Abstract：Objective: To investigate the role of naringin in promoting osteogenic differentiation of human adipose derived mesenchymal stem cells through the cyclic adenosine phosphate (cAMP)/protein kinase A (PKA)/cAMP response element binding protein (CREB) pathway. Methods: The cells were divided into control group,naringin low,medium and high dose group,cAMP inhibitor (SQ22536) group and osteogenic differentiation induction group (induction group).12.5 μmoL/L,25 μmoL/L and 50 μmoL/L naringin were added to the naringin low,medium and high dose groups,respectively,and 100 μmoL/L SQ22536 was added to the SQ22536 group.L SQ22536,10~8 moL/L dexamethasone,10 mmoL/L sodium β-glycerophosphate and 50mg/L ascorbic acid were added to the induction group; only equal volumes of culture solution were added to the control group,and six replicate wells were set up for each group.After 48h of incubation,ALP activity was measured by alkaline phosphatase (ALP) staining and p-nitrophenyl phosphate (PNPP); calcium deposition was observed by Alizarin Red S (ARS) staining; cAMP level was measured by ELISA; PKA,CREB,RUNX2,Osteocalcin (OCN),OPN messenger ribonucleic acid (mRNA) expression was measured by RT-qPCR.The expression of PKA,CREB,RUNX2,osteocalcin (OCN) and osteopontin (OPN) messenger ribonucleic acid (mRNA) was measured by RT-qPCR,and the expression of PKA,CREB,RUNX2,OCN,OPN and p-PKA and p-CREB were measured by WB. Results: ALP staining showed a small amount of blue precipitation in the control group,with the least amount of blue precipitation in the SQ22536 group and gradually increasing in the induction and naringin 3 kinds of dose groups.ALP activity was significantly decreased in the SQ22536 group compared to the control group (P<0.01); ALP activity was significantly increased in the induction and naringin 3 dose groups compared to the control and SQ22536 groups (P<0.01).A small amount of calcium deposition was present in the control group,significantly reduced in the SQ22536 group and gradually increased in the induction and naringin 3 dose groups; cellular cAMP levels,PKA,CREB,RUNX2,OCN,OPN mRNA and protein expression and p-PKA and p-CREB levels were significantly reduced in the SQ22536 group compared to the control group (P<0.01).Compared with the control and SQ22536 groups,the cAMP level,PKA,CREB,RUNX2,OCN,OPN mRNA and protein expression and p-PKA and p-CREB levels increased in the induction and naringin 3 dose groups,and the differences were statistically significant (P<0.01); the differences between the above indices in the induction and naringin low dose groups were not statistically significant ( P>0.05); the above indicators naringin showed a dose dependence. Conclusion: Naringin can promote osteogenic differentiation of human adipose derived mesenchymal stem cells,which may be related to the activation of cAMP/PKA/CREB signaling pathway,promotion the expressions of RUNX2,OCN and OPN,and up-regulation of ALP activity.
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2022, Vol. 28 
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