Effects of Total Flavonoids of Epimedium on Bone Marrow Adipose Tissue in Ovariectomized Rats

Figures

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  Figure 1. Example of a 2-point Dixon sequence for measuring fat fraction. The region of interest was manually drawn in the distal femur in a fat-only image, avoiding the cortical bone, and directly copied onto a water-only image.

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  Figure 2. Body weights of rats in all treatment groups throughout the experimental period. Values are presented as mean ± SD (Sham, n = 11; OVX, n = 10; TFE, n = 11). OVX, ovariectomy; Sham, sham-operation; TFE, total flavonoids of Epimedium. P values represent Bonferroni-corrected p-values (two-way repeated-measures ANOVA). **P <0.001 between the OVX and Sham controls and between the OVX controls and TFE-treated group at week 12.

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  Figure 3. Effects of TFE treatment on (A) uterine wet weight and (B) uterine index. Data are presented as mean ± SD (Sham, n = 11; OVX, n = 10; TFE, n = 11). OVX, ovariectomy; Sham, sham-operation; TFE, total flavonoids of Epimedium; Ve, vehicle. **P<0.001 by One-way ANOVA with Bonferroni-correction.

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  Figure 4. Effects of TFE treatment on (A) 17β-estradiol, (B) P1NP, and (C) CTX-I. Values are presented as mean (SD; Sham, n = 11; OVX, n = 10; TFE, n = 11). CTX-I, C-terminal telopeptides of type I collagen; P1NP, type 1 collagen amino propeptide; OVX, ovariectomy; Sham, sham-operation; TFE, total flavonoids of Epimedium; Ve, vehicle. TFE treatment inhibited bone turnover in OVX rats. Serum 17β-estradiol was significantly reduced while levels of CTX-I were higher in OVX rats than in Sham rats. *P<0.05 and **P<0.001 by One-way ANOVA with Bonferroni-correction.

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  Figure 5. Effects of TFE treatment on bone trabeculae microstructure in the tibia in OVX rats. Sagittal views of micro-CT images at the proximal tibia region (A) showed deterioration of trabecular microstructure in OVX rats, and TFE treatment improved bone trabeculae microstructure. TFE-treated rats showed responses to the OVX-induced losses of (B) BV/TV, (C) Tb.N, (D) Tb.Th, (E) SMI, and Tb.Sp (F). BV/TV, bone volume fraction; Tb. Sp, trabecular separation; Tb.N, trabecular number; Tb.Th, trabecular thickness; SMI, structure model index; OVX, ovariectomy; Sham, sham-operation; TFE, total flavonoids of Epimedium; Ve, vehicle. Data are shown as mean ± SD (Sham, n = 11; OVX, n = 10; TFE, n = 11). *P<0.05 and **P<0.001 between groups analyzed by One-way ANOVA with Bonferroni-correction.

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  Figure 6. Changes in the marrow adipose (A) and adipocyte quantitative parameters (B–D). Sections of decalcified bone were stained with H&E (×200). OVX markedly increased infiltration of marrow fat (arrow). This increase in marrow adiposity was restored by TFE treatment. Ad.D, adipocyte density; Ad.MD, adipocyte mean diameter; Ad.V/Ma.V, percentage adipocyte volume per marrow volume; OVX, ovariectomy; Sham, sham-operation; TFE, total flavonoids of Epimedium; Ve, vehicle. Data are shown as mean ± SD (Sham, n = 11; OVX, n = 10; TFE, n = 11). **P<0.001 between groups analyzed by One-way ANOVA with Bonferroni-correction.

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  Figure 7. Effects of TFE treatment on marrow white and brown adipocyte gene markers in whole tibiae of OVX rats. Gene expression markers of Prdm16 and Ucp1 were normalized to FABP4 expression in the same sample. OVX, ovariectomy; Sham, sham-operation; TFE, total flavonoids of Epimedium; Ve, vehicle. Results are presented as means ± SD (Sham, n = 11; OVX, n = 10; TFE, n = 11). *P<0.05 and **P<0.001 between groups analyzed by One-way ANOVA with Bonferroni-correction.

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  Figure 8. Representative microscopic features of PPARγ2 and FABP4 expression in bone marrow (200x magnification). Quantification of PPARγ2 expression by immunofluorescence (A) and FABP4 expression by immunohistochemistry (B) in the bone marrow showed a significant increase in PPARγ2 and FABP4 protein expression in proximal tibiae of OVX rats, which were restored to normal levels after TFE treatment. For PPARγ2, the positively staining pixels were presented as a percentage of the total pixels (C); for FABP4, semi-quantification is expressed as the relative integrated intensity of intramarrow staining (D). FABP4, fatty acid-binding protein 4; PPARγ2, peroxisome proliferator-activated receptor gamma-2; OVX, ovariectomy; Sham, sham-operation; TFE, total flavonoids of Epimedium; Ve, vehicle. Data are shown as mean ± SD (Sham, n = 11; OVX, n = 10; TFE, n = 11). **P<0.001 between groups analyzed by One-way ANOVA with Bonferroni-correction.