Protective effect of equol on mitochondrial damage induced by high glucose and fat in osteoblasts

Abstract

Objective To observe the effect of equol (Eq) intervention on osteoblasts induced by high glucose and fat, and to explore its mechanism of action. Methods After ROS1728 osteoblasts were treated with different concentrations of Eq, sodium palmitate (PA) and glucose (GLU) for 48 h, nuclear apoptosis was observed by HE staining and cell survival rate was detected by CCK-8 assay in order to determine the optimal concentrations of GLU, PA and Eq interventions to induce cellular model of diabetic osteoporosis. ROS1728 cells were then divided into control group (5.5 mmol/L GLU), model group (30 mmol/L GLU+250 μmol/L PA), and low, medium and high dose Eq treatment groups (model+1×10-8, 1×10-7 and 1×10-6 mol/L Eq). CCK8 assay was used to detect cell viability, and flow cytometry for cell apoptosis. Hoechst33342 staining and HE staining were employed to observe apoptotic nuclei. Mitochondrial structure, mitochondrial membrane potential (MMP) changes, and protein levels of Sirt1, PGC-1α, Bcl-2, Bax and Cleaved caspase-3 were observed by transmission electron microscopy, Mito-Tracker Red CMXRos (mitochondrial red fluorescent probe) staining, and Western blotting, respectively. Results Compared with the control group, treatment of 30 mmol/L GLU and 250 μmol/L PA significantly reduced the cell viability rate (P < 0.05) and increased the apoptotic rate (P < 0.05), and the optimal concentrations were determined to induce high glucose and fat environment of osteoblasts. While Eq intervention significantly increased the survival rate and decreased the apoptotic rate (P < 0.05), attenuated the damage of mitochondrial structure, elevated MMP (P < 0.05) and decreased ROS level (P < 0.05), as well as up-regulated the expression levels of Sirt1, PGC-1α and Bcl-2 and down-regulated those of Bax and Cleaved caspase-3 (P < 0.05). Conclusion Eq intervention could effectively reduce mitochondrial damage, oxidative stress and apoptosis of osteoblasts induced by high glucose and fat, which might be partially related to its activation of Sirt1 and PGC-1α.

Keywords

• equol
• ros1728 cells
• high-glucose and high-fat
• mitochondria
• oxidative stress
• apoptosis