2023, Vol. 27 ›› Issue (19): 2968-2974

Effect of glycogen synthase kinase-3 beta inhibitor Tideglusib on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells under high glucose microenvironment

Luo Wenhao1, 2, Feng Le2, 3, Huang Haixia1, 2, Liu Min1, 2, Wang Pin1, 2   

Abstract: BACKGROUND: High glucose condition can inhibit the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and how to improve the biological activity of osteoblasts in high glucose conditions is the key to improve osseointegration in diabetic patients.  
OBJECTIVE: To investigate the effect of Tideglusib, a non-ATP-competitive specific glycogen synthase kinase-3β (GSK-3β) inhibitor, on the osteogenic differentiation of rat bone marrow mesenchymal stem cells under high glucose conditions.
METHODS: Rat bone marrow mesenchymal stem cells were cultured and purified by whole bone marrow apposition method. Bone marrow mesenchymal stem cells were divided into four groups as follows: normal control group (5.5 mmol/L glucose), Tideglusib group (5.5 mmol/L glucose + 20 nmol/L Tideglusib), high glucose group (25.5 mmol/L glucose), and Tideglusib + high glucose group (25.5 mmol/L glucose+20 nmol/L Tideglusib). (1) The proliferation activities of cells were detected by CCK-8 assay. (2) Alkaline phosphatase activity was detected at 4 and 7 days after osteogenic differentiation in each group. (3) Calcium nodules were detected by alizarin red staining at 21 days after osteogenic differentiation in each group. (4) Phalloidin staining was used to observe the adhesion morphology of each group after 24 hours of inoculation. (5) RT-qPCR was used to detect the mRNA expression of osteogenic genes Runx2 and OPN. Western blot assay was used to detect the protein expression of β-catenin and p-GSK-3β.  
RESULTS AND CONCLUSION: (1) Tideglusib (20 nmol/L) not only significantly promoted the proliferation of bone marrow mesenchymal stem cells (P < 0.05), but also reversed the inhibitory effect of the high glucose on the proliferation of bone marrow mesenchymal stem cells (P < 0.05). (2) Tideglusib (20 nmol/L) significantly reduced the inhibitory effect of high glucose conditions on alkaline phosphatase activity (P < 0.05) and promoted the formation of calcified nodules under high glucose conditions (P < 0.05). (3) Tideglusib (20 nmol/L) could improve the adhesion morphology of bone marrow mesenchymal stem cells damaged by high glucose conditions. (4) Compared with the high glucose group, Tideglusib (20 nmol/L) up-regulated the mRNA expression of osteogenic genes Runx2 and OPN under the high glucose conditions (P < 0.05). (5) Compared with the high glucose group, Tideglusib (20 nmol/L) upregulated the expression of Wnt/β-catenin pathway related target β-catenin protein and down-regulated the expression of p-GSK-3β protein under high glucose conditions (P < 0.05). (6) The results showed that Tideglusib (20 nmol/L) could reverse the damage of high glucose conditions on the initial adhesion morphology, cell proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by activating Wnt/β-catenin signaling pathway.

Key words: Tideglusib, bone marrow mesenchymal stem cell, high glucose microenvironment, cell proliferation, osteogenic differentiation, Wnt/β-catenin, adhesion morphology, osteopontin