人参皂苷Rg1通过miR-138-5p/SIRT1轴诱导骨髓间充质干细胞向髓核样细胞增殖分化

Ginsenoside Rg1 Induces the Proliferation and Differentiation of Bone Marrow Mesenchymal Stem Cells into Nucleus Pulposusk-like Cells Through the miR-138-5p/SIRT1 Axis

目的探讨人参皂苷Rg1诱导大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向髓核样细胞(nucleus pulposus cells,NPCs)分化的机制。方法从成年SD大鼠中分离培养BMSCs。通过CCK-8法检测不同浓度人参皂苷Rg1(0,2.5,5,10,20μmol·L^(–1))对BMSCs生存活性的影响。将细胞分为空白组,5μmol·L^(–1)人参皂苷Rg1干预组和10μmol·L^(–1)人参皂苷Rg1干预组。培养48 h后通过流式细胞术检测细胞凋亡率;Western blotting检测NPCs标志蛋白胶原蛋白2型(collagen type 2,COL2)、Aggrecan和配对盒基因(paired box gene 1,Pax1)的表达;将细胞分为空白组、人参皂苷Rg1干预组、人参皂苷Rg1干预+NC mimic组和人参皂苷Rg1干预+miR-138-5p mimic组;通过以上方法进行进一步检测miR-138-5p对人参皂苷Rg1诱导BMSCs分化的影响;双荧光素酶报告基因和RNA结合蛋白免疫沉淀法检测miR-138-5p和sirtuin-1(SIRT1)的靶向关系;转染pcDNA-SIRT1检测其对miR-138-5p诱导BMSCs分化的影响。结果2.5,5,10μmol·L^(–1)人参皂苷Rg1呈剂量依赖性增强BMSCs活力(P<0.05);与空白组比较,5μmol·L^(–1)和10μmol·L^(–1)人参皂苷Rg1干预组呈剂量依赖性抑制细胞凋亡(P<0.05),促进NPCs标志蛋白表达(P<0.05);与人参皂苷Rg1干预组相比,人参皂苷Rg1+miR-138-5p组BMSCs凋亡率增加(P<0.01),NPCs标志蛋白表达减少(P<0.01);此外,SIRT1是miR-138-5p的靶基因;与miR-138-5p mimic+pcDNA-3.1组相比,miR-138-5p mimic+pcDNA-SIRT1组细胞凋亡率降低(P<0.01),NPCs标志蛋白表达增加(P<0.01)。结论人参皂苷Rg1可通过调控miR-138-5p/SIRT1轴诱导BMSCs向NPCs增殖和分化。

OBJECTIVE To explore the mechanism of ginsenoside Rg1 induced differentiation of rat bone marrow mesenchymal stem cells(BMSCs) into nucleus pulposus like cells(NPCs).METHODS Isolation and culture of BMSCs from adult SD rats.The effects of different concentrations of ginsenoside Rg1(0,2.5,5,10,20μmol·L^(–1)) on the viability of BMSCs were detected by CCK-8 assay.The cells were divided into blank group,5μmol·L^(–1) ginsenoside Rg1 intervention group and 10μmol·L^(–1) ginsenoside Rg1 intervention group.After 48 h of culture,and then the apoptosis rate was determined by flow cytometry,the expression of NPCs marker proteins collagen type 2(COL2),Aggrecan and paired box gene 1(Pax1) was detected by Western blotting.BMSCs were divided into blank group,ginsenoside Rg1 group,ginsenoside Rg1+NC mimic group,and ginsenoside Rg1+miR-138-5p mimic groups,followed by the detection of the effect of miR-138-5p on the BMSC differentiation induced by ginsenoside Rg1.The target relationship between miR-138-5p and sirtuin-1(SIRT1) was detected by dual luciferase reporter and RNA binding protein immunoprecipitation assays.pcDNA-SIRT1 was transfected into BMSCs to detect its effect on the differentiation of BMSCs induced by miR-138-5p.RESULTS The 2.5,5 and 10μmol·L^(–1) ginsenoside Rg1 enhanced BMSCs viability in a dose-dependent manner(P<0.05).Compared with the blank group,5μmol·L^(–1) and 10μmol·L^(–1) ginsenoside Rg1 intervention groups inhibited cell apoptosis(P<0.05) and promoted the expression of NPCs marker proteins in a dose-dependent manner(P<0.05).Compared with the ginsenoside Rg1 intervention group,the apoptosis rate of BMSCs in the ginsenoside Rg1+miR-138-5p group was increased(P<0.01) and the expression of NPC marker proteins was decreased(P<0.01).Moreover,SIRT1 was a target gene of miR-138-5p.Compared with the miR-138-5p mimic+pcDNA-3.1group,the apoptosis rate of BMSCs in the miR-138-5p mimic+pcDNA-SIRT1 group was decreased(P<0.01),and the expression of NPCs marker proteins was increased(P<0.01).CONCLUSION Ginsenoside Rg1 can induce BMSCs to proliferate and differentiate into NPCs by regulating the miR-138-5p/SIRT1 axis.