Crataegus pinnatifida Bunge Inhibits RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells and Prevents Bone Loss in an Ovariectomized Rat Model

Abstract

Osteoporosis is characterized by a decrease in bone microarchitecture with an increased risk of fracture. Long-term use of primary treatments, such as bisphosphonates and selective estrogen receptor modulators, results in various side effects. Therefore, it is necessary to develop alternative therapeutics derived from natural products. Crataegus pinnatifida Bunge (CPB) is a dried fruit used to treat diet-induced indigestion, loss of appetite, and diarrhea. However, research into the effects of CPB on osteoclast differentiation and osteoporosis is still limited. In vitro experiments were conducted to examine the effects of CPB on RANKL-induced osteoclast differentiation in RAW 264.7 cells. Moreover, we investigated the effects of CPB on bone loss in the femoral head in an ovariectomized rat model using microcomputed tomography. In vitro, tartrate-resistant acid phosphatase (TRAP) staining results showed the number of TRAP-positive cells, and TRAP activity significantly decreased following CPB treatment. CPB also significantly decreased pit formation. Furthermore, CPB inhibited osteoclast differentiation by suppressing NFATc1, and c-Fos expression. Moreover, CPB treatment inhibited osteoclast-related genes, such as Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2. In vivo, bone mineral density and structure model index were improved by administration of CPB. In conclusion, CPB prevented osteoclast differentiation in vitro and prevented bone loss in vivo. Therefore, CPB could be a potential alternative medicine for bone diseases, such as osteoporosis.

Figure 1

Quantitative HPLC of (a) chlorogenic acid standard and (b) CPB. The HPLC-analysis for standards and sample solutions. (a) Chlorogenic acid standard solution; (b) CPB samples were detected at 330 nm.

Figure 2

Effect of CPB on cell viability, osteoclast differentiation, and bone formation. (a) RAW 264.7 cells were measured by MTS assay of CPB treatment for 24 h. (b) After differentiation into osteoclasts for 5 days, cytotoxicity was measured using MTS. (c) TRAP-positive cells and pit area were captured using an inverted microscope (100x, Scale bars: 200 μm). (d) TRAP-positive cells were counted with an inverted microscope. (e) TRAP activity was measured with an ELISA reader (405 nm). (f) The pit area was measured with ImageJ version 1.46 (100x, Scale bars: 200 μm). The results are presented as the mean ± SEM (n = 3). ^##p < 0.01 compared to the normal group (untreated cells), and ^∗∗p < 0.01, ^∗p < 0.05 compared to the control group (only-RANKL treated cells).

Figure 3

Effect of CPB extract on transcription factor such as NFATc1 and c-Fos. (a) RAW 264.7 cells were treated with RANKL (100 ng/mL) and CPB treatment for 24 h. The expressions of NFATc1 and c-Fos were determined by western blotting. (b) NFATc1 and c-Fos were normalized to Actin with ImageJ version 1.46. The results are presented as the mean ± SEM (n = 3). ^##p < 0.01 compared to the normal group (untreated cells), and ^∗∗p < 0.01, ^∗p < 0.05 compared to the control group (only-RANKL treated cells).

Figure 4

Effect of CPB extract on osteoclast-related genes. (a) RAW 264.7 cells were treated with RANKL (100 ng/mL) and CPB treatment for 4 days. RT-qPCR was used to determine the mRNA levels of osteoclast-related genes. (b) The levels of mRNA were normalized to GAPDH. The results are presented as the mean ± SEM (n = 3). ^##p < 0.01, ^#p < 0.05 compared to the normal group (untreated cells), and ^∗∗p < 0.01, ^∗p < 0.05 compared to the control group (only-RANKL treated cells).

Figure 5

Effect of CPB on OVX-induced model. (a) The body weight was measured once a week. (b) Uterus weight, (c) femurs weight, (d) tibia weight, and (e) tibia ash was measured after sacrifice. The results are presented as the mean ± SEM of each experimental group (n = 8). ^##p < 0.01, ^#p < 0.05 compared to the normal group (sham group), and ^∗∗p < 0.01, ^∗p < 0.05 compared to the control group (OVX group).

Figure 6

Effect of CPB on an osteoporosis rat model. (a) Analysis of micro-CT in the femoral head. The bone microstructure parameters, such as (b) BMD, (c) BV/TV, and (d) SMI, were measured by micro-CT. The results are presented as the mean ± SEM for each experimental group (n = 8). ^##p < 0.01, ^#p < 0.05 compared to the normal group (sham group), and ^∗∗p < 0.01, ^∗p < 0.05 compared to the control group (OVX group).

Figure 7

Effect of CPB on OVX-induced bone loss model. (a) The histology of the bone tissues was examined using H&E staining, and (b) IHC staining. (c) The trabecular area was measured using ImageJ version 1.46. (d) CTK-positive cells were counted using ImageJ version 1.46. CTK-positive cells are indicated by red arrows. The results are presented as the mean ± SEM for each experimental group (n = 8). ^##p < 0.01, ^#p < 0.05 compared to the normal group (sham group), and ^∗∗p < 0.01, ^∗p < 0.05 compared to the control group (OVX group).