沉默信息调节因子2同源蛋白1通过Akt/PKB通路抑制人退变椎间盘髓核细胞凋亡

Sirtuin 1 Maitains Survival via PKB Signaling in Degenerative Human Disc Nucleus Pulposus Cells

沉默信息调节因子2同源蛋白1(silent mating type information regulation 2 homolog 1,SIRT1/sirtuin 1)是组蛋白去乙酰化酶,参与表观遗传修饰调节,促进多种细胞的生存,但目前对椎间盘髓核细胞的作用未见研究.为了阐明临床不同来源的椎间盘髓核手术标本SIRT1的表达变化,用免疫组化、定量RT-PCR、Western blot方法对中老年腰椎间盘突出症病人及青壮年腰椎骨折病人术中髓核标本进行研究,表明老年人髓核SIRT1的mRNA及蛋白质水平均显著低于青壮年的髓核.同时,用resveratrol(SIRT1激动剂)、烟碱(SIRT1抑制剂)、SIRT1-siRNA对培养的退变髓核细胞进行处理或转染后,用流式细胞仪检测凋亡率变化,结果表明resveratrol能显著促进退变髓核细胞生存,相反,当烟碱或SIRT1-siRNA转染后则显著促进髓核细胞凋亡.为了进一步分析SIRT1抑制退变髓核细胞凋亡的分子机制,应用Western blot及抑制剂方法研究表明,当SIRT1-siRNA转染髓核细胞后能降低磷酸化Akt蛋白的表达,而白藜芦醇处理则促进磷酸化Akt蛋白的表达,当用LY294002(PI3K抑制剂)或Akt-siRNA转染后显著抑制髓核细胞的生存率.研究结果表明,SIRT1通过Akt通路能显著抑制髓核细胞凋亡,为深入揭示退变性椎间盘疾病的病理生理及生物治疗提供新的思路和靶点.

Many studies have demonstrated that Sirtuin 1, an NAD＋-dependent deacetylase, reduces apoptosis in several different cells. However, the role of Sirtuin 1 in apoptosis of disc nucleus pulposus （NP） cells remains unclear. The present study was performed to determine whether degenerative human NP would express Sirtuin 1, and to investigate the role of Sirtuin 1 in NP cells apoptosis. Here we show that Sirtuin 1 mRNA and protein levels in disc NP from patients （〈 25 years） with lumbar vertebra fracture （LVF） are greater than those in degenerative disc NP from patients （〉 55 years） with lumbar disc herniation （LDH）. The rate of apoptosis was far fewer in resveratrol-treated NP cells than that in siRNA transfected or nicotinamide-treated NP cells. We also tried to explore the signaling molecules that mediate the protective property of Sirtuin 1 by Western blot and inhibitor analysis. After Sirtuin 1 siRNA transfected, NP cells decreased phosphorylation of Akt, while resveratrol phosphorylated Akt. Treatment with LY294002 or Akt siRNA increased the rate of apoptosis. Our results indicate that Sirtuin 1 plays a critical role in survival of degenerative human NP cells through the Akt anti-apoptotic signaling pathway.