| Periodontitis affects about 15%of adults,it is a chronic inflammatory disease in which periodontal support tissues are progressively destroyed and,if left unchecked,can lead to loosening,displacement of teeth and,in severe cases,eventually tooth loss.Periodontitis seriously affects the oral aesthetics and oral function of patients,in recent years,orthodontic auxiliary periodontal treatment is chosen by more and more patients and doctors,which aims to align teeth through orthodontic methods,close the space,restore the occlusal relationship,eliminate the damage of abnormal occlusal force to periodontal tissue,restore the oral function and aesthetic problems of patients caused by periodontitis,but for patients with periodontitis,because of its unstable periodontal tissue,so the time after correction is longer,and some patients even need to maintain it for life.This brings a lot of distress to the patient’s life.Stabilizing orthodontic teeth and shortening the retention time of periodontitis patients after correction have become hot issues that orthodontists have paid attention to in recent years.Human periodontal ligament stem cells(hPDLSCs)have the ability to form bone,dentin,and periodontal ligaments-like structures.Compared with pulp stem cells and apical tooth cyst stem cells,hPDLSCs have stronger regenerative ability,and studies have shown that transplantation of PDLSCs into rats changes the immune microenvironment,thereby enhancing periodontal tissue regeneration.hPDLSCs are seed cells with potential for periodontal tissue regeneration.Panax notoginseng is a time-honored traditional Chinese herbal medicine that has a long history as a hemostatic agent in China,and at the same time,it has also been used to treat cardiovascular diseases,and chronic hepatitis,in addition,Panax notoginseng has antioxidant,anti-inflammatory and neuroprotective effects.Ginsenosides Rd,Re,Rg1,Rb1 and Panax notoginseng saponin R1(NG-R1)are the main active ingredients of Panax notoginseng,NGR1 is a kind of phytoestrogen isolated from Panax notoginseng,which has neuroprotective,anti-tumor and anti-inflammatory effects,in vitro experiments have proved that NG-R1 can promote in vitro osteogenesis of a pre-osteoblast cell line(MC3T3-E1).In addition,studies have shown that NG-R1 can promote osteogenesis through the estrogen receptor signaling pathway and can be used as a potential drug for the treatment of osteoporosis in postmenopausal women,but there are no studies to explore whether NG-R1 can promote osteogenic differentiation of hPDLSCs and its possible mechanism.Wnt signaling pathway plays a controlling role in many processes of biological growth and development,the pathway is essential for maintaining homeostasis in the human skeletal environment,pathological mutations in genes related to the Wnt signaling pathway can lead to severe osteopenia,and a large number of studies have shown that there are many traditional Chinese medicines that can enhance the osteogenic effect of hPDLSCs by activating the classical Wnt signaling pathway.Objectives:The purpose of this study is to explore the effect of NG-R1 on the proliferation and osteogenic differentiation of hPDLSCs,and to explore the role of Wnt/β-catenin signaling pathway in this process,so as to provide a theoretical basis for periodontal tissue regeneration.Materials and methods:(1)Isolation,culture and identification of hPDLSCs:hPDLSCs were isolated and cultured by tissue block method,and the isolated primary hPDLSCs were subcultured.The expression of intereschymal-related markers CD90 and hematopoietic markers CD34 and CD45 was detected by flow cytometry,and the multi-directional differentiation ability of hPDLSCs was verified by osteogenesis and lipogenesis induction experiments.(2)Effect of NG-R1 on the proliferation of hPDLSCs:The effects of different concentrations of NG-R1 on the proliferation of hPDLSCs were explored through Cellcounting kit-8(CCK-8)experiments and cell cloning experiments,and the optimal concentration was screened.(3)Effect of NG-R1 on osteogenic differentiation of hPDLSCs:The effect of NG-R1 on osteogenic differentiation of hPDLSCs was explored by alkaline phosphatase(ALP)activity and staining,alizarin red staining and quantitative experiments.The effects of NG-R1 osteogenic correlated factors ALP,RUNX2,COL-1,and β-catenin expression were detected by qRT-PCR and Westerm Blot.(4)Effect of Wnt/β-catenin signaling pathway on NG-R1 to promote osteogenic differentiation of hPDLSCs:small molecule inhibitors of Wnt/β-catenin signaling pathway were used to block Wnt/β-catenin signaling pathway,and the role of Wnt/β-catenin in promoting osteogenic differentiation of hPDLSCs by NG-R1 was explored.Results:(1)In this experiment,primary hPDLSCs were successfully isolated by tissue block method,and P3-P5 generation cells were selected for subsequent experiments after subculture.Flow cytometry showed positive CD90 expression and negative CD34 and CD45 expression in isolated cells,indicating that the cells that we cultured were mesenchymal sources.(2)CCK-8 and cloning results showed that 20 μmol/L NG-R1 could significantly promote the proliferation of hPDLSCs.(3)ALP activity and alizarin red staining and quantitative experiments showed that 20μmol/L NG-R1 could significantly promote the ALP activity of hPDLSCs and the formation of mineralized nodules.qRT-PCR and Westerm Blot experiments showed that 20μmol/L NG-R1 could significantly increase the expression of osteogenic correlated factors ALP,RUNX2,COL-1 and β-catenin.(4)After the addition of XAV-939,the expression of osteogenic genes ALP,RUNX2,COL-1 and pathway-related genes GSK3β,P-GSK3β(Ser9),β-catenin and LEF decreased compared with the control group,and after the addition of NG-R1 and XAV-939,the protein expression level increased compared with the XAV-939 group,indicating that NG-R1 partially reversed the inhibitory effect of XAV-939 on the Wnt signaling pathway.Conclusion:(1)NG-R1 can promote the proliferation and osteogenic differentiation of hPDLSCs,and 20 μmol/L is the optimal concentration.(2)NG-R1 promotes osteogenic differentiation of hPDLSCs by activating the Wnt/βcatenin signaling pathway. |
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