Gossypol Promotes Bone Formation in Ovariectomy-Induced Osteoporosis through Regulating Cell Apoptosis

Figures

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  Figure 1. Gossypol promotes bone formation in ovariectomy-induced osteoporosis in vivo. (a) Mice underwent ovariectomy in the presence or absence of gossypol injection and the thighbone of each mouse was collected for H&E staining. Red arrow means trabeculae of epiphyseal and black arrow means trabeculae in metaphyseal regions. (b) Serum osteocalcin level of each mouse in control and gossypol injection groups was determined with Elisa methods. (c) Serum OPG levels were assessed with Elisa methods for the mice injected with solvent or gossypol. (d) Serum RANKL levels of mice from two groups were measured with Elisa methods. (e) Serum RANKL/OPG levels were assessed by the ratio of serum RANKL and serum OPG levels. Each experiment was repeated for three times. Data were presented as mean ±SEM. ∗P<0.05 versus control.

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  Figure 2. Gossypol increased bone strength after ovariectomy. (a) MicroCT was used to scan the thighbone of each mouse in control and gossypol group. (b) Bone density of each mouse was assessed in control and gossypol group. (c) Ultimate strength of each mouse was determined in control and gossypol group. (d) Postyield displacement of each mouse was measured in control and gossypol group. Each experiment was repeated for three times. Data were presented as mean ±SEM. ∗P<0.05 versus control.

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  Figure 3. Gossypol promotes cell viability in osteoblast cell line MC3T3-E1 in vitro. (a) Cell viability was assessed in cells treated with DMSO or gossypol at different time intervals. (b) Cell viability was assessed in cells treated with DMSO or gossypol with various concentrations. Each experiment was repeated for three times. Data were presented as mean ±SEM. ∗P<0.05 versus control.

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  Figure 4. Gossypol inhibits cell apoptosis in osteoblast cells in vitro. (a) Cell apoptosis was determined in MC3T3-E1 cells treated with gossypol for 48h with a concentration of 20nm. (b) TUNEL assay was performed to assess the cell apoptosis in cells treated with gossypol. Five random fields were selected for calculation. (c) Relative activities of caspase-3, caspase-8, and caspase-9 in cells treated with gossypol. (d) Western blot assays were performed to examine the expression of caspase-3, caspase-8, and caspase-9 in MC3T3-E1 cells in response to gossypol treatments. Each experiment was repeated for three times. Data were presented as mean ±SEM. ∗P<0.05 versus control.

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  Figure 5. Gossypol treatments in MC3T3-E1 cells increases Wnt/ β -catenin signaling pathway. (a) Western blot assays examined the protein levels of Wnt, β-catenin, GSK-3β, and TGFβ1 in cells treated with gossypol. (b) qRT-PCR assays examined the mRNA levels of Wnt, β-catenin, GSK-3β, and TGFβ1 in cells treated with gossypol. Each experiment was repeated for three times. Data were presented as mean ±SEM. ∗P<0.05 versus control.

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  Figure 6. Inhibition of Wnt// β -catenin signaling pathway reversed the effects caused by gossypol in vitro. (a) mRNA levels of bone formation related genes in MC3T3-E1 cells treated with gossypol in presence or absence of ICG-001. ∗P<0.05, gossypol versus control. #P<0.05, gossypol+ ICG-001 versus gossypol. (b) Protein levels of bone formation related genes (osteocalcin, osterix, Runx2, ODF, OPG, AKP, TGFβ1, and COL1A1) in MC3T3-E1 cells treated with gossypol in presence or absence of ICG-001. Each experiment was repeated for three times. Data were presented as mean ±SEM.