Rutin Promotes The Proliferation And Osteogenic Differentiation Of Periodontal Ligament Stem Cells Through The GPR30-mediated PI3K/AKT/mTOR Signaling Pathway

Objectives:Rutin is one of the common flavonoids in vegetables and fruits.Recent studies have shown that flavonoids play an important role in bone development and metabolism.Rutin,as a representative of common flavonoids.has an important impact on cell proliferation and bone development.The purpose of this study was to investigate the effects of rutin on the proliferation and osteogenic differentiation of periodontal ligament stem cells,and to explore the mechanisms involved in these processes.In order to provide basic theory and guidance for the regeneration of periodontal tissue and bone tissue by rutin in periodontal tissue engineering,and to provide reference for the application of flavonoids in periodontal tissue regeneration.Methods:Periodontal ligament stem cells(PDLSCs)were isolated by enzymatic digestion and cultured in vitro.The sternness of periodontal ligament stem cells(PDLSCs)were tested by flow cytometric analysis and multipotential differentiation assays.Cell counting kit-8(CCK-8)was used to detect the proliferation activity of PDLSCs.Alkaline phosphatase(ALP)activity assay,alkaline phosphatase(ALP)staining and alizarin red staining were used to test osteogenic differentiation of PDLSCs.What is more,the mRNA and protein levels of osteogenic genes were evaluated by real-time polymerase chain reaction(RT-PCR)and Western blot.In order to elucidate the mechanism of this process,the PI3K/AKT/mTOR signaling pathway-related proteins AKT,p-AKT,mTOR and p-mTOR,and G protein-coupled receptor 30(GPR30)were detected by Western blot.LY294002(PI3K signaling pathway inhibitor)and G15(a selective antagonist of GPR30)were used to further verify the function of GPR30/PI3K/mTOR signaling pathway in promoting bone function of rutin.Results:Periodontal ligament stem cells were successfully isolated by enzymatic digestion.PDLSCs were positive for specific surface markers of mesenchymal stem cells(MSCs)and had multiple differentiation potential.CCK-8 proliferation test showed that rutin promoted the proliferation of PDLSCs.ALP activity test.ALP staining and alizarin red staining results showed that rutin increased ALP activity,secretion of mineralized matrix and formation of mineralized nodules of periodontal ligament stem cells.In addition,rutin significantly enhanced the expression of osteogenic genes and proteins in periodontal ligament stem cells.and increased the phosphorylation levels of AKT and mTOR.The expression of p-AKT,p-mTOR and osteogenic genes and proteins decreased significantly in periodontal ligament stem cells with the treatment of PI3K signaling pathway inhibitor LY294002.In addition,rutin could promote the expression of GPR30.The selective antagonist G15 of GAPR30 could reduce the stimulating effect of rutin and block the activation of PI3K/AKT/mTOR signal pathway.GPR30 is an upstream regulator of PI3K/AKT/mTOR signal.Conclusions:Our study suggested that rutin promoted the proliferation and osteogenic differentiation of PDLSCs by activating the GPR30/PI3K/AKT/mTOR signaling pathway and might play an important role in periodontal tissue engineering and bone regeneration.