Atorvastatin Increases Trabecular Bone Volume and Bone Formation in Aged apoE−/− Mice
To determine the effects of atorvastatin on the bone phenotype in aged apoE−/− mice, we first performed micro-CT reconstruction on the distal metaphyseal region of the femurs (Fig. 1). Relative to the control group, femurs from the atorvastatin group exhibited marked increases in trabecular BMD (+22.57%, p< 0.01), trabecular bone volume (BV/TV, +26.93%, p< 0.05), trabecular thickness (Tb.Th, +19.25%, p< 0.01), trabecular number (Tb.N, +31.24%, p< 0.05) and decreased trabecular spacing (Tb.sp, -17.87%, p< 0.05) (Fig. 1a,b) after adjustment for body weight. No significant changes in the cortical parameters of femurs were observed between the atorvastatin and control groups (Fig. 1c,d)
Atorvastatin Improved the Balance of Bone Turnover in Aged apoE−/− Mice
To further understand the changes in bone turnover in aged apoE−/− mice after treatment with atorvastatin, the levels of serum TRAP5b and OCN were also measured between the two groups. Compared to the control group, the level of serum OCN significantly increased (+66.2%, ** p< 0.01, Fig. 2d) in aged apoE−/− mice treated with atorvastatin, accompanied by a decrease in serum total cholesterol (TC, -51.6%, ** p< 0.01, Fig. 2b) and oxidized low-density lipoprotein (ox-LDL, -48.4%, ** p< 0.01, Fig. 2c). There were no remarkable changes in body weight or serum tartrate-resistant acid phosphatase 5b (TRAP5b) between the two groups (Fig. 2a,e).
Atorvastatin Increased Sirt1 Expression in the Bone Tissue of Aged apoE−/− Mice
To further analyse the mechanisms of the increase in bone mass and bone formation in aged apoE−/− mice after atorvastatin treatment, we measured Sirt1 and Runx2 expression in the bone tissue of the two groups (Fig. 3). When aged apoE−/− mice were treated with atorvastatin (10 mg kg−1 day−1) for 12 weeks, the mRNA expression of Sirt1 in bone tissue increased by 82.1% compared with expression in the control group (p< 0.05) (Fig. 3a). Similarly, the mRNA expression of Runx2, ALP, and OCN was significantly higher in the atorvastatin group after normalization (+72.6%, +68.6%, and +82.8%, respectively, p< 0.05) (Fig. 3a). Western blot analysis showed that the levels of Sirt1 and Runx2 increased by 65.8% and 105.6% in the atorvastatin group compared to the control group (p< 0.05) (Fig. 3b-d).
Atorvastatin Increases the Expression of Sirt1 and Runx2 in BMSCs from apoE−/− Mice In Vitro
To comprehensively determine the mechanism for atorvastatin-induced osteogenesis in apoE−/− mice, we investigated the effect of atorvastatin on the expression of Sirt1 and Runx2 in BMSCs in vitro. We treated BMSCs from 18-week-old apoE−/− mice exposed to different concentrations of atorvastatin (0, 10, 100, and 1000 nmol/L). The results showed that the mRNA and protein expression of Sirt1 and Runx2 increased significantly with the concentration of atorvastatin, reaching a maximum at a concentration of 100 nmol/L (p< 0.05 and p< 0.01, compared to 0 nmol/L) (Fig. 4a). The mRNA expression of ALP and OCN in BMSCs showed the same trend (p< 0.01) (Fig. 4a). The protein expression of Sirt1 and Runx2 also increased significantly with the concentration of atorvastatin, reaching a maximum at a concentration of 100 nmol/L (p< 0.05, compared to 0 nmol/L) (Fig. 4b-d).
Atorvastatin Promoted Osteogenesis in BMSCs from apoE−/− Mice through Activation of Sirt1
Based on the preceding observations, we then searched for the mechanism underlying the effect of atorvastatin on Sirt1–Runx2-mediated osteogenesis. The mRNA expression of Runx2, ALP, and OCN in BMSCs increased significantly (+86.1%, +111.7%, and +258.9%, respectively, p< 0.05, p< 0.01) after atorvastatin (100 nmol/L) treatment. However, when the BMSCs were pre-treated with EX527 (10 μM), the atorvastatin-induced increase in the mRNA expression of Runx2, ALP, and OCN was abolished and even decreased significantly (p< 0.05) or showed no difference (Fig. 5a). The protein expression of Runx2 increased by 136.1% after atorvastatin treatment. When BMSCs were pre-treated with EX527, the expression of Runx2 decreased by 15.2% and 18.9% after treatment without or with atorvastatin, respectively, compared with the control (p< 0.05) (Fig. 5b,c).