人参皂苷Rg1通过抑制Wnt/β-catenin通路促进退变人腰椎间盘髓核细胞的生长及胞外基质合成

Ginsenoside Rg1 promotes growth and extracellular matrix synthesis in degenerative human lumbar nucleus pulposus cells by inhibiting Wnt/β-catenin pathway

目的:探讨人参皂苷Rg1对退变人腰椎间盘髓核细胞(HNPCs)生长的调控及其作用机制。方法:培养正常HNPCs,构建HNPCs的氧糖剥夺(OGD)模型模拟退变HNPCs的微环境。光镜观察正常HNPCs和OGD组细胞的形态。分别给予25、50和100μmol/L人参皂苷Rg1后,用real-time PCR和Western blot法分别检测II型胶原(collagen II)和聚集蛋白聚糖(aggrecan)的表达,CCK-8法检测细胞活力的变化,real-time PCR法检测增殖蛋白Ki67的转录水平,流式细胞术检测细胞凋亡水平,caspase-3试剂盒检测caspase-3活性,Western blot法检测Wnt/β-catenin通路蛋白的表达。加入Wnt/β-catenin通路激活剂Li Cl后,检测其对Wnt/β-catenin通路蛋白、细胞活性、细胞凋亡及基质合成蛋白表达的影响。结果:正常HNPCs细胞贴壁快,形态呈类圆形,胞质丰富;OGD组细胞贴壁较慢,形态多为长梭形,胞质减少,初步证实了退变HNPCs模型构建成功。与HNPCs组相比,OGD组中collagen II和aggrecan的m RNA和蛋白表达均显著降低(P<0.05),人参皂苷Rg1可升高collagen II和aggrecan的表达(P<0.05)。与HNPCs组相比,OGD组的细胞活力显著降低(P<0.05),Ki67表达下调(P<0.05),而不同浓度的人参皂苷Rg1可增加细胞活力和上调Ki67的表达(P<0.05)。同时,不同浓度的人参皂苷Rg1可减少OGD增加的细胞凋亡和caspase-3活性(均P<0.05)。此外,100μmol/L人参皂苷Rg1可显著减弱OGD触发的Wnt/β-catenin通路的活化(P<0.05)。而用Wnt通路激动剂Li Cl处理后可明显减弱人参皂苷Rg1对OGD细胞的保护作用(P<0.05),提示该通路参与人参皂苷Rg1对退变HNPCs的保护作用。结论:人参皂苷Rg1可通过抑制Wnt/β-catenin通路促进HNPCs的生长和胞外基质合成。本研究将为退变HNPCs的防治提供新的思路。

AIM:To explore the role of ginsenoside Rg1 in the growth of degenerative human lumbar nucleus pulposus cells(HNPCs).METHODS:Cultured HNPCs were subjected to oxygen-glucose deprivation(OGD)to mimic the micro-environment of degenerative HNPCs.The morphological changes of the cells in control group and OGD group were observed under optical microscope.The cells were treated with ginsenoside Rg1 at concentrations of 25,50 and 100μmol/L.The expression of collagen II and aggrecan at mRNA and protein levels was determined by real-time PCR and Western blot analysis.The cell viability was measured by CCK-8 assay.The mRNA level of Ki67 was detected by real-time PCR.The apoptosis was analyzed by flow cytometry.The activity of caspase-3 was measured by a caspase-3 kit.The expression of Wnt/β-catenin pathway-related proteins was determined by Western blot.Furthermore,the expression of Wnt/β-catenin pathway-related proteins,the cell viability and apoptosis,and the expression of extracellular matrix synthesis proteins were assessed after the cells were co-treated with LiCl and 100μmol/L ginsenoside Rg1.RESULTS:Normal HNPCs attached on the cell culture plate faster,and were almost round with rich cytoplasm.However,the cell adherence was slower,and the cells were long fusiform with decreased cytoplasm after OGD treatment,indicating that the model of degenerative HNPCs was successfully established.Compared with normal HNPCs,the expression of collagen II and aggrecan at mRNA and protein levels was decreased in OGD group(P<0.05),which was then increased after the cells were treated with ginsenoside Rg1 at 25,50 and 100μmol/L(P<0.05).Compared with normal HNPCs,the cell viability and Ki67 expression were decreased in OGD group(P<0.05),which were increased after treatment with ginsenoside Rg1(P<0.05).Meanwhile,the apoptotic rate and caspase-3 activity were significantly increased in OGD-treated cells(P<0.05),which were decreased after treatment with ginsenoside Rg1(P<0.05).In addition,the activation of Wnt/β-catenin pathway was also inhibited by ginsenoside Rg1 treatment at dose of 100μmol/L(P<0.05).LiCl,a Wnt/β-catenin pathway agonist,obviously decreased the protective effects of ginenoside Rg1 on OGD-induced cells(P<0.05),indicating that the Wnt/β-catenin pathway was involved in the protective effects of ginenoside Rg1 on degenerative HNPCs.CONCLUSION:Ginsenoside Rg1 promotes growth and extracellular matrix synthesis of degenerative HNPCs through inhibiting Wnt/β-catenin pathway.This study will provide a new idea for prevention and treatment of degenerative HNPCs.