Protective effect of Korean Red Ginseng against glucocorticoid-induced osteoporosis in vitro and in vivo

Abstract

Background: Glucocorticoids (GCs) are commonly used in many chemotherapeutic protocols and play an important role in the normal regulation of bone remodeling. However, the prolonged use of GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. In this study, effects of Korean Red Ginseng (KRG) on GC-treated murine osteoblastic MC3T3-E1 cells and a GC-induced osteoporosis mouse model were investigated.

Methods: MC3T3-E1 cells were exposed to dexamethasone (Dex) with or without KRG and cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Real-time polymerase chain reaction was performed to evaluate the apoptotic gene expression; osteogenic gene expression and alkaline phosphatase (ALP) activity were also measured. Western blotting was performed to evaluate the mitogen-activated protein kinase (MAPK) proteins. A GC-induced osteoporosis animal model was used for in vivo study.

Results and conclusion: The MTT assay revealed that Korean Red Ginseng (KRG) prevents loss of cell viability caused by Dex-induced apoptosis in MC3T3E1 cells. Real-time polymerase chain reaction data showed that groups treated with both Dex and KRG exhibited lower mRNA levels of caspase-3 and -9, whereas the mRNA levels of Bcl2, IAPs, and XIAP increased. Moreover, groups treated with both Dex and KRG demonstrated increased mRNA levels of ALP, RUNX2, and bone morphogenic proteins as well as increased ALP activity in MC3T3-E1 cells, compared to cells treated with Dex only. In addition, KRG increased protein kinase B (AKT) phosphorylation and decreased c-Jun N-terminal kinase (JNK) phosphorylation. Moreover, microcomputed tomography analysis of the femurs showed that GC implantation caused trabecular bone loss. However, a significant reduction of bone loss was observed in the KRG-treated group. These results suggest that the molecular mechanism of KRG in the GC-induced apoptosis may lead to the development of therapeutic strategies to prevent and/or delay osteoporosis.

Keywords: Korean Red Ginseng; Panax ginseng; dexamethasone; osteoblast; osteoporosis.

Fig. 1

Effects of KRG on the viability of MC3T3-E1 cells. Cells were treated with KRG for 48 h. Cell viability was determined using the MTT assay. The experiment was run in triplicate. KRG, Korean Red Ginseng; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Fig. 2

Effect of Dex on the viability of MC3T3-E1 cells. Cells were treated with Dex (0μM, 50μM, 125μM, and 250μM) for 48 h. Treatment with Dex reduced cell viability, which was determined using the MTT assay. *p < 0.05 versus control. **p < 0.01 versus control. Dex, dexamethasone; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Fig. 3

Effect of KRG on the viability of Dex-exposed MC3T3-E1 cells. Cells were treated with KRG at various concentrations and/or cotreated with Dex (100μM) for 48 h. Cell viability was determined using the MTT assay. The experiment was run in triplicate. *p < 0.05 versus Dex control without KRG. **p < 0.01 versus Dex control without KRG. Dex, dexamethasone; KRG, Korean Red Ginseng; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Fig. 4

Effect of KRG on mRNA expression level of caspase-3, -6, -7, and -9 in Dex-exposed MC3T3-E1 cells. RNA was extracted from MC3T3-E1 cells treated with various doses of KRG and cotreated with Dex (100μM) for 48 h. The mRNA levels were analyzed using quantitative real-time PCR, and relative expression levels were calculated by comparison with the internal β-actin control. All experiments were performed in triplicate and values are presented as mean ± SD. *p < 0.5 versus control. **p < 0.01 versus control. ***p < 0.5 versus Dex control without KRG. ****p < 0.01 versus Dex control without KRG. Dex, dexamethasone; KRG, Korean Red Ginseng; PCR, polymerase chain reaction; SD, standard deviation.

Fig. 5

Effect of KRG on mRNA expression level of antiapoptotic genes in Dex-exposed MC3T3-E1 cells. RNA was extracted from MC3T3-E1 cells treated with various doses of KRG and cotreated with Dex (100μM) for 48 h. mRNA levels were analyzed using quantitative real-time PCR, and relative expression levels were calculated by comparison with the internal β-actin control. All experiments were performed in triplicate, and values are presented as mean ± SD. *p < 0.5 versus Dex control without KRG. Dex, dexamethasone; KRG, Korean Red Ginseng; PCR, polymerase chain reaction; SD, standard deviation.

Fig. 6

Effect of KRG on mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) signaling. MC3T3-E1 cells were treated with Dex (100μM) in the presence or absence of KRG (1 mg/mL) for 24 h. After 24-h treatment, Western blot analysis using p-P38, p-JNK, and p-AKT antibody was performed; β-actin was used as a loading control. Dex, dexamethasone; KRG, Korean Red Ginseng; p-AKT, phospho-AKT; p-P38, phospho-p38 mitogen-activated protein kinase; p-JNK, phospho-c-Jun N-terminal kinase.

Fig. 7

Effect of KRG and 100μM Dex on (A) ALP activity and (B) mRNA expression of MC3T3-E1 cell differentiation. Cells were treated with KRG at various concentrations and/or cotreated with Dex (100μM) for (A) 7 d and (B) 5 d. The mRNA levels were analyzed using quantitative real-time PCR, and relative expression levels were calculated by comparison with the internal β-actin control. *p < 0.05 versus Dex. **p < 0.01 versus Dex. ***p < 0.05 versus DC. ****p < 0.01 versus DC. ALP, alkaline phosphatase; BMP, bone morphogenic protein; DC, differentiation control; Dex, dexamethasone; KRG, Korean Red Ginseng; OCN, osteocalcin; OPN, osteopontin; PCR, polymerase chain reaction.

Fig. 8

Effect of KRG on glucocorticoid-induced osteoporosis mice model. Slow release pellets of prednisolone (PDS) (2.1 mg/kg/d) were administrated for 35 d by subcutaneous implantation. One wk after implantation, the mice were orally administrated with KRG extract (100 mg/kg and 500 mg/kg) or saline. After 4 wks, the mice were euthanized for bone analysis. The femurs were imaged with a micro-CT machine. CT, computed tomography; KRG, Korean Red Ginseng; PDS.