金雀异黄素调控miR-21表达促进LPS活化的RAW264.7细胞凋亡

Genistein regulates miR-21 to promote the apoptosis in LPS-activated RAW264.7 cells

本文旨在研究金雀异黄素(genistein, GEN)对脂多糖(lipopolysaccharide, LPS)活化的RAW264.7细胞凋亡的影响,探讨GEN抗动脉粥样硬化的药理学作用机制。应用LPS活化RAW264.7细胞, qRT-PCR检测肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)和白介素-6 (interleukin 6, IL-6) mRNA表达, Western blot检测环氧合酶-2(cyclooxygenase-2, COX-2)和诱导型一氧化氮合酶(inducible nitric oxide synthases, iNOS)蛋白表达。使用GEN预处理细胞2 h,再与LPS共孵育24 h后, CCK8检测细胞活力, Annexin V-FITC/PI法检测细胞凋亡, qRT-PCR检测CHOP、caspase-3和miR-21基因表达, Western blot检测CHOP和caspase-3蛋白表达。结果显示, 1 000 ng·mL-1 LPS上调RAW264.7细胞TNF-α、IL-6、COX-2和iNOS基因或蛋白表达; GEN呈浓度依赖性下调LPS活化的RAW264.7细胞活力,增加凋亡率,上调CHOP和caspase-3表达,并下调miR-21表达;慢病毒介导的miR-21 up抑制LPS活化的RAW264.7细胞CHOP和caspase-3表达,与GEN作用相反;慢病毒介导的miR-21 down促进LPS活化的RAW264.7细胞CHOP和caspase-3表达,与GEN具有协同作用。这些结果提示,金雀异黄素能够促进LPS活化的RAW264.7细胞凋亡,其作用机制可能与下调miR-21表达,激活内质网应激反应性凋亡途径有关。

The research is aimed to investigate the effect of genistein(GEN) on the apoptosis in lipopolysac‐charide(LPS)-activated RAW264.7 cells and explore the pharmacological mechanism of GEN anti-atherosclerosis(AS). RAW264.7 cells were activated by LPS, the level of TNF-α and IL-6 mRNA were detected by qRT-PCR, the expression of COX-2 and iNOS were detected by Western blot. RAW264.7 cells were pretreated with GEN for 2 h,and then incubated with LPS for 24 h. After that, CCK8 kit was used for the cell viability, Annexin V-FITC/PI kit for the apoptosis of cell. qRT-PCR was used to detect the level of CHOP, caspase-3 and miR-21. Western blot was used to detect the expression of CHOP and caspase-3. Results showed that LPS(1 000 ng · mL-1) increased the expression of TNF-α, IL-6, COX-2 and iNOS in RAW264.7 cells compared with that in control group. GEN inhibited the cell activity and the level of miR-21, promoted the expression of CHOP and caspase-3 in LPS-activated RAW264.7 cells in a dose-dependent manner. miR-21 up inhibited the expression of CHOP and caspase-3 in LPS-activated RAW264.7 cells and this process was reversed by GEN treatment. miR-21 down promoted the expression of CHOP and caspase-3, which were further enhanced by GEN. These results indicate that GEN promotes the apoptosis of RAW264.7 cells activated by LPS through down regulating miR-21 and activating endoplasmic reticulum(ER) stress pathway.