Geniposide Ameliorated Dexamethasone-Induced Cholesterol Accumulation in Osteoblasts by Mediating the GLP-1R/ABCA1 Axis

Abstract

Background: Overexposure to glucocorticoid (GC) produces various clinical complications, including osteoporosis (OP), dyslipidemia, and hypercholesterolemia. Geniposide (GEN) is a natural iridoid compound isolated from Eucommia ulmoides. Our previous study found that GEN could alleviate dexamethasone (DEX)-induced differentiation inhibition of MC3T3-E1 cells. However, whether GEN protected against Dex-induced cholesterol accumulation in osteoblasts was still unclear.

Methods: DEX was used to induce rat OP. Micro-CT data was obtained. The ALP activity and mineralization were determined by the staining assays, and the total intracellular cholesterol was determined by the ELISA kits. The protein expression was detected by western blot.

Results: GEN ameliorated Dex-induced micro-structure damages and cell differentiation inhibition in the bone trabecula in rats. In MC3T3-E1 cells, Dex enhanced the total intracellular cholesterol, which reduced the activity of cell proliferation and differentiation. Effectively, GEN decreased DEX-induced cholesterol accumulation, enhanced cell differentiation, and upregulated the expression of the GLP-1R/ABCA1 axis. In addition, inhibition of ABAC1 expression reversed the actions of GEN. Treatment with Exendin9-39, a GLP-1R inhibitor, could abrogate the protective activity of GEN.

Conclusions: GEN ameliorated Dex-induced accumulation of cholesterol and inhibition of cell differentiation by mediating the GLP-1R/ABCA1 axis in MC3T3-E1 cells.

Keywords: ABCA1; GLP-1R; cholesterol; geniposide; glucocorticoid; osteoporosis.

Figure 1

GEN protected against DEX-induced OP in rat models (n = 6, repeated twice). (A) The HE staining of the bone trabecula on the femoral neck of the proximal femur (×40 magnification). (B) 3D reconstruction and 2D reconstruction of micro-CT on the proximal femur. (C) Bone mineral density (BMD) of the proximal femur. The values of Tb.Sp (D), SMI (E), Tb.Th (F), BS (G), BV (H), and BV/TV (I) were detected. All experiments were implemented separately in triplicate. * p < 0.05. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN.

Figure 2

GEN ameliorated DEX-induced inhibition of osteoblast differentiation. (A) The immunohistochemical study of RUNX2 and OPN in the bone trabecula of the rat proximal femurs (×40 magnification). The staining intensity of RUNX2 (B) and OPN (C) was evaluated. (D) The ALP staining and the Alizarin Red S staining assays were conducted (×100 magnification). The protein expression of RUNX2 (E,F) and OPN (E,G) were determined by western blot. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN.

Figure 3

GEN ameliorated DEX-induced cholesterol accumulation in MC3T3-E1 cells. (A) MTT assays were conducted to investigate the effects of cholesterol on cell viability. (B) The level of intracellular cholesterol after administration with exogenous cholesterol. (C–E) The protein expression of RUNX2 and OPN in CHO (50 μM)-treated MC3T3-E1 cells was determined by western blot. (F) The level of the total intracellular cholesterol was measured by the ELISA kit. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. OIM, osteogenic induction medium; CHO, cholesterol.

Figure 4

GEN ameliorated DEX-induced cholesterol accumulation by increasing ABCA1 expression. (A,B) The in vivo immunohistochemical examination of ABCA1 expression was conducted. (C–E) The protein expression of ABCA1 and ApoA-I was detected by western blot in DEX-treated MC3T3-E1 cells. (F) The level of the total intracellular cholesterol was measured using the ELISA kit in MC3T3-E1 cells co-treated with DEX and DIDS (200 μM). (G–I) The protein expression of ABCA1 and ApoA-I was detected by western blot in DEX/DIDS-treated MC3T3-E1 cells. (J) The ALP staining and the Alizarin Red S staining assays were conducted (×100 magnification). (K–M) The protein expression of RUNX2 and OPN was detected by western blot in DEX/DIDS-treated MC3T3-E1 cells. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. OIM, osteogenic induction medium. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN.

Figure 5

GEN promoted ABCA1-mediated cholesterol metabolism in a GLP-1R-dependent manner. (A,B) The in vivo immunohistochemical examination of GLP-1R expression was conducted. (C,D) The protein expression of GLP-1R was determined by western blot in DEX-treated MC3T3-E1 cells. (E) The ALP staining and the Alizarin Red S staining assay were performed in EX-treated MC3T3-E1 cells (×100 magnification). (F) The level of the total intracellular cholesterol was measured using the ELISA kit. (G–K) The protein expression of GLP-1R, ABCA1, RUNX2, and OPN was detected by western blot. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN; EX, Exendin9-39.