人参皂苷Rg1促进人牙龈成纤维细胞增殖、迁移及成骨分化的研究

Ginsenoside Rg1 promotes proliferation,migration and osteogenic differentiation of human gingival fibroblasts

目的探讨人参皂苷Rg1(GsRg1)对人牙龈成纤维细胞(HGFs)增殖、迁移及成骨分化的影响及分子机制。方法采用组织块法分离培养HGFs,并进行形态学和免疫荧光鉴定;取第3代HGFs,CCK-8法检测6种浓度的GsRg1(0、6.25、12.5、25、50、100 mg/L)对HGFs增殖的影响;Transwell实验检测不同浓度GsRg1对HGFs迁移能力的影响;碱性磷酸酶(ALP)染色检测成骨能力;茜素红染色观察并定量钙结节;qRT-PCR检测COL-Ⅰ、OCN和OPN成骨基因表达;Western blot检测OCN、OPN和COL-Ⅰ以及PI3K/AKT信号通路相关分子的蛋白表达情况。结果与对照组比较,浓度为12.5、25、50、100mg/L GsRg1可明显提高HGFs增殖能力(P<0.05),其中100 mg/L GsRg1促增殖作用最强,6.25 mg/L GsRg1组与对照组比较差异无统计学意义;GsRg1处理后HGFs迁移能力增强,且呈浓度依赖。与对照组比较,100 mg/L GsRg1组ALP活性明显增加(P<0.01);茜素红染色钙结节定量明显增多(P<0.01);成骨相关基因OCN、OPN和COL-Ⅰ的mRNA和蛋白表达水平升高(P<0.05);p-PI3K、p-AKT蛋白表达水平随时间上调明显(P<0.05),而PI3K、AKT蛋白表达水平无明显变化。结论GsRg1可以促进HGFs增殖和迁移,100 mg/L GsRg1能够促进HGFs的成骨分化,可能与激活PI3K/AKT信号通路有关。

Objective To investigate the effect of Ginsenoside Rg1(GsRg1)on proliferation,migration and osteogenic differentiation of human gingival fibroblasts(HGFs)and its molecular mechanism.Methods Human gingival fibroblasts(HGFs)were isolated and cultured by tissue block method,and identified by morphology and immunofluorescence.The effect of six concentrations of GsRg1(0,6.25,12.5,25,50,100 mg/L)on the proliferation of HGFs was detected by CCK-8 method.Transwell assay was used to detect the effects of different concentrations of GsRg1 on the migration ability of HGFs.Alkaline phosphatase(ALP)staining was used to detect osteogenic ability.Alizarin red staining was used to observe and quantify calcium nodules.The expression of COL-Ⅰ,OCN and OPN osteogenic genes was detected by qRT-PCR.Western blot was used to detect the protein expression of OCN,OPN,COL-Ⅰand PI3K/AKT signaling pathway.Results Compared with the control group,the proliferation ability of HGFs was significantly improved at the concentrations of 12.5,25,50 and 100 mg/L GsRg1(P<0.05),and the proliferation promotion effect of 100 mg/L GsRg1 was the strongest.There was no significant difference between the 6.25 mg/L GsRg1 group and the control group.After GsRg1 treatment,the migration ability of HGFs was enhanced and showed concentration dependence.Compared with the control group,the activity of ALP in 100 mg/L GsRg1 group significantly increased(P<0.01).Alizarin red staining showed a significant increase in the number of calcium nodules(P<0.01).The mRNA and egg white expression levels of osteogenic genes OCN,OPN and COL-Ⅰincreased(P<0.05).The expression levels of p-PI3K and p-Akt were significantly up-regulated with time(P<0.05),while the expression levels of PI3K and AKT had no significant changes.Conclusion GsRg1 can promote the proliferation and migration of HGFs,and 100 mg/L GsRg1 can promote the osteogenic differentiation of HGFs,which may be related to the activation of PI3K/AKT signaling pathway.