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- 2016
黄芪甲苷通过SIRT1/PGC-1α通路促进退变的髓核细胞增殖
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Abstract:
摘要：目的 探究黄芪甲苷对退变髓核细胞功能的影响及其作用机制。方法 原代培养正常与退变髓核细胞并观察，免疫组化检测细胞中胶原蛋白Ⅱ(collagenⅡ)的表达；分别采用20、40、60、80μg/mL的黄芪甲苷处理细胞，通过CCK-8和MTT试剂盒检测细胞活力和增殖，流式细胞术检测细胞凋亡情况；Western blotting检测凋亡相关基因Bcl-2、Bax和沉默信息调节因子2同源蛋白1(SIRT1)、过氧化物酶体增殖物激活受体γ辅助激活因子-1(PGC-1α)的表达及其乙酰化和磷酸化水平；比色法检测丙二醛(MDA)、超氧化物岐化酶(SOD)、β-galactosidase和三磷酸腺苷(ATP)的含量。结果 与正常髓核细胞相比，退变髓核组织中坏死细胞更多，且collagen Ⅱ表达降低；细胞经不同浓度黄芪甲苷处理后，40μg/mL黄芪甲苷不仅能显著提高退变髓核细胞活力，促进细胞增殖并抑制其凋亡，而且能上调细胞中SIRT1、PGC-1α的蛋白表达和PGC-1α磷酸化水平，下调PGC-1α乙酰化水平，增加SOD和ATP含量，降低MDA和β-galactosidase含量。结论 黄芪甲苷可能通过激活SIRT1/PGC-1α信号通路促进髓核细胞的增殖和存活。
ABSTRACT: Objective To explore the effects and mechanisms of astragaloside on degenerative nucleus pulposus cells. Methods Normal and degenerative nucleus pulposus cells were cultured and observed, collagen Ⅱ was detected by immunohistochemical staining. Degenerative nucleus pulposus cells were cultured with astragaloside at the concentrations of 20, 40, 60 and 80μg/mL, respectively. The cell viability and proliferation were detected using CCK-8 assay and MTT. Meanwhile, cell apoptosis was determined by flow cytometry analysis; Western blotting was then used to determine the expressions of Bcl-2, Bax, SIRT1 and PGC-1α as well as the level of acetylation and phosphorylation for PGC-1α; MDA, SOD, β-galactosidase and ATP were measured by colorimetry. Results Compared with the normal nucleus pulposus cells, the degenerative nucleus pulposus cells showed more patches of necrosis and lower expression of collagen Ⅱ. The cell viability and proliferation were increased after treatment with 40μg/mL of astragaloside. However, cell apoptosis was inhibited. Moreover, the astragaloside-treated group showed increased SIRT1 and PGC-1α, phosphorylation of PGC-1α, SOD and ATP; but decreased acetylation of PGC-1α, MDA and β-galactosidase. Conclusion Low-dose astragaloside promotes the proliferation and survival of degenerative nucleus pulposus cells through activating SIRT1/PGC-1α pathway
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