PTEN对ox-LDL诱导的巨噬细胞炎性因子的影响及机制研究

Effect of phosphatase and tensin homolog deleted on chromosome ten on ox-LDL-induced inflammatory cytokine secretion from macrophages and its underlying mechanism

目的:分析第10号染色体缺失的磷酸酶和张力蛋白同源基因(PTEN)对氧化低密度脂蛋白(ox-LDL)诱导的巨噬细胞炎症因子影响及其作用机制。方法:将构建pc DNA3.1(+)-PTEN(r PTEN)重组表达载体及PTEN siRNA转染小鼠巨噬细胞系RAW 264.7,用制备的50 mg/L的ox-LDL孵育24 h,RT-PCR及Western blot分析PTEN的mRNA及蛋白表达水平;ELISA检测炎性因子TNF-α和IL-6的水平;同时Western blot分析对Toll样受体4(TLR4)及转录因子-κB(NF-κB)的影响及其作用机制。结果:PTEN过表达增高了ox-LDL诱导的TNF-α和IL-6的水平;PTEN沉默抑制了ox-LDL诱导的TNF-α和IL-6炎性因子水平。进一步分析表明,PTEN过表达加强了巨噬细胞中ox-LDL诱导的TLR4及其下游NF-κB通路的活化,而抑制其表达后,TLR4-NF-κB通路明显受到抑制;用TLR4特异性抗体预处理后,PTEN过表达诱导的TNF-α和IL-6的水平明显下降。进一步机制分析证实,磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)抑制剂LY294002(1μmol/L)预处理后,PTEN沉默抑制的TLR4-NF-κB通路的活性明显增加,且伴随有TNF-α和IL-6水平的上调。结论:PTEN有可能通过负向调节PI3K/AKT抗炎通路来影响TLR4-NF-κB炎性通路,进而参与巨噬细胞介导的炎症进程。因此,本研究将为心脑血管疾病的防治提供新的靶标。

AIM：To investigate the effect of phosphatase and tensin homolog deleted on chromosome ten （PTEN） on ox-LDL-induced inflammatory cytokine levels in macrophages and its underlying mechanism. METHODS： The constructed pcDNA3.1（＋）-PTEN （rPTEN） recombinant vector and PTEN siRNA were transfected into RAW 264.7 followed by stimulation with 50 mg/L ox-LDL for 24 h. Real-time PCR （RT-PCR） and Western blotting were used to analyze the expression levels of PTEN mRNA and protein. ELISA was used to detect the inflammatory cytokine levels of TNF-α and IL-6. The expression level of TLR4-NF-κB and its underlying mechanism were also explored. RESULTS： Overexpression of PTEN enhanced ox-LDL-induced inflammatory cytokine levels of TNF-α and IL-6, whereas its silencing attenuated this progress. PTEN upregulation increased the expression of ox-LDL-induced TLR4 and its downstream p65 NF-κB. However, silencing PTEN expression with PTEN siRNA significantly decreased ox-LDL-triggered activation of TLR4-NF-κB signaling. Further mechanism analysis demonstrated that preconditioning with PI3K/AKt specific inhibitor LY294002 （1 μmol/L） dramatically augmented PTEN silencing-decreased activation of TLR4-NF-κB pathway, accompanied by the increase in TNF-α and IL-6 levels. CONCLUSION： PTEN may regulate macrophage inflammatory by PI3K/AKt-TLR4-NF-κB pathway. This study thus provides a potential target for the treatment of cardiovascular diseases.