The Role Of H2S And TGF-? Signaling In HGMSCs Osteogenic Differentiation And Primary Mechanism Exploration

[Objective]The purpose of this work is to investigate endogenous H2S levels in human gingiva-derived mesenchymal stem cells(hGMSCs)osteogenesis process;to explore the role of H2S and TGF-? signaling in hGMSCs osteogenic differentiation;And further explore the relationship between H2S and TGF-? signaling in hGMSCs osteogenic differentiation[Methods]hGMSCs were obtained in clinic and characterized by morphological observation,surface markers detection.After 21 days induction,alizarin red S staining was performed to evaluate hGMSCs osteogenic differentiation capacity.Additionally,H2S fluorescent probe CouMC and flow cytometry analyses were used to detect hGMSCs endogenous H2S level in osteogenesis group and SB431542 group.In order to further explore the influence of the exogenous H2S and TGF-?signaling on osteogenic differentiation,100?M H2S and 1?M SB431542 were used to treat hGMSCs.According to culture medium,cells were randomized into six groups:control group,osteogenesis group,SB431542 group,H2S+control group,H2S+osteogenesis group,H2S+SB431542 group.After 21 days osteogeneic induction,alizarin red S staining was used to investigate hGMSCs osteogenesis.At day 7,the expression of Runx-2.CBS,CSE,Smad2 and Smad3 were assessed by RT-qPCR and Western blotting[Results]Primary cells from gingiva and periodontal tissues were spindle morphology and had clonal growth ability.hGMSCs were positive for mesenchymal stem cells surface markers CD73(99.94%),CD90(99.91%)and negative for CD34(0.26%)and CD45(0.21%).hGMSCs had the capacity of osteogenic differentiation and enhanced by SB431542 treatment.The staining of H2S fluorescent probe CouMC with endogenous H2S is sensitive.As detected by H2S fluorescent probe CouMC and FCM,hGMSCs endogenous H2S level was increased in SB431542 group than osteogenesis group After treated by 100?M H2S and 1?M SB431542 for 21 days,alizarin red S staining showed that alcification nodules formation in SB431542 group and H2S+osteogenesis group were higher than that in osteogenesis group,and H2S+osteogenesis group had the biggest and most mineralization nodule.Expression of Runx-2 protein was enhanced by 100?M H2S and 1?M SB431542.just like the result of alizarin red S staining.H2S synthesized enzymes CBS and CSE and TGF-? signaling related protein Smad2.Smad3 were increased by SB431542 but decreased by exogenous H2S treatment in either osteogenesis group or SB431542 group,as assessed by Western Blotting and RT-qPCR.[Conclusion]Exogenous H2S and TGF-? signaling inhibitor SB431542 synergeticlly improve hGMSCs osteogenesis in vitro.Exogenous H2S could inhibit CBS,CSE expression in hGMSCs.SB431542 could promote CBS,CSE expression in hGMSCs and improve exdogenous H2S level.Exogenous H2S decreased Smad,Smad3 expression in hGMSCs,while SB431542 increased Smad,Smad3 expression,these data indicated that H2S and TGF-? signaling had certain relations.This study need further experiments to explore the mechanism that how TGF-? signaling and H2S involve in hGMSCs osteogenic differentiation in vitro and in vivo.