Abstract:

Objective To investigate the effects of sodium palmitate （PA）-induced lipotoxicity on mitochondrial function and apoptosis in mouse L929 fibroblasts， and to clarify its associations with mitofusin 2 （MFN2） expression and mitochondria-associated endoplasmic reticulum membrane （MAMs） structure. Methods Mouse L929 fibroblasts were used as the model，which were treated with different concentrations of PA （0.1， 0.2， 0.3， 0.4， 0.5， and 0.6 mmol·L^-1） for 12， 24， 36， and 48 h. Cell counting kit-8 （CCK-8） method was used to detect the survival rates of the cells in various groups after treatment， so as to screen the optimal intervention condition of PA. The cells were then divided into control group and PA group. Flow cytometry method was used to detect the apoptotic rates in the cells in two groups. Oil red O staining method was used to observe the lipid deposition in the cells in two groups. Transmission electron microscopy （TEM） was used to observe the structural characteristics of mitochondria and MAMs in the cells in two groups. Flow cytometry was used to detect the levels of reactive oxygen species （ROS） in the cells in two groups. JC-1 method was used to detect the mitochondrial membrane potential in the cells in two groups. Real-time fluorescence quantitative PCR （RT-qPCR） method and Western blotting method were used to detect the expression levels of MFN2 mRNA and protein in the cells in two groups. Results The CCK-8 assay results showed that at 12， 24， 36， and 48 h post treatment， compared with control group， the survival rates of L929 cells in groups treated with PA at concentrations ≥0.2 mmol·L^-1 were significantly decreased （P<0.05 or P<0.001）， showing a concentration- and time-dependent effect. The survival rate of L929 cells was approximately 60% in 0.3 mmol·L^-1 PA group after 24 h of treatment， which was used as the subsequent PA intervention condition. The flow cytometry and Oil red O staining results showed that compared with control group， the apoptotic rate of L929 cells in PA group was significantly increased （P<0.001） and the level of lipid deposition in the cells was significantly increased （P<0.001）. The TEM results showed that the mitochondria in L929 cells in PA group exhibited swelling， disruption of cristae structure， and fragmentation， while the number of MAMs contact sites was reduced and the coupling distance was increased. The flow cytometry， JC-1 method， and RT-qPCR method results showed that compared with control group， the level of ROS in L929 cells in PA group was significantly increased （P<0.001），the mitochondrial membrane potential of the cells was significantly decreased （P<0.01）， and the expression levels of MFN2 mRNA and protein in the cells were significantly decreased （P<0.01）. Conclusion PA-induced lipotoxicity downregulates MFN2 expression and disrupts MAMs structure， thereby promoting oxidative stress， mitochondrial depolarization， and cell apoptosis. These findings suggest that under PA-induced lipotoxic conditions， the downregulation of MFN2 expression and the damage to MAMs structure are closely associated with mitochondrial homeostasis imbalance and cell apoptosis.

Key words: Pelvic floor dysfunction, Lipotoxicity, Mitofusin 2, Mitochondria-associated endoplasmic reticulum membrane, Mitochondrial dysfunction