高迁移率族蛋白1对Aβ_(25-35)刺激下BV-2细胞NF-κB表达的作用

Eeffect of HMGB1 on expression of NF-κB in BV-2 cells stimulated with Aβ_(25-35)

目的:探讨高迁移率族蛋白1(HMGB1)对β-淀粉样蛋白(Aβ)_(25-35)刺激下的小鼠小胶质细胞系BV-2中核因子-κB(NF-κB)表达的影响。方法:将对数期生长的BV-2细胞分为4组:正常细胞组(不做任何处理)、模型组(加入40μmol/L Aβ_(25-35))、RNA干扰组(采用RNA干扰HMGB1后加入40μmol/L的Aβ_(25-35))和溶剂对照组(加入终浓度为0.1%的DMSO)。各组细胞孵育72 h后(Aβ_(25-35)处理24 h)采用Western blot法检测HMGB1和NF-κB p65的蛋白表达情况。结果:CCK-8结果显示40μmol/L Aβ_(25-35)为最佳造模浓度。选择30 nmol/L带GFP荧光基团的HMGB1-siRNA转染BV-2细胞,转染效率约为80%~90%。Western blot实验结果显示,HMGB1-siRNA片段干扰后BV-2细胞中HMGB1的表达明显降低(P<0.05);Aβ_(25-35)刺激BV-2细胞后,胞内HMGB1和核内NF-κB p65表达均明显升高(P<0.05),HMGB1-siRNA作用后,HMGB1和核内NF-κB p65表达均明显下降(P<0.05)。结论:RNA干扰HMGB1表达可减少Aβ_(25-35)刺激的BV-2细胞核内NF-κB的表达。

AIM: To investigate the effect of high mobility group box-1 protein( HMGB1) on the expression of nuclear factor-κB( NF-κB) in BV-2 cells stimulated with amyloid β-protein( Aβ)_(25-35). METHODS: Cultured BV-2 cells in logarithmic growth phase were divided into 4 groups: normal cell group( without any treatment),model group( treated with Aβ_(25-35) at 40 μmol/L),RNA interference( RNAi) group( conducted with HMGB1-siRNA followed by Aβ_(25-35) stimulation) and solvent control group( treated with 0. 1% DMSO). After treatment with Aβ_(25-35) for 24 h,the protein levels of HMGB1 and NF-κB in BV-2 cells were determined by Western blot. RESULTS: Aβ_(25-35) at 40 μmol/L was used to stimulate BV-2 cells. The GFP fluorescence-tagged HMGB1-siRNA( 30 nmol/L) was used to transfect BV-2 cells and its transfection efficiency was about 80% ~ 90%. The results of Western blot showed that the protein level of HMGB1 was significantly decreased after the interference of siRNA fragment( P < 0. 05). The protein levels of HMGB1 and nucleic NF-κB p65 were dramatically increased in BV-2 cells stimulated with Aβ_(25-35)( P < 0. 05). After RNA interference with HMGB1,the expression of HMGB1 and nucleic NF-κB p65 were significantly decreased in BV-2 cells stimulated with Aβ_(25-35)( P < 0.05). CONCLUSION: RNA interference with HMGB1 reduces the expression of nucleic NF-κB in BV-2 cells stimulated with Aβ_(25-35).