The Protective Effect of Luteolin in Glucocorticoid-Induced Osteonecrosis of the Femoral Head

Abstract

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a frequently occurring type of nontraumatic osteonecrosis. A failure of the timely treatment can eventually result in the collapse of the subchondral bone structure. Luteolin (Lut), a compound extracted from Rhizoma Drynariae, is reported to possess multiple pharmacological properties including anticancer, antioxidant, antiapoptosis, and antiinflammatory properties. However, whether Lut has a protective effect on the development of GIONFH remains unclear. In this study, we evaluated the effect of Lut on Dexamethasone (Dex)-induced STAT1/caspase3 pathway in vitro and evaluated GIONFH model in vivo. In vitro, Lut inhibited the upregulation of Dex-induced phospho-STAT1, cleaved caspase9, and cleaved caspase3. In addition, Lut inhibited Dex-induced expression of Bax and cytochrome c and increased the expression of B cell lymphoma-2(Bcl-2). In vivo, Lut decreased the proportion of empty lacunae in rats with GIONFH. Taken together, these findings indicate that Lut may have therapeutic potential in the treatment of GIONFH. Further, this effect might be achieved by suppressing mitochondrial apoptosis of osteoblasts via inhibition of STAT1 activity.

Keywords: STAT1/caspase3 pathway; apoptosis; glucocorticoid-induced osteonecrosis of the femoral head; luteolin; mitochondrial pathway.

Figure 1

Potential Cytotoxicity of Lut on osteoblasts. Chemical structure of Lut (A). The cytotoxic effects of Lut on osteoblasts were determined with increasing concentrations (0, 1, 10, 50 and 100 μM) for 24 and 48 hours using a CCK8 assay (B, C). And the viability of osteoblasts induced by Dex was determined by ELISAs and LDH release assay (D, E). Values represent the averages ± S.D. Significant differences between different groups are indicated as ^##P < 0.01, vs control group; *P < 0.05, **P < 0.01, vs Dex alone treatment group, n = 5.

Figure 2

Effects of Lut on Dex-induced STAT1/caspase3 pathway in Rat Osteoblasts. The osteoblasts treated with or without Lut (1、10 μM) for 2 h and then coincubated in the absence or presence of Dex (1 μM) for 24 h. The protein expression of STAT1, P-STAT1, caspase3, cleaved-caspase3, caspase9 and cleaved-caspase9 were measured by western blot (A) and quantification of the resulting bands (B). Significant differences between different groups are indicated as ^##P < 0.01, vs. control group; *P < 0.05, **P < 0.01 vs. Dex alone treatment group, n = 5.

Figure 3

Up-regulation STAT1 and cleaved caspase-3 expression in osteoblasts induced by Dex. The osteoblasts treated with or without Lut (10 μM) for 2 h and then coincubated in the absence or presence of Dex (1 μM) for 24 h. Typical P-STAT1 (A) and cleaved caspase‐3 (B) were detected by immunofluorescence combined with DAPI staining for nuclei (scale bar: 10 μm). n = 5.

Figure 4

Effect of Lut on mitochondrial apoptosis pathway of rat osteoblasts induced by Dex. The protein expressions of Bcl-2, Bax and cytochrome c in osteoblasts treated above were detected by Western blotting (A) and quantification of the resulting bands (B). Apoptotic osteoblasts were detected by utilizing with a Tunel Assay Kit. TUNEL assay was performed in osteoblast cells. The green fluorescence represents the apoptosis of osteoblasts and blue fluorescence represents the nucleus (C) (scale bar: 200 μm). Image J software 2.1 to evaluate the number of TUNEL positive cells (D). Significant differences between different groups are indicated as ^#P < 0.01, vs. control group; *P < 0.05, **P < 0.01, vs. Dex alone treatment group, n = 5.

Figure 5

The surface and histological analysis of glucocorticoid-induced osteonecrosis of the femoral head in vivo. In the control group, the femoral head had a white appearance. However, the femoral head surface was cyanotic in the model group. In the Lut group, the appearance of femoral head was between that of the control and model groups (A). And there were no empty lacunae in the femoral head of the control group. A large number of empty bone lacunae and necrotic bone marrow cells were seen in the model group, while there were few empty bone lacunae in the Lut group (B) (scale bar: 100 μm). The proportion of empty bone lacunae in the model group was significantly higher than that in the control group and Lut group (C). Values represent the averages ± S.D. Significant differences between different groups are indicated as ^##P < 0.01 vs. the Control group and ^**P < 0.01 vs. the Model group.

Figure 6

Analysis of bone structure of rat femoral head by Micro-CT scanning. The Micro-CT scanning of the femoral head and quantitative analysis of bone trabeculae was compared among the control group, the model group and the Lut group (A, B). Values represent the averages ± S.D. Significant differences between different groups are indicated as ^##P < 0.01 vs. the Control group and ^**P < 0.01 vs. the Model group.

Figure 7

The effect of Lut on the activation of C-caspase3 in vivo Immunohistochemical staining of C-caspase3 expression in the samples. Caspase3 is an aspartic acid-specific cysteine protease, the activation of which could participate in apoptotic cell death. Positive expressions was found in the model groups (scale bar: 10 μm) (A). The percentages of Cleaved-caspase3 positive cells in each section were quantified by Image J software (B). Values represent the averages ± S.D. Significant differences between different groups are indicated as ^##P < 0.01 vs. the Control group and **P < 0.01 vs. The Model group.