Succinate Supplement Promoted Proliferation,Migration And Osteogenesis Of Periodontal Ligament Cells

[Objective]Most mesenchymal stem cells reside in a niche of low oxygen tension.Iron-chelating agents such as Co Cl2 and deferoxamine(DFO)have been utilized to mimic hypoxia and promote cell growth.The purpose of present study was to explore whether supplement of succinate,a natural metabolite of the tricarboxylic acid(TCA)cycle,can mimic hypoxia condition to promote proliferation,migration,and osteogensis of human periodontal ligament cells(h PDLSCs).[Methods]The effects of hypoxia and exogenous succinate on proliferation,migration,and osteogenesis of human periodontal stem cells(h PDLSCs)were investigated by cell proliferation assay,scratch assay,and alkaline phosphatase(ALP)/Alizarin Red S(ARS)staining.We used q PCR and Western blot to analyze the proliferation and osteogenesis-related genes and proteins in normoxia and hypoxia,such as Cyclin D1(CCND1),Cyclin D3(CCND3),Cyclin B1(CCNB1),osteocalcin(OCN),alkaline Phosphatase(ALP)and collagenase I(COL-1).In order to confirm intracellular succinate accumulation in hypoxic and exogenous succinate-treatment condition,the succinate colorimetric assay kit was used to measure intracellular succinate concentration at 2 h,6 h,12 h,and 24 h,respectively.To investigate the metabolic changes of h PDLSCs under hypoxic conditions,succinate dehydrogenase(SDH)activity was detected by ELISA.Moreover,glycolysis and TCA cycle-related genes including hexokinase 2(HK2),6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3)and SDH weredetected by q PCR and Western blot.To explore whether intracellular accumulation of succinate can stabilize hypoxia inducible factor-1α(HIF-1α)by inhibiting prolyl hydroxylases(PHD),protein levels of PHD2 and HIF-1αwere detected by Western blot.Cells were pretreated with the inhibitor BAY 87-2243 of HIF-1α for 3 h,and the expression of cell cycle-related genes in the control group,hypoxic group,and exogenous succinate-treated group were detected by q PCR.In order to analyze the effect of succinate receptor 1(SUCNR1)on the proliferation of h PDLSCs under hypoxic and exogenous succinate-treated conditions,the extracellular succinate concentration in the control group,hypoxic group and exogenous succinate treatment group were detected by succinate colorimetric assay.The expression of SUCNR1 gene in the control group,hypoxic group and exogenous succinate treatment group were detected by q PCR.After transfection of h PDLSCs with sh SUCNR1,cell cycle-related genes in control group,hypoxic group,and exogenous succinate treatment group were detected by q PCR.[Results]The cell proliferation assay,scratch assay,and ALP/ARS staining assay of hypoxic group and exogenous succinate-supplementation group showed that hypoxia and exogenous succinate supplement increased proliferation,migration,and osteogensis of h PDLSCs,respectively.q PCR and Western Blot analysis suggested cell-cycle related genes and osteogensis genes were elevated in hypoxia or succinate-treated h PDLSCs.Intracellular succinate concentration of h PDLSCs increased significantly after 12 h and 6 h respectively under hypoxic condition and exogenous succinate supplementation condition.SDH enzyme activity was impaired under hypoxic conditions and exogenous succinate supplementation condition.m RNA transcription and protein expression of glycolysis-related genes increased significantly.Gene transcription of SDH was increased at 4 h,while the protein expression of SDH was decreased at 24 h.Meanwhile,the PHD protein level in hypoxic group and exogenous succinate supplementation group decreased,while the HIF-1 α protein level increased.After the addition of HIF-1 α inhibitors,compared with the control group,the expression of cell cycle-related genes was decreased under hypoxic and exogenous succinate conditions.Compared with the control group,the extracellular succinate supplement was increased under hypoxic conditions and exogenous succinate supplementation.SUCNR1 gene expression was increased in both groups.After sh SUCNR1 was transfected,the expression of cell cycle-related genes in h PDLSCs was decreased significantly under hypoxic conditions.However,the expression of the cell-cycle related genes between the exogenous succinate group and control si RNA group had no significant difference.[Conclusions]Culturing hPDLSCs in both hypoxic and condition promoted cell proliferation,migration and osteogenic differentiation;moreover,hypoxia shifted cell metabolism from oxidative phosphorylation to glycolysis with accumulation of succinate in the cytosol resulting in its release into culture supernatants.Succinate supplement enhanced h PDLSCs proliferation,migration,and osteogenesis,inducing metabolic reprogramming from oxidative phosphorylation to glycolysis in a normal oxygen condition.Succinate supplement in cell cultures promoted intracellular succinate accumulation while stabilized HIF-1 α,leading to a state of pseudohypoxia.Moreover,we demonstrate hypoxia-induced proliferation was SUCNR1-dependent,while exogenous succinate-elicited proliferation involved SUCNR1-dependent and-independent pathway.