Effect Of Sophora Polysaccharide On Proliferation And Osteogenic Differentiation Of Rat Dental Pulp Stem Cells

Objective: To determine the effects of sophora flavescens polysaccharide(SFRP)on the proliferation,migration,and osteogenic differentiation of dental pulp stem cells(DPSCs).Methods: Flow cytometry was used to identify the surface biomarkers of dental pulp stem cells isolated from rat dental crow tissue.In this study,cell viability was assessed using Cell Counting Kit-8(CCK-8)after DPSCs were treated with different doses of SFRP over a period of time.Transwell and scratch assays were used to determine cell migration ability.The osteogenic differentiation ability of different concentrations of SFRP was detected using alizarin red staining and alkaline phosphatase staining.To determine the effect of different concentrations of SFRP on osteogenic differentiation ability,Alizarin red s and alkaline phosphatase staining were performed.Real-time quantitative PCR(qRT-PCR)and Western Blotting assay were performed to detect the expression of Runt-related transcription factor 2(RUNX2),β-catenin,osteocalcin,and collagen I.Results: it showed high expression of CD90 and low expression of CD45 on the surface of the isolated dental pulp stem cells.The experimental results of CCK8 indicate that: DPSC proliferation was inhibited by high concentrations of SFRP(P<0.01),with 10 mg/ml of SFRP having the strongest inhibitory effect.Based on the Transwells experiment,it was found that: SFRP at a low concentration(0.2 mg/m L)promoted the migration of dental pulp stem cells,while high concentrations(1 mg/ml and 10 mg/m L respectively)inhibited the migration of dental pulp stem cells(P<0.01).Based on scratch assays,it appears that: after 24 hours and 48 hours of intervention,the migration ability of dental pulp stem cells was promoted by low concentrations of SFRP(0.2 mg/m L)(p<0.01);however,the migration ability of dental pulp stem cells was inhibited by high concentrations of SFRP(10 mg/m L)(P<0.01).The results of the alizarin red s were as follows: After 21 days of intervention,with a low concentration of SFRP(0.2 mg/m L),the largest mineralized nodules have been formed,as have the greatest numbers of nodules;however,with increasing concentrations,the mineralized nodules have become smaller and fewer in number.Alkaline phosphatase activity showed that: After 21 days of intervention,the alkaline phosphatase activity of DPSCs was significantly increased when SFRP was present at low concentrations(0.2 mg/m L),and the activity was inhibited when SFRP was present at high concentrations(1 mg/ml and 10 mg/m L respectively).Results of q RT-PCR showed that: After 21 days of intervention,RUNX2,(?)-catenin,osteocalcin,and collagen I protein levels increased in the low and medium concentration SFRP experimental groups(0.2 mg/m L and 1 mg/m L,respectively).The difference between Collage I and the control group in high-concentration SFRP(10 mg/m L)was statistically significant(P<0.01).At high concentrations,there were no statistically significant differences between RUNX2,β-catenin,and osteocalcin target proteins(P > 0.05).Results of Western Blot showed that: In the low-medium concentration SFRP experimental group(0.2 mg/m L and 1mg/ml respectively),the protein level of RUNX2,β-catenin,osteocalcin,and collagen I increased(P < 0.05).While high levels of SFRP(10 mg/m L)downregulated related protein levels(P<0.01),which was statistically significant.Conclusion: SFRP at low concentrations(0.2 mg/m L)can significantly promote the migration of DPSCs and osteogenic differentiation,as well as up-regulate osteogenic gene expression.