miR-129-5p对LPS引起星形胶质细胞炎性介质释放的影响和机制

The effects and action mechanism of miR-129-5p on the release of inflammatory mediators from astrocytes induced by LPS

目的探讨miR-129-5p对脂多糖(LPS)引起星形胶质细胞炎性介质释放的作用和机制。方法分离培养大鼠星形胶质细胞,分成Control组(空白对照)、LPS组(LPS处理)、miR-NC+LPS组(转染mimicscontrol后用LPS处理)、miR-129-5p+LPS组(转染miR-129-5pmimics后用LPS处理),用qRT-PCR方法测定miR-129-5p表达,以ELISA法测定IL-6、IL-1β、TNF-α水平,以Griess法检测NO水平,Western blot检测高迁移率族蛋白B1(HMGB1)蛋白表达。利用生物信息学软件(targetscan)预测miR-129-5p的靶基因,荧光素酶报告系统鉴定二者的靶向关系。将miR-129-5pmimics、pcDNA3.1-HMGB1共转染到星形胶质细胞中,然后以LPS细胞培养液培养,同样测定IL-6、IL-1β、TNF-α、NO水平。结果与Control组比较,LPS组细胞中miR-129-5p水平降低,培养液上清中IL-6、IL-1β、TNF-α、NO水平升高,HMGB1蛋白水平升高(P<0.05)。与miR-NC+LPS组比较,miR-129-5p+LPS组细胞中miR-129-5p水平升高,培养液上清中IL-6、IL-1β、TNF-α、NO水平降低,HMGB1蛋白水平下降(P<0.05)。miR-129-5p和HMGB1互为靶向关系。pcDNA3.1-HMGB1可以逆转miR-129-5p mimics对LPS引起的星形胶质细胞IL-6、IL-1β、TNF-α、NO释放影响。结论miR-129-5p抑制LPS引起的星形胶质细胞炎性介质释放,机制与下调HMGB1有关。

Objective To investigate the effects and action mechanism of miR-129-5p on release of lipopolysaccharide(LPS)-induced inflammatory mediator from astrocytes.Methods The rat astrocytes were isolated,cultured,and divided into Control group(blank control),LPS group(LPS treatment),miR-NC+LPS group(transfection with mimicscontrol and treatment with LPS),miR-129-5p+LPS group(transfection with miR-129-5p mimics and treatment with LPS).The expression of miR-129-5p was measured by qRT-PCR method,the levels of IL-6,IL-1β,and TNF-αwere measured by ELISA,and the level of NO was measured by Griess method.Western blot was used for detection of expression of high mobility group protein B1(HMGB1).Bioinformatics software(targetscan)was used to predict the target genes of miR-129-5p,and the luciferase reporter system was used to identify the target relationship between the two.The miR-129-5 pmimics and pcDNA3.1-HMGB1 were co-transfected into astrocytes,and then cultured with LPS cell culture medium,and the levels of IL-6,IL-1β,TNF-α,and NO were also measured.Results Compared with those in control group,the levels of miR-129-5p in LPS group were decreased,but,the levels of IL-6,IL-1β,TNF-α,and NO in the culture supernatant were increased,and the protein levels of HMGB1 were increased(P<0.05).Compared with those in miR-NC+LPS group,the miR-129-5p levels in miR-129-5p+LPS group were increased,however,the levels of IL-6,IL-1β,TNF-α,and NO in culture supernatant were decreased,and the protein levels of HMGB1 were significantly decreased(P<0.05).And miR-129-5p and HMGB1 were mutually targeted.Moreover pcDNA3.1-HMGB1 could reverse the effect of miR-129-5p mimics on the release of IL-6,IL-1β,TNF-αand NO in astrocytes caused by LPS.Conclusion The miR-129-5p can inhibit the release of inflammatory mediators in astrocytes caused by LPS,and its action mechanism may be related to the down-regulation of HMGB1.