Geniposide suppressed OX-LDL-induced osteoblast apoptosis by regulating the NRF2/NF-κB signaling pathway

Abstract

Background: Osteoporosis (OP), due to microarchitectural alterations, is associated with decreased bone mass, declined strength, and increased fracture risk. Increased osteoblast apoptosis contributes to the progression of OP. Natural compounds from herbs provide a rich resource for drug screening. Our previous investigation showed that geniposide (GEN), an effective compound from Eucommia ulmoides, could protect against the pathological development of OP induced by cholesterol accumulation.

Methods: The rat OP models were duplicated. Dual-energy X-ray absorptiometry, hematoxylin and eosin staining, and immunohistochemistry were used to evaluate bone changes. TUNEL/DAPI staining assays were used for cell apoptosis detection. Protein expression was determined by western blotting assays.

Results: A high-fat diet promoted OP development in vivo, and OX-LDL stimulated osteoblast apoptosis in vitro. GEN exhibited protective activities against OX-LDL-induced osteoblast apoptosis by increasing the NRF2 pathway and decreasing the NF-κB pathway. PDTC, an NF-κB inhibitor, could further promote the biological functions of GEN. In contrast, ML385, an NRF2 inhibitor, might eliminate GEN's protection.

Conclusion: GEN suppressed OX-LDL-induced osteoblast apoptosis by regulating the NRF2/NF-κB signaling pathway.

Keywords: Apoptosis; Geniposide; NRF2/NF-κB; Osteoblast; Osteoporosis.

Fig. 1

GEN ameliorated HF-induced OP in rat models (n = 6). A–F The serum levels of OX-LDL, TC, TG, LDL, HDL, and body weight were detected. (G) HE staining of the proximal femoral trabeculae was evaluated (40 × magnification). H The proximal femurs were scanned by DEXA. I The values of BMD in the femoral neck were analyzed. J The correlation between weight and BMD was analyzed. *p < 0.05, **P˂0.01. NC, negative control; HF, high fat; 50 mg/kg, HF + 50 mg/kg GEN; 100 mg/kg, HF + 100 mg/kg GEN

Fig. 2

The inhibitory effects of GEN against HF-induced osteoblast apoptosis in vivo and in vitro. The changes of Bcl-2 (A, B) and caspase3 (C, D) in IHC (× 40 magnification) were analyzed. E CCK-8 assays were performed. F TUNEL staining assays were performed for detection of cell apoptosis. The protein expression of Bcl-2 (G, H) and cleaved-caspase3 G, I in OX-LDL-treated MC3T3-E1 cells was determined by western blot. *p < 0.05, **p˂0.01. NC, negative control; HF, high fat; 50 mg/kg, HF + 50 mg/kg GEN; 100 mg/kg, HF + 100 mg/kg GEN

Fig. 3

GEN inhibited OX-LDL-induced osteoblast apoptosis by down regulating NF-κB pathway. A, B The changes of p65 in IHC (× 40 magnification) were analyzed. The protein expression of Bcl-2 (C, D), cleaved-caspase3 (C, E), and p-p65/p65 C, F were detected by western blot in HF/PDTC-treated MC3T3-E1 cells. G TUNEL staining assays were performed for detection of cell apoptosis. *p < 0.05; **p < 0.01, NC, negative control; HF, high fat; 50 mg/kg, HF + 50 mg/kg GEN; 100 mg/kg, HF + 100 mg/kg GEN

Fig. 4

GEN inhibited OX-LDL-induced osteoblast apoptosis by up regulating NRF2 pathway. A, B The changes of NRF2 in IHC (× 40 magnification) were analyzed. The protein expression of NRF2 (C, D), HO-1 (C, E), Bcl-2 (C, F), cleaved-caspase3 (C, G), p-p65/p65 (C, H) were detected by western blot in OX-LDL/ML385-treated MC3T3-E1 cells. I TUNEL staining assays were performed for detection of cell apoptosis. *p < 0.05; **p < 0.01, NC, negative control; HF, high fat; 50 mg/kg, HF + 50 mg/kg GEN; 100 mg/kg, HF + 100 mg/kg GEN