Abstract: BACKGROUND: Cuproptosis is a newly identified copper-dependent cell death. It has attracted more attention to various diseases in recent years. Cuproptosis has been studied in cancer, but its role in osteoblasts remains unclear.
OBJECTIVE: To investigate the mechanism of high glucose-induced cuproptosis in osteoblasts.
METHODS: (1) MC3T3-E1 cells were cultured in four groups: 20 μmol/L CuCl2 group, 40 μmol/L CuCl2 group, 20 μmol/L CuCl2+high glucose group, and 40 μmol/L CuCl2+high glucose group. The high glucose environment was simulated by adding 25 mmol/L glucose and 200 mmol/L sodium palmitate. After 48 hours of osteogenic induction, western blot assay was used to detect the protein expression of copper ion transporters (copper ion influx transporter SLC31A1 and copper ion efflux transporter ATP7B). (2) MC3T3-E1 cells were cultured in four groups: the normal group without any treatment, the high glucose group with the addition of 25 mmol/L glucose and 200 mmol/L sodium palmitate, the CuCl2 group with the addition of 20 μmol/L CuCl2, and the high glucose + CuCl2 group with the simultaneous addition of 25 mmol/L glucose, 200 mmol/L sodium palmitate, and 20 μmol/L CuCl2. After osteogenic induction, mitochondrial alterations were observed via transmission electron microscopy. The osteoblast differentiation and mineralization were evaluated using alkaline phosphatase staining and alizarin red staining. qRT-PCR was used to detect osteoblast-specific gene expression, and western blot was used to detect osteoblast- and cuproptosis-related protein expression.
RESULTS AND CONCLUSION: (1) Western blot analysis showed that the expression of SLC31A1 protein was higher in the 20 μmol/L CuCl2+high glucose group than the 20 μmol/L CuCl2 group (P < 0.05), while the expression of ATP7B protein was lower (P < 0.05). In the 40 μmol/L CuCl2+high glucose group, the expression of SLC31A1 protein was higher than in the 40 μmol/L CuCl2 group (P < 0.05), and the expression of ATP7B protein was lower (P < 0.05). (2) Under the transmission electron microscopy, CuCl2 induced changes in the mitochondrial structure of MC3T3-E1 cells, primarily characterized by the disappearance of mitochondrial cristae, mitochondrial shrinkage, reduced volume, and disruption of mitochondrial membrane structure. The combined exposure to high glucose exacerbated these structural alterations. Alkaline phosphatase and alizarin red staining results indicated that CuCl2 inhibited osteogenic differentiation and mineralization of MC3T3-E1 cells, and the inhibitory effect of CuCl2+high glucose environment on osteogenic differentiation and mineralization was stronger than that of CuCl2 alone. qRT-PCR and western blot analysis revealed that the CuCl2+high glucose environment significantly inhibited the expression of osteogenic-specific gene at mRNA and protein levels, inducing osteoblast cuproptosis. These findings suggest that high glucose may induce and exacerbate osteoblast cuproptosis by disrupting copper transport and cuproptosis-related protein expression. 

Key words: cuproptosis, osteoblasts, high glucose, SLC31A1/FDX1 axis, MC3T3-E1 cell, copper transporter