Effect Of A151 On The Polarization Of Microglia Induced By Glucose And Oxygen Deprivation And Lipopolysaccharide

BackgroundCerebral infarction(CI)is a clinical disease that is caused by disease and thrombosis of the main arterial vascular wall supplying brain tissue,and hypoxia and necrosis of the corresponding brain area,followed by functional impairment.The necrotic cells,fragments and oxygen-active substances appearing after cerebral infarction can stimulate the apoptosis and activation of microglia and trigger neuro-inflammatory response.Microglia belong to the mononuclear-phagocytic cell family.They originate from monocytes in the blood system and then settle in the central nervous system.They become immune cells that can swallow and remove foreign bodies and promote tissue repair.After cerebral infarction occurs,the necrotic cells,oxygen free radicals and other substances in the brain rapidly activate microglia.There are two types of activated microglia,one is called classical activation type(M1 type),which Typical features are high expression of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),nitric oxide synthase(iNOS),CD16/CD32,etc.Another type called selective activation type(M2 type),M2 type microglia is characterized by high expression of arginase-1(Arg-1),CD206,interleukin-10(IL-10),Interleukin-4(IL-4)and so on.Studies have found that after cerebral infarction occurs,it promotes the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype,plays a role in regulating inflammation,thereby reducing brain damage.The inhibitory oligonucleotide A151 is derived from the structure of telomeres.Studies have suggested that the release of telomeres from host cells can reduce inflammation.The structure of A151 is composed of a repeating sequence of 4 TTAGGG bases,bound to the phosphorothioate(PO)backbone.In recent years,A151 has been found to have the ability to regulate inflammation,including regulating the production of tumor necrosis factor-α(TNF-α)and interleukin 1β.Current studies have found that intraperitoneal injection of A151 in cerebral infarction rats can effectively reduce the volume of cerebral infarction,reduce cerebral edema,and promote functional recovery.In vitro studies have confirmed that A151 intervention significantly reduces the release of inflammatory factors from bone marrow-derived macrophages.Based on the results of previous studies,this article mainly studies the effect of A151 on the M1/M2 polarization of BV-2 microglia induced by glucose and oxygen deprivation(OGD)and lipopolysaccharide(LPS),and preliminary discussion the mechanism of action.Method1.ReagentA151 purchased from invivogen,USA2.CellBV-2 microglia come from the research group3.CCK-8 testWhen the BV-2 microglia are in good condition,resuspend the cells and seed them in a 96-well plate(1x104/well),and place them in an incubator overnight.BV-2 microglia were incubated with different concentrations of A151 or Control ODN(0μg/ml、25μg/ml、50μg/ml、75μg/ml、100μg/ml)for 24 hours.Adding 10 μ1 CCK-8 and incubating for 1-4 hours.Setting the microplate reader to 450 nm wavelength to measure the absorbance.4.Experimental groupingThere are four groups of experiments:Control group,LPS+OGD group,A151 group and Control ODN group.The cells in the Control group were not treated.In LPS+OGD group,cells were stimulated with LPS(10 ng/ml)for 24 hours and OGD for 4 hours.After the cells were treated with LPS(10ng/ml)stimulation for 24 hours and OGD stimulation for 4 hours in the A151 group and Control ODN group,the cells were treated with 50μg/ml A151 and 50μg/ml Control ODN for 24 hours,respectively.5.Experimental method5.1 Observation of cell morphologyBV-2 microglia were seeded on 6-well plate(2×105/well)and divided into Control group,LPS+OGD group,A151 group and Control ODN group,with 3 parallel wells in each group.The cells were processed according to the method in the experimental group,and the morphological changes of the cells were observed using an inverted microscope.5.2 ELISABV-2 microglia were seeded on 6-well plate(2×105/well),and divided into Control group,LPS+OGD group,A151 group and Control ODN group.The cells were treated according to the above experimental grouping method,and the supernatant was collected.The contents of TNF-α、IL-1β、IL-4、IL-10 were detected5.3 RT-PCRCell grouping and treatment were the same as ELISA.Take each group of cells after treatment to extract mRNA,and perform RT-PCR according to the one-step RT-PCR kit.RT-PCR reaction conditions:pre denaturation at 94℃ for 2min→[denaturation at 94℃ for 30s→annealing at 56℃ for 30s→extension at 72℃for 1 min]×35 cycles→extension at 72℃ for 5min.The relative expression of mRNA was 2-ΔΔCT,ΔCT=CT value of target gene-CT value of internal reference gene,-ΔΔCT=average value of ΔCT of Control group-ΔCT value of each sample.5.4 Western BlottingCell grouping and treatment were the same as ELISA.Collect the cells of each group and lyse the cells with RIPA lysate to obtain total protein.Take 10ug of protein for electrophoresis and electrophoresis under constant pressure 80v.After the marker runs out of the concentrated gel,switch to constant pressure 100v electrophoresis for 