HMGB1诱导的小鼠系膜细胞增殖与Akt/NF-κB信号通路激活的相关性

Correlation of HMGB1-induced proliferration of MMC cells and activation of PI3K/Akt signal pathway

目的探讨Akt/NF-κB信号途径激活在高迁移率族蛋白1(high mobility group protein box 1,HMGB1)诱导小鼠系膜细胞增殖中的作用及意义。方法将常规培养的小鼠系膜细胞随机分为正常对照组和100μg/L HMGB1刺激组;HMGB1刺激+sh-Akt组(质粒转染组)、HMGB1刺激+sh-NC组(空白质粒组)分别培养10 min、20 min、30 min、40 min、50 min、1 h和8 h收集细胞,采用Real-time PCR及Western blot技术检测Akt、p-Akt、PTEN、PCNA、Cyclin D1、CDK4、p16的表达,免疫荧光技术检测BrdU和NF-κB p65蛋白表达。结果 (1)重组的HMGB1能够促进小鼠系膜细胞Akt ser473位点磷酸化和NF-κB核转位;(2)沉默细胞中Akt表达能够显著降低HMGB1诱导的系膜细胞增殖;BrdU阳性率降低;(3)沉默Akt表达显著降低HMGB1诱导的系膜细胞中Cyclin D1、CDK4的表达,上调p16表达;(4)沉默Akt表达能够降低HMGB1诱导的NF-κB核转位,细胞核内NF-κB p65阳性表达降低。结论 PI3K/Akt激活参与HMGB1介导的小鼠系膜细胞增殖,促进NF-κB核转录,激活Cyclin D1/CDK4/p16信号途经可能是其重要的机制之一。

Purpose To explore the effect of Akt/NF-κB signal pathway on cell proliferation of MMC cells induced by HMGB1. Methods MMC was randomly divided into four groups： normal control, HMGB1, HMGB1 plus sh-Akt and HMGB1 plus sh-NC groups. The cells were collected at 10 min, 20 min, 30 min, 40 min, 50 min, 1 h and 8 h, respectively. RNAi method was used to knock down the expression of Akt. qPCR and Western blot were used for the detection of Akt, p-Akt, PTEN, PCNA, cyclinD1, CDK4 and pl6 expression. Immunofluorescent staining was used to detect the BrdU and NF-κB p65 expression. Results （ 1 ） HMGB1 induced Akt phosphorylation and NF-κB p65 nuclei dislocation. （2） Knockdown of Akt expression markedly prevented HMGBl-induced cell proliferation. The positive ratio of BrdU and PCAN expression decreased as compared with HMGBl-treated group. （3） The sh-Akt vector effectively decreased cyclin D1 and CDK4 expression and up-regulated pl6 expression as compared with HMGBl-treated group; （4） Knock-down of Akt downregulated NF-κB p65 nuclei dislocation induced by HMGB1. Conclusion Activation of PI3K/Akt signal pathway could induce NF-κB p65 nuclei dislocation and activate cyclinD1/CDK4/pl6 signal pathway, and thus mediate HMGBI-induced MCC cells proliferation.