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基质细胞衍生因子1α通过Akt信号通路抑制软骨细胞凋亡并促进自噬

Stromal Cell-Derived Factor 1α Inhibits Chondrocyte Apoptosis and Promotes Autophagy Through the Akt Signaling Pathway

    摘要
  • 摘要:
    目的 探究基质细胞衍生因子1α（stromal cell-derived factor 1α, SDF-1α）对软骨细胞凋亡及自噬的影响及其潜在机制。
    方法 从新生小鼠的膝关节中分离提取软骨细胞，以0（对照组）、50、100和200 ng/mL的SDF-1α刺激软骨细胞，CCK-8实验检测SDF-1α刺激24 h、48 h、72 h对软骨细胞活性的影响，划痕实验检测SDF-1α刺激12 h、24 h对软骨细胞迁移能力的影响，Western blot实验测定SDF-1α作用后软骨细胞内Akt信号通路相关蛋白表达变化。以0（对照组）、200 ng/mL的SDF-1α刺激软骨细胞，流式细胞术检测SDF-1α对软骨细胞凋亡的影响，透射电镜下检测SDF-1α对软骨细胞自噬的影响，并利用免疫荧光染色实验直观显示SDF-1α作用后软骨细胞内p-Akt蛋白表达及分布的差异。
    结果 与对照组相比，50、100和200 ng/mL SDF-1α在各时点并不降低软骨细胞活性（P<0.01），且均能在24 h促进软骨细胞迁移（P<0.05）。Western blot实验结果显示，与对照组相比，50、100和200 ng/mL SDF-1α能够显著上调软骨细胞内p-Akt的蛋白表达量，Akt表达量未见明显差异。与对照组相比， 流式细胞术示SDF-1α能够抑制软骨细胞凋亡（P<0.05），透射电镜观察到SDF-1α促进软骨细胞自噬（P<0.05），免疫荧光染色实验结果显示，p-Akt在软骨细胞的表达主要集中在细胞核周，SDF-1α作用后软骨细胞内p-Akt表达进一步在细胞核周部位增强。
    结论 SDF-1α通过激活Akt信号通路，抑制软骨细胞凋亡，促进软骨细胞迁移和自噬。

     

    Abstract:
    Objective To investigate the effects of stromal cell-derived factor 1α (SDF-1α) on the apoptosis and autophagy of chondrocytes and the underlying mechanisms.
    Methods Chondrocytes were isolated from the knee joints of neonatal mice. The chondrocytes were then stimulated with 0 (the control group), 50, 100, and 200 ng/mL of SDF-1α. CCK-8 assay was performed to determine the effects of SDF-1α stimulation for 24 h, 48 h, and 72 h on the viability of the chondrocytes. Wound healing assay was conducted to determine the effects of SDF-1α stimulation for 12 h and 24 h on chondrocyte migration. The changes in the expression of Akt signaling pathway proteins in chondrocytes were determined by Western blot assay. Chondrocytes were stimulated with 0 (the control group) and 200 ng/mL of SDF-1α. Flow cytometry was performed to determine the effect of SDF-1α on the apoptosis of chondrocytes. Transmission electron microscope was used to examine the effect of SDF-1α on chondrocyte autophagy. Immunofluorescence staining assays were performed to visualize the differences in p-Akt expression and distribution in chondrocytes treated with SDF-1α.
    Results Compared with the control group, findings for the experimental groups showed that SDF-1α at the concentrations of 50, 100, and 200 ng/mL did not decrease chondrocyte activity at any time point (P<0.01) and it consistently promoted chondrocyte migration at 24 h (P<0.05). Western blot results revealed that, in comparison to the control group, SDF-1α at concentrations of 50, 100, and 200 ng/mL significantly up-regulated the protein expression of p-Akt in chondrocytes, while no significant difference in Akt expression was observed. Flow cytometry demonstrated that SDF-1α could inhibit chondrocyte apoptosis (P<0.05) and transmission electron microscopic observation showed that SDF-1α promoted chondrocyte autophagy (P<0.05). Immunofluorescence staining showed that the expression of p-Akt in chondrocytes was concentrated in the perinuclear area of the cells and this expression was further enhanced in the perinuclear area of the chondrocytes after treatment with SDF-1α.
    Conclusion SDF-1α inhibits chondrocyte apoptosis and promotes chondrocyte migration and autophagy through activating the Akt signaling pathway.

     

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