Effects of Sparganii Rhizoma on Osteoclast Formation and Osteoblast Differentiation and on an OVX-Induced Bone Loss Model

Abstract

Postmenopausal osteoporosis is caused by an imbalance between osteoclasts and osteoblasts and causes severe bone loss. Osteoporotic medicines are classified into bone resorption inhibitors and bone formation promoters according to the mechanism of action. Long-term use of bisphosphonate and selective estrogen receptor modulators (SERMs) can cause severe side effects in postmenopausal osteoporosis patients. Therefore, it is important to find alternative natural products that reduce osteoclast activity and increase osteoblast formation. Sparganii Rhizoma (SR) is the dried tuberous rhizome of Sparganium stoloniferum Buchanan-Hamilton and is called "samreung" in Korea. However, to date, the effect of SR on osteoclast differentiation and the ovariectomized (OVX)-induced bone loss model has not been reported. In vitro, tartrate-resistant acid phosphatase (TRAP) staining, western blots, RT-PCR and other methods were used to examine the effect of SR on osteoclast differentiation and osteoblasts. In vivo, we confirmed the effect of SR in a model of OVX-induced postmenopausal osteoporosis. SR inhibited osteoclast differentiation and decreased the expression of TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells 1 (NFATc1) and c-Fos pathway. In addition, SR stimulates osteoblast differentiation and increased protein expression of the bone morphogenetic protein 2 (BMP-2)/SMAD signaling pathway. Moreover, SR protected against bone loss in OVX-induced rats. Our results appear to advance our knowledge of SR and successfully demonstrate its potential role as a osteoclastogenesis-inhibiting and osteogenesis-promoting herbal medicine for the treatment of postmenopausal osteoporosis.

Keywords: bone remodeling; osteoblast; osteoclast; ovariectomized; sparganii rhizoma.

FIGURE 1

The effects of SR on osteoclast differentiation and bone resorption in RANKL-induced RAW 264.7 cells. (A) RAW 264.7 cell viability was measured using a CCK-8 assay kit. (B) The effects of SR (0, 125, 250, 500, 1,000 μg/ml) on the viability of osteoclasts induced by RANKL using a CCK-8 assay kit. (C) In vitro experimental design to investigate the effect of SR on osteoclast differentiation. (D) TRAP-positive cells were stained using a TRAP kit. (E) A number of TRAP-positive cells with >3 nuclei were counted using an inverted microscope (magnification, ×100). (F) TRAP activity was measured using an ELISA reader. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^# p < 0.05, ^## p < 0.01 vs. the normal group (untreated cells); ^* p < 0.05, ^** p < 0.01 vs. the RANKL only treatment group.

FIGURE 2

The effects of SR on bone resorption and F-actin ring formation in RANKL-induced RAW 264.7 cells. (A) Pit formation was captured using an inverted microscope (magnification, ×100; scale bar, 200 µm). (B) F-actin rings were stained with fluorescent phalloidin (magnification, ×100; scale bar, 200 µm). (C) The pit area was measured using ImageJ software. Data are presented as the mean ± SD (standard error of the mean) of three independent experiments. (D) The number of F-actin rings was counted. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^## p < 0.01 vs. the normal group (untreated cells); ^* p < 0.05, ^** p < 0.01 vs. the RANKL only treatment group.

FIGURE 3

The effects of SR on the expression of TRAF6, NF-κB and MAPK signaling pathway. (A) Protein expression levels of TRAF6, p-NF-κB, NF-κB, p-IκB and IκB were measured by western blotting. (B) TRAF6, p-IκB and IκB were normalized to that of β-actin, which was used as a loading control. p-NF-κB and NF-κB were normalized to that of Lamin B, which was used as a loading control. (C) Protein expression levels of p-ERK, p-JNK, p-p38 were measured by western blotting. The bands were normalized to that of ERK, JNK and p38. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^# p < 0.05, ^## p < 0.01 vs. the normal group (untreated cells); ^* p < 0.05, ^** p < 0.01 vs. the RANKL only treatment group.

FIGURE 4

The effects of SR on NFATc1 and c-Fos levels in RAW 264.7 cells. (A) Protein expression levels of NFATc1 and c-Fos were measured by western blotting. (B) The bands were normalized to that of β-actin, which was used as a loading control. (C) mRNA expression levels in Nfatc1 and Fos were measured by RT-PCR. The bands were normalized to that of β-actin (Actb). Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^## p < 0.01 vs. the normal group (untreated cells); ^* p < 0.05, ^** p < 0.01 vs. the RANKL only treatment group.

FIGURE 5

The effects of SR on the expression of osteoclast-related genes in RANKL-induced RAW 264.7 cells. (A) Protein expression of MMP-9 was determined using Western blotting and (B) mRNA expression of MMP-9 (Mmp9) was determined using RT-PCR. The bands were normalized to that of β-actin (Actb). (C) mRNA expression of RANK (Tnfrsf11a), TRAP (Acp5), CA2 (Ca2), OSCAR (Oscar), ATP6v0d2 (Atp6vod2) and DC-STAMP (Dcstamp) was determined using RT-PCR. (D) The bands were normalized to that of β-actin (Actb). Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^# p < 0.05, ^## p < 0.01 vs. the normal group (untreated cells); ^* p < 0.05, ^** p < 0.01 vs. the RANKL only treatment group.

