牛膝总皂苷对大鼠椎间盘髓核细胞外基质合成的影响

Effects of Achyranthes bidentata saponins on extracellular matrix synthesis in rat nucleus pulposus

目的探讨牛膝总皂苷(ABS)对大鼠椎间盘髓核细胞外基质合成的影响。方法体外培养大鼠椎间盘髓核细胞,并建立氧糖剥夺(OGD)模型模拟退变微环境。采用不同浓度ABS单独或加入SIRT1抑制剂EX527、自噬抑制剂3-MA干预细胞。CCK-8法检测细胞存活率,流式细胞术检测细胞凋亡,caspase-3试剂盒检测caspase-3活性,实时定量PCR检测细胞外基质collagen II、Aggrecan mRNA表达, Western blot检测SIRT1、collagen II、Aggrecan及自噬相关蛋白ATG5、LC3II/LC3Ⅰ和p62的蛋白表达。结果 ABS(40μg/mL)可促进大鼠椎间盘髓核细胞存活(P<0.01);ABS干预可使OGD环境中的细胞存活率增加,细胞凋亡、caspase-3活性降低,collagen II、Aggrecan mRNA和蛋白表达均上调,SIRT1、ATG5和LC3II/LC3Ⅰ蛋白表达上调,p62蛋白表达下调(P<0.05);EX527、3-MA干预可抑制OGD环境中ABS介导的细胞增殖增加、凋亡减少,collagen II和Aggrecan的蛋白表达上调(P<0.05)。结论 ABS可通过SIRT1/自噬信号通路促进大鼠椎间盘髓核细胞存活和细胞外基质合成。

AIM To investigate effects of Achyranthes bidentata saponins(ABS) on extracellular matrix synthesis in rat nucleus pulposus.METHODS Upon rat nucleus pulposus cells of intervertebral disc cultured in vitro,oxygen glucose deprivation(OGD)model was established to mimic the microenvironment of intervertebral disc degeneration.Cells were cultured with different concentrations of ABS, with or without SIRT1 inhibitor EX527 or autophagy inhibitor 3-MA. Subsequently cells were subjected to detection of cellular proliferation by CCK-8 assay, cellular apoptosis by flow cytometry, caspase-3 activity by its detection kit, the expression of collagen II and Aggrecan mRNA by quantitative real time PCR, the protein expression of SIRT1, collagen II, Aggrecan and autophagy-related protein ATG5, LC3 II/LC3Ⅰ and p62 by Western blot.RESULTS ABS(40 μg/mL) promoted the survival of rat nucleus pulposus cells(P<0.01). ABS intervention significantly increased cell viability;reduced cell apoptosis and caspase-3 activity, upregulated the mRNA and protein expression of collagen II and Aggrecan, as well as the protein expression of SIRT1, ATG5 and LC3 II/LC3Ⅰ;and down-regulated the protein expression of p62 in OGD-treated cells(P<0.05). Moreover, treatment with EX527 or 3-MA dramatically reversed ABS-mediated increase of cell proliferation, reduction of cell apoptosis and upregulation of the protein expression of collagen II and Aggrecan in OGD-treated cells(P<0.05).CONCLUSIONS ABS can promote the survival of rat intervertebral disc nucleus pulposus cells and its synthesis of extracellular matrix via activating SIRT1/autophagy signaling pathway.