Abstract: BACKGROUND: Matrix metalloproteinase 9 affects osteogenic and myogenic differentiation, but its specific regulatory mechanism is not well understood.
OBJECTIVE: To investigate the effects of oxidative stress injury on mitophagy in mouse MC3T3-E1 and C2C12 cells, and to examine the role of matrix metalloproteinase 9 as a critical regulator in regulating mitophagy and affecting osteogenic and myogenic differentiation via the PTEN-induced kinase 1/Parkin signaling pathway.
METHODS: Mouse C2C12 and MC3T3-E1 cells were selected for differentiation and culture. Each cell line was divided into four groups: control, model (hydrogen peroxide-induced oxidative damage), GM6001 (matrix metalloproteinase 9 inhibitor), and 3-methyladenine (PTEN-induced kinase 1/Parkin pathway inhibitor) groups, respectively. Cells in each group were treated for 24 hours. Flow cytometry was used to detect intracellular mitochondrial membrane potential and reactive oxygen species levels. Transmission electron microscopy was used to observe cellular mitochondrial damage and mitophagy. Western blot was used to detect matrix metalloproteinase 9 protein expression. Quantitative reverse transcription polymerase chain reaction and western blot were used to detect the mRNA and protein expressions of P62, microtubule-associated protein light chain 3, PTEN-induced kinase 1, and parkin in MC3T3-E1 cells. Western blot was used to detect the protein expressions of osteogenic proteins osteocalcin, osteopontin, and Runt-related transcription factor 2 in MC3T3-E1 cells. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of myogenic genes myogenic factor 5, myogenic factor 6, myogenic differentiation 1, and myogenin in C2C12 cells.
RESULTS AND CONCLUSION: (1) In MC3T3-E1 and C2C12 cells, mitochondrial membrane potential was decreased and reactive oxygen species levels were increased in the model, GM6001, and 3-methyladenine groups compared with the control group. Reactive oxygen species levels were decreased in the GM6001 group compared with the model group, and reactive oxygen species levels were increased in the 3-methyladenine group compared with the GM6001 group. (2) Transmission electron microscopy showed that mitochondrial damage was more severe and mitophagy was formed in the model group. Mitochondrial damage was improved in the GM6001 group. Mitochondrial damage and mitophagy were weaker in the 3-methyladenine group compared with the model group. (3) Compared with the control group, matrix metalloproteinase 9 protein expression was increased in the model, GM6001, and 3-methyladenine groups. Compared with the model group, matrix metalloproteinase 9 protein expression was significantly decreased in the 3-methyladenine group. Compared with the 3-methyladenine group, matrix metalloproteinase 9 protein expression was significantly decreased in the GM6001 group. (4) Compared with the control group, the mRNA and protein expressions of P62 were decreased in the model group, further decreased in the GM6001 group, and increased in the 3-methyladenine group. Compared with the control group, the mRNA and protein expressions of microtubule-associated protein light chain 3 II, microtubule-associated protein light chain 3 I, PTEN-induced kinase 1, and Parkin were elevated in the model group, further elevated in the GM6001 group, and decreased in the 3-methyladenine group. (5) In the MC3T3-E1 cells, compared with the control group, the protein expressions of osteocalcin, osteopontin, and Runt-related transcription factor 2 were decreased in the model, GM6001, and 3-methyladenine groups. Compared with the model group, the protein expressions of osteocalcin, and osteopontin were increased in the GM6001 group. Compared with the GM6001 group, the protein expressions of osteocalcin and osteopontin were decreased in the 3-methyladenine group. (6) In the C2C12 cell line, compared with the control group, the mRNA expressions of myogenic factor 5, myogenic factor 6, myogenic differentiation 1, and myogenin were decreased in the model, GM6001, and 3-methyladenine groups. Compared with the model group, the expressions of the above myogenic genes were elevated in the GM6001 group, and compared with the GM6001 group, the above myogenic gene expressions were decreased in the 3-methyladenine group. The results showed that matrix metalloproteinase 9 exhibits high expression levels in oxidative injury models, and its inhibitors enhance mitophagy in the oxidative damage environment. This regulation was based on the activation of PTEN-induced kinase 1/Parkin pathway, thereby promoting osteogenic and myogenic differentiation.

Key words: osteoporosis, sarcopenia, osteosarcopenia, matrix metalloproteinase 9, mitophagy, PTEN-induced kinase 1/Parkin pathway, osteogenic differentiation, myogenic differentiation