90 minutes,Transfer the film for 90min under the condition of constant current 210 mA,and then blocked with 5%skimmed milk powder for 90min.Then the first antibody was blocked at 4℃ overnight:Anti-β-actin(1:2000),anti-Arg-1(1:500),anti iNOS(1:1000),anti-CD16/CD32(1:1000),anti-CD206(1:500),anti-p-NF-κB p65(1:1000),anti-NF-κB p65(1:1000),anti-IκBα(1:1000),anti-p-IκBα(1:1000),anti-NLRP3(1:1000),anti-Caspase-1(1:500)were blocked for 45 minutes at room temperature the next day.Results Image J software was used for gray analysis.5.5 ImmunofluorescenceCoat the cell slide with polylysine and place it in a 24-well plate.Add 500ul cell suspension(3×104 cells/well)to each well and set up the Control group,LPS+OGD group,A151 group,and Control ODN group.After the cells were treated according to the method in the experimental group,the cell slide was taken out.After washing with PBS,4%paraformaldehyde was added to cover the cells sufficiently for fixation.After washing with PBS,0.1%Triton X-100 was added to the cell slide for permeability,and then blocked with 10%goat serum.Add a sufficient amount of diluted primary antibody to each slide:anti-CD 16/CD32(1:200),anti-CD206(1:200)and incubate overnight at 4℃ in a humid box.The next day,after PBS washing,add appropriate amount of fluorescent secondary antibody,incubate in wet box at room temperature for 45min,add a drop of anti-quenching agent containing DAPI.After mounting the slides with glycerol,observe the collected images under an inverted fluorescence microscope,and analyze the fluorescence images with Image J software.6.Statistical AnalysisAll data were analyzed by graphpad prism 8.0.1 software.All data were in accordance with normal distribution by Shapiro-Wilk test,expressed as x±s.One way ANOVA was used to analyze the differences between the groups,Tukey’smultiple comparison analysis was used to compare the pairwise multiple within the group,P<0.05 was considered as statistically significant(two tailed)Result1.CCK-8 results showed that 0-100μg/ml of A151 had no inhibitory effect on the viability of BV-2 microglia,and 0-75μg/ml of Control ODN did not decrease the viability of BV-2 microglia,but 100μg/ml of Control ODN significantly decreased the viability of BV-2 microglia.Referring to the results of CCK-8 and other literatures,50ug/ml was used as the experimental concentration of A151 and Control ODN.2.Observation of BV-2 microglial morphology by inverted phase contrast optical microscope.It was found that BV-2 microglia in Control group were in resting state,and the cell size was small,round or oval.In LPS+OGD group,most of the cells showed activation characteristics,and the cell morphology showed obvious long shuttle shape,large and round cell body,and the cell body appeared fine and long processes around.The cell state of A151 group was significantly improved than that of LPS+OGD group,and the protuberance became shorter and the cell body was shorter and the cell body was in the shape of long shuttle ellipse.The cells of Control ODN group were not significantly different from LPS+OGD group.3.ELISA and RT-PCR showed that tnf-α,IL-1β release and gene transcription increased in LPS+OGD group,and the release level and gene transcription of IL-10 and IL-4 decreased or not significantly changed.A151 group inhibited tnf-α、IL-1βrelease and gene transcription,promoted the release and gene transcription of IL-10 and IL-4 in supernatant.However,the Control ODN group had no obvious effect on the release and gene transcription of cytokines TNF-α、IL-1β、IL-10、IL-4.4.Western blotting and immunofluorescence results showed that,compared with the Control group,the protein expressions of the M1 phenotype surface markers iNOS、CD16/CD32 were up-regulated in the LPS+OGD group,and the M2 phenotype surface markers Arg-1、CD206 protein expression were no significant change or decrease in expression.A151 inhibited the protein expression of iNOS and CD16/CD32,and promoted the protein expression of Arg-1 and CD206.The Control ODN group had no obvious effect on the expression of surface markers of M1 and M2 phenotypes.5.Western blotting showed that compared with the Control group,the expressions of NF-κB signaling pathway proteins and NLRP3 inflammasome in the LPS+OGD group were increased:p-NF-κB p65、p-IκBα、NLRP3、Caspase-1,while the A151 group inhibited the expression of p-NF-κB p65、p-IκBα、NLRP3、Caspase-1,while Control ODN has no obvious effect on the expression of p-NF-κB p65、p-IκBα、NLRP3、Caspase-1.Conclusion1.A151 improves the morphology of BV-2 microglia induced by LPS+OGD.2.A151 inhibits the release of pro-inflammatory cytokines and promotes the release of anti-inflammatory cytokines in BV-2 microglia induced by LPS+OGD.3.A151 inhibits the gene transcription of pro-inflammatory cytokines and promotes the gene transcription of anti-inflammatory cytokines in BV-2 microglia induced by LPS+OGD.4.A151 inhibits the M1 phenotype of BV-2 microglia induced by LPS+OGD and promotes its polarization to the M2 phenotype.5.A151 inhibits the activation of NF-κB signaling pathway in BV-2 microglia induced by LPS+OGD.6.A151 inhibits the activation of NLRP3 inflammasome in BV-2 microglia induced by LPS+OGD.