FIGURE 6

The effects of SR on osteoblast differentiation. (A) MC3T3-E1 cell viability was analyzed by CCK-8 assays at 3, 7 and 14 days. (B) Calcified nodules produced by osteoblasts were stained with Alizarin Red S for 14 and 21 days. (C) The absorbance of Alizarin Red S staining dye was measured using an ELISA reader at 450 nm. (D) Protein expression levels of BMP-2, p-Smad 1/5, RUNX-2 and Osterix were measured by western blotting. (E) BMP-2, RUNX-2 and Osterix levels were normalized to that of β-actin. p-Smad 1/5 levels was normalized to that of Smad 1/5/9. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^## p < 0.01 vs. the normal group (untreated cells); ^* p < 0.05, ^** p < 0.01 vs. the osteogenic medium-treated cells.

FIGURE 7

Change in body weight and serum levels of TRAP, ALP, AST and ALT in the OVX model. (A) Body weight was measured once a week for 8 weeks. (B) The uterine weight was measured after sacrifice. The serum levels of (C) TRAP, (D) ALP, (E) AST, and (F) ALT were measured using an ELISA reader. The results are presented as the mean ± SEM of each experimental group (n = 8). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^# p < 0.05, ^## p < 0.01 vs. the normal group (sham-operation group); ^* p < 0.05, ^** p < 0.01 vs. the control group (OVX-induced group).

FIGURE 8

Effects of SR on bone loss in the OVX-induced model. (A) Micro-CT analysis of the femurs. (B) BMD, (C) BV/TV, (D) Tb.Th and (E) Tb. sp of femurs were measured using micro-CT. The results are presented as the mean ± SEM of each experimental group (n = 8). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^# p < 0.05, ^## p < 0.01 vs. the normal group (sham-operation group); ^* p < 0.05, ^** p < 0.01 vs. the control group (OVX-induced group).

FIGURE 9

Effect of SR on trabecular area, osteoclasts and osteoblast in the OVX-induced rats. (A) The trabecular area was measured using H&E staining of femur tissue. (B) The number of osteoclasts (purple) in the femoral tissue was verified through TRAP staining. (C) The number of osteoblasts (yellow) in the femoral tissue was measured through masson-goldner’s trichrome staining. (D) Trabecular area, (E) Oc.N /BS (/mm). (F) Oc.S /BS (%). (G) Ob. N/BS and (F) Ob. S/BS were measured using ImageJ version 1.46. The results are presented as the mean ± SEM of each experimental group (n = 8). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^## p < 0.01, ^# p < 0.05 vs. the normal group (sham-operation group); ^* p < 0.05, ^** p < 0.01 vs. the control group (OVX-induced group).

FIGURE 10

Effect of SR on histopathological examination in the OVX-induced rats. (A) CTK-, (B) NFATc1- and (C) BMP-2-positive cells were measured using IHC staining. (D) The number of CTK- (E) NFATc1- and (F) BMP-2-positive cells was counted using ImageJ version 1.46. The results are presented as the mean ± SEM of each experimental group (n = 8). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^## p < 0.01, ^# p < 0.05 vs. the normal group (sham-operation group); ^* p < 0.05, ^** p < 0.01 vs. the control group (OVX-induced group).

FIGURE 11

LC-MS analysis was carried out using the A Waters e2695 system. Kaempferol was confirmed in the (A) standard and (B) SR extracts.

FIGURE 12

Comparative analysis of pharmacological effects of SR and kaempferol on the differentiation of osteoclasts and osteoblasts. (A) TRAP-positive cells were stained using a TRAP kit. (B) TRAP activity was measured using an ELISA reader. (C) A number of TRAP-positive cells with >3 nuclei were counted using an inverted microscope (magnification, ×100). (D) RAW 264.7 cell viability was measured using a CCK-8 assay kit. (E) Protein expression levels of NFATc1 and c-Fos were measured by western blotting. (F) The bands were normalized to that of β-actin, which was used as a loading control. (G) MC3T3-E1 cell viability was analyzed by CCK-8 assays at 3 and 7 days. (H) Protein expression levels of BMP-2, p-Smad 1/5, RUNX-2 and Osterix were measured by western blotting. (I) BMP-2, RUNX-2 and Osterix levels were normalized to that of β-actin. p-Smad 1/5 levels was normalized to that of Smad 1/5/9. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ^## p < 0.01 vs. the normal group (untreated cells, RAW 264.7 cells); ^* p < 0.05, ^** p < 0.01 vs. the RANKL only treatment group. ^† p < 0.05, ^†† p < 0.01 vs. the normal group (untreated cells, MC3T3-E1 cells); ^$ p < 0.05 vs. the osteogenic medium-treated cells.

FIGURE 13

Schematic diagram of SR in bone metabolism.