Mechanisms Of MiR-199a-5p/SIRT1 Mediated NLRP3 Inflammasomes To Regulate LPS-Induced Inflammatory Injury In BV-2 Cells In Vitro

Objective: The purpose of this study is to explore the biological regulatory function of miR-199a-5p on SIRT1 by constructing the inflammation model of BV-2 cells in vitro,and to preliminarily explain the molecular mechanism of miR-199a-5p regulating inflammatory injury in the inflammation model of BV-2 cells in vitro,so as to provide a new theoretical basis for the intervention and treatment of secondary inflammatory injury after Intracerebral Hemorrhage.Methods:1.Establishment and validation of BV-2 cells inflammation model in vitro,detection of miR-199a-5p and SIRT1 expression,subcellular localization of SIRT1: CCK-8 test was used to detect cell viability,qRT-PCR,ELISA and Western blot were used to detect the relative expression of miR-199a-5p,SIRT1 and pro-inflammatory factors(IL-1 ?,IL-6).Immunofluorescence staining showed that SIRT1 was located in the subcellular localization of BV-2 cells.2.To verify the binding site and regulatory relationship of miR-199a-5a to SIRT1: The binding site of miR-199a-5a and SIRT1 was predicted by TargetScan,miRTarBase and other bioinformatics software,and was verified by Dual-luciferase reporter gene experiment.BV-2 cells were treated with biosynthetic miR-199a-5p mimic/inhibitor and negative control respectively,and the relative expression levels of miR-199a-5p and SIRT1 were determined by qRT-PCR and Western Blot methods.3.Research on the mechanism of miR-199a-5p in the inflammation model of BV-2 cells: CCK-8 experiment was used to determine cell viability,qRT-PCR,ELISA and Western Blot methods were used to determine SIRT1,NLRP3,Cleared-Caspase1,ASC and the relative expression of pro-inflammatory factors(IL-1?,IL-6).Results:1.Lipopolysaccharide(LPS)stimulated BV-2 cells,the morphology of BV-2 cells changed,the viability of BV-2 cells decreased significantly compared with the control group(P < 0.05),and the expression of pro-inflammatory factors IL-1? and IL-6 increased significantly compared with the control group(P<0.05).2.In the inflammation model of BV-2 cells,the expression level of miR-199a-5p was significantly increased compared with the control group(P<0.05);the expression levels of SIRT1 mRNA and protein were significantly lower than that of the control group(P<0.05).3.BV-2 cells were transfected with miR-199a-5p mimic/inhibitor and negative control respectively.The relative expression of miR-199a-5p in miR-199a-5p mimic group was significantly higher than that of mimic NC group(P<0.05),The expression level in the miR-199a-5p inhibitor group was significantly lower than the inhibitor NC group(P<0.05);In the BV-2 cell inflammation model,the LPS+miR-199a-5p mimic group was compared with the LPS+mimic NC group,cell viability was decreased significantly(P<0.05),the relative expression of pro-inflammatory factors IL-1? and IL-6 increased significantly(P<0.05),and the concentration of IL-1? and IL-6 in the culture supernatant increased significantly(P<0.05);LPS+miR-199a-5p inhibitor group compared with LPS+inhibitor NC group,cell viability was significantly increased(P<0.05),the expression levels of pro-inflammatory factors IL-1? and IL-6 were significantly reduced(P<0.05),the concentrations of IL-1? and IL-6 in the culture supernatant were significantly reduced(P<0.05).4.Cell immunofluorescence staining showed that SIRT1 was mainly expressed in the nucleus of BV-2 cells.5.The results of the dual-luciferase test showed that the fluorescence intensity of the wild-type reporter gene co-transformed was significantly lower than that of the mutant reporter gene(P<0.05).miR-199a-5p mimic/inhibitor and negative control were transfected into BV-2 cells respectively.Compared with mimic NC group,miR-199a-5p mimic group had significantly lower SIRT1 mRNA and protein expression(P<0.05);in miR-199a-5p inhibitor group compared with inhibitor NC group,the expression of SIRT1 mRNA and protein was significantly increased(P<0.05).7.BV-2 cells were transfected with miR-199a-5p mimic/miR-199a-5p inhibitor and mimic NC/inhibitor NC respectively,and then BV-2 cell inflammation model was constructed.LPS group compared with Control group,SIRT1 protein expression level was significantly reduced(P<0.05),NLRP3,ASC,Cleaved-Caspase1 protein expression levels were significantly increased(P<0.05);LPS+miR-199a-5p mimic group compared with LPS+mimic NC group,SIRT1 protein expression level was significantly reduced(P<0.05),NLRP3,ASC,Cleaved-Caspase1 protein expression levels were significantly increased(P<0.05);LPS+miR-199a-5p inhibitor group compared with LPS+inhibitor NC group,SIRT1 protein expression level was significantly increased(P<0.05),NLRP3,ASC,Cleaved-Caspase1 protein expression levels were significantly reduced(P<0.05).Conclusion: In the inflammation model of BV-2 cells,miR-199a-5p can target SIRT1 and inhibit the expression of SIRT1,thereby activating NLRP3 inflammasome,which further aggravating the LPS-induced inflammatory injury of BV-2 cells.