柚皮苷通过Fas膜受体通路调控髓核细胞凋亡的机制研究

Mechanism of naringin on the apoptosis of nucleus pulposus cells through Fas membrane receptor pathway

关键词:  柚皮苷  人髓核细胞  凋亡  椎间盘  退变

Key words:  naringin  human nucleus pulpous  apoptosis  intervertebral disc  degeneration

摘要:

[目的]观察中药骨碎补单体柚皮苷（Naringin）对人退变椎间盘髓核细胞凋亡的影响，为治疗椎间盘退变提供理论依据。[方法]对体外培养的人退变椎间盘髓核细胞利用番红O染色和甲苯胺蓝染色进行鉴定，并对P3代细胞分别用柚皮苷0 g/mL （对照组）、柚皮苷20 g/mL、柚皮苷40 g/mL、柚皮苷80 g/mL的培养基培养人髓核细胞48 h，噻唑蓝（MTT）法选择其抑制人退变椎间盘髓核细胞凋亡的最佳浓度，并用流式细胞仪测定各组细胞凋亡率。实时定量聚合酶链锁反应（qRT-PCR）检测Fas、FasL、Caspase-8基因表达。蛋白免疫印迹法（Western-blot）检测Fas蛋白表达。[结果]MTT法和流式细胞检测均测定20 g/mL柚皮苷为抑制髓核细胞凋亡的最佳浓度（P<0.05）。qRT-PCR检测20 g/mL柚皮苷能够抑制Fas、FasL、Caspase-8 mRNA表达（P<0.05）。Western-blot检测显示20 g/mL柚皮苷能够抑制Fas、FasL、Caspase-8蛋白表达。[结论]柚皮苷可能通过调控Fas/Fasl死亡受体凋亡通路抑制人退变椎间盘髓核细胞凋亡。

Abstract:

[Objective] To study the effects of naringin on the apoptosis of nucleus pulposus cells of human degenerative intervertebral disc.[Methods] Nucleus pulposus cells were cultured in vitro and were identified via the safranin O and toluidine blue. Next, P3 cells were cultured with naringin 0 g/mL (control group), naringin group (20 g/mL, 40 g/mL, 80 g/mL). After 48 hours, the best concentration of naringin was selected by MTT for inhibition apoptosis of human degenerated nucleus pulposus cells and the rate of apoptosis in each group was measured by flow cytometry.[Results] The results of cells staining were positive, and it was proved that the cells cultured in the experiment were nucleus pulposus cells. MTT and flow cytometry were used to determine the optimum concentration of naringin (20 g/mL) to inhibit the apoptosis of nucleus pulposus cells(P<0.05). Naringin(20 g/mL) could inhibit the expression of Fas, FasL, Caspase-8 mRNA by qRT-PCR (P<0.05). Western-blot detection showed that 20 g/mL naringin could inhibit the expression of Fas, FasL, Caspase-8 protein.[Conclusion] Naringin may inhibit the apoptosis of nucleus pulposus cells by regulating the Fas/FasL death receptor apoptosis pathway.