP3=84.25;P_TNF-α<0.001,F_TNF-α=60.10).Conclusion Silencing of the P2X4R gene in microglia can down-regulate the expression of mRNA and protein in activated glial cells NLRP3 and TNF-α,which may impair the inflammatory response of microglia.This will provide new therapeutic prospects for neurodegenerative diseases caused by inflammation.