Inhibition Of The Expression Of MKP-1 In BV-2 Microglia On Inflammatory Mediators And Its Mechanisms

BackgroundPsychiatric diseases are often accompanied by abnormal expression of the immuneneuro-endocrine system,which activates peripheral mononuclear cells/ macrophages and intracerebral microglia associated with mental illness,resulting in abnormally elevated levels of inflammatory factors in the body.Inflammatory activation of microglia in patients with depression,and inflammatory activation of microglia with mitogen-activated protein kinase phosphatase-1(MKP-1),mitogen-activated kinase(MAPKs),NF-?B-P65 signal path changes.In this study,BV-2 microglia were used as the research object,and LPS and MKP-1 inhibitors(Ro-318220)were used to induce BV-2 microglia to construct an inflammatory model by detecting MKP-1 protein.And the expression of genes,MAPKs,NF-?B-P65 signaling pathway proteins and microglia inflammatory levels,explore the effect of MKP-1 expression on the inflammatory activation level of microglia and its mechanism.ObjectivesTo investigate the relationship between MKP-1,MAPK family proteins and NF-?B-P65 signaling pathway protein changes and microglial inflammatory activation by detecting LPS inflammatory induced BV-2 microglia and administering Ro-318220,and to understand the MKP-1 and MAPK families.Mechanism of action of protein,NF-?B-P65 signaling pathway and microglial inflammatory factor release.It is preliminarily clarified that MKP-1 mediates the inflammatory activation of microglia through P38,JNK and NF-?B-P65 signaling pathways,which may provide a basis for the hypothesis of depression neurosis.Methods1.Experimental groupingBV-2 microglia were divided into Control group,Ro-318220 group,LPS group and LPS+Ro-318220 group.2.Processing methodBV-2 microglia were given LPS(1ug/ml)to make an inflammatory model,and different concentrations of Ro-318220 were given to inhibit the expression of MKP-1.3.Western blotting was used to detect MKP-1,P-ERK,ERK,P-JNK,JNK,P-P38 and P38,P-P65,P65 and other proteins in four groups of BV-2 microglia.expression.4.The expression of ionized calcium binding adaptor molecule-1(Iba-1)in each group was detected by immunofluorescence technique.5.Quantitative RT-PCR was used to verify the expression of inflammatory cytokines in BV-2 microglia.6.The expression of inflammatory cytokines in BV-2 microglia was verified by ELISA.7.Statistical methodsMeasurement data are expressed as mean ± standard deviation(x±S).Comparison between groups was performed by one-way analysis of variance.Statistical analysis was performed between the two groups.Independent sample t test was used.Two-sided test was used.<0.05 expression was statistically significant.Results1 Detection of MKP-1 expression in BV-2 microglia under different conditions(1)With the increase of drug concentration of Ro-318220(0uM,2.5uM,5uM,10uM),the expression of MKP-1 in microglia gradually decreased.When the drug concentration reached 5uM,MKP-1 in BV-2 microglia The expression decreased,the difference was statistically significant(P<0.05).When the drug concentration reached 10 uM,thedifference was the most significant(P<0.01).(2)When Ro-318220(5uM)was applied to BV-2 microglia for 3h,the expression of MKP-1 in BV-2 microglia decreased,the difference was statistically significant(P<0.05).(3)When LPS(1ug/ml)induced BV-2 microglia(0?30min,1h,3h,6h,12 h,24h),the protein expression level of MKP-1 in BV-2 microglia at 1h The highest was reached,and the difference was statistically significant(P<0.05).2.LPS induces the activation of BV-2 microglia activation markersAfter induction of BV-2 microglia by LPS for 1 h,compared with the control group,the Ro group staining was weak,and the LPS group staining was stronger.Compared with the LPS group,the staining intensity of the LPS+Ro group was decreased,indicating that the expression of Iba-1 was different.3.Western blot analysis of MAPK signaling pathway results(1)The effect of MKP-1 protein level: Compared with the control group,the expression level of MKP-1 protein in LPS group was increased,the difference was statistically significant(P<0.05).Compared with LPS group,MKP-1 protein expression in LPS+Ro group The level was decreased and the difference was statistically significant(P<0.05).Real-time quantitative PCR showed that compared with the control group,the expression level of MKP-1 gene in LPS group was increased,the difference was statistically significant(P<0.05).Compared with LPS group,the expression level of MKP-1 gene in LPS+Ro group was decreased.The difference was statistically significant(P<0.05).(2)Effects of P-P38/P38 protein levels: There were no significant differences in P-P38/P38 protein expression levels between the control group,the Ro group,and the LPS+Ro group.Compared with the LPS group,the expression of P-P38/P38 protein in the LPS+Ro group was increased,and the difference was statistically significant(P<0.05).(3)Effects of P-JNK/JNK protein levels: Compared with the control group,the P-JNK/JNK protein levels in the Ro group were increased,and the difference wasstatistically significant(P<0.05).Compared with the LPS group,the expression level of P-JNK/JNK protein in the LPS+Ro group was increased,and the difference was statistically significant(P<0.05).(4)Effects of P-ERK/ERK protein levels: Compared with the control group,the P-ERK/ERK protein levels in the Ro group were increased,and the difference was statistically significant(P<0.05).There were no significant differences in P-ERK/ERK protein levels between the Ro group,the LPS group,and the LPS+Ro group.4.The effect of P-P65/P65 protein levelCompared with the control group,the expression level of P-P65/P65 protein in microglia in Ro group decreased,and the expression level of P-P65/P65 protein in microglia in LPS group.The difference was statistically significant(P<0.05).Compared with the LPS group,the expression of P-P65/P65 protein in the LPS+Ro group was decreased,and the difference was statistically significant(P<0.05).5.Real-time quantitative PCR was used to detect inflammatory factors.Compared with the control group,the expression levels of IL-6,IL-1?,TNF-? and COX-2 in microglia in the Ro group were decreased,and in the LPS group.The expression levels of IL-6,IL-1?,TNF-? and COX-2 were increased,and the difference was statistically significant(P<0.05).Compared with LPS group,the expression levels of IL-6,IL-1?,TNF-? and COX-2 in LPS+Ro group were significantly lower(P<0.05).6.ELISA was used to detect inflammatory factors.Compared with the control group,the levels of IL-6,IL-1?,TNF-? and secretion in microglia in the Ro group were decreased,and IL-6 and IL-1?in the microglia in the LPS group.The level of TNF-?secretion increased,the difference was statistically significant(P<0.05).Compared with the LPS group,the levels of IL-6,IL-1?and TNF-? in the LPS+Ro group were decreased,and the difference was statistically significant(P<0.05).Conclusions1.MKP-1 may be an important gene in the development of neuroinflammation.2.MAPK-P38,JNK,NF-?B-P65 signaling pathway may be involved in the pathogenesis of neuroinflammation.3.MKP-1 may affect the changes of neuroinflammatory factors through P38,JNK and NF-?B-P65 signaling pathways.