?-Ketoglutarate Regulates Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells Through UCHL1

Objective: New regenerative cell therapies for bone diseases and trauma often include the use of patient derived or xenogeneic adult stem cells.Bone marrow mesenchymal stem cells(BMSCs)show excellent osteogenic and chondrogenic abilities under the standard differentiation scheme.They have good clinical application prospects,such as good biocompatibility with scaffold materials,but there are also corresponding practical difficulties.The biochemical and molecular interactions in a specific niche of MSCs are very complex,and it is difficult to recombine by cell culture and tissue regeneration strategy in vitro.Therefore,understanding and using MSCs differentiation related factors for treatment will be the key to improve the success rate of adult stem cell therapy.?-ketoglutarate(AKG)is an important participant in the tricarboxylic acid cycle and an important regulator of glutamine metabolic pathway.Its function is the precursor of energy supply,amino acid biosynthesis,the necessity of collagen synthesis,and the essential substance and key regulatory site of ?-ketoglutarate dependent dioxygenase activity.It is easy to obtain,good storage,good solubility,and has been widely used in clinical practice.In vitro,?-ketoglutarate can promote the osteogenic differentiation of osteoblasts,and has good performance in anti-aging and anti osteoporosis.Therefore,our study aims to explore the effect of ?-ketoglutarate on the aging of human bone marrow mesenchymal stem cells and its possible mechanism.Research methods: In this study,alizarin red staining was used to detect the effect of AKG on calcium deposition during osteogenic differentiation of human BMSCs The effects of AKG on the expression levels of osteogenic related genes and proteins(Runx2,OPN,Col I)in the process of osteogenic differentiation of human BMSCs were detected by blot;the effects of AKG on the expression levels of uchl1 in the process of osteogenic differentiation of BMSCs were further studied,and the expression levels of uchl1 in the gene and protein levels were detected by real-time PCR and Western blotting The effects of AKG on the expression of TET1 and TET2 proteins during the osteogenic differentiation of human BMSCs were detected by blot,and the effects of AKG on the expression of UCHL-1,RUXN2 and COL ? proteins during the osteogenic differentiation of human BMSCs were detected after pretreatment with LDN57444,an inhibitor of UCHL-1.Results: Compared with the control group,the amount of calcium deposition detected by alizarin red staining increased in accordance with the AKG concentration gradient after 7 days of osteogenic induction of AKG.AKG at low concentration(2mmol/L)had no significant effect on the gene expression levels of Col I,OPN and Runx2(P>0.05),but the protein expression levels of Col I and RUNX2 were increased.With the increase of AKG concentration,AKG up-regulated the gene and protein expression levels of Col I,OPN and Runx2 during the osteogenic differentiation of BMSCs AKG upregulated the expression of UCHL1 gene and protein during the osteogenic differentiation of BMSCs.At the same time,LDN57444,a specific inhibitor of UCHL1,was used for osteogenesis induction and AKG treatment of human BMSCs.It was found that the inhibition of UCHL1 expression level had an effect on RUNX2 and Col I(P<0.01),and the addition of AKG(8mmol/L)partially reversed LDN57444's inhibition process of UCHL1,Runx2 and Col I protein expression.Compared with the control group,the expression levels of TET1 and TET2 increased with the concentration gradient of AKG.Conclusion: AKG can promote the differentiation of BMSCs and up regulate the expression of osteogenic related genes and proteins.AKG also promoted the expression levels of UCHL1,TET1 and TET2 in this process,and inhibiting the expression of UCHL1 would decrease the expression levels of RUNX2 and COL I,but AKG reversed this process.These results suggest that the mechanism of AKG promoting osteogenic differentiation of BMSCs is related to the role of UCHL1,proteins.TETs may also participate in this process.