[논문]Stewartia pseudocamellia and Torilis japonica Extracts Inhibit RANKL-induced Osteoclastogenesis in RAW 264.7 Cells

Ki-Young Kim; Anh-Thu Nguyen; Chun Soo Na

doi:10.5012/jkcs.2023.67.2.120

[국내논문] Stewartia pseudocamellia and Torilis japonica Extracts Inhibit RANKL-induced Osteoclastogenesis in RAW 264.7 Cells 원문보기

대한화학회지 = Journal of the Korean Chemical Society, v.67 no.2, 2023년, pp.120 - 128

Ki-Young Kim (Department of Genetics and Biotechnology, Kyung Hee University) , Anh-Thu Nguyen (Department of Genetics and Biotechnology, Kyung Hee University) , Chun Soo Na (Lifetree Biotech Co., Ltd)

Abstract ▼ AI-Helper

Osteoporosis is a disease that causes the weakening of bone by increasing porosity, which often results in fractures. Osteoporosis treatment measures include the use of Bisphosphonates and estrogen. However, these treatments cannot be used in the long term as these treatments have adverse side effects. Therefore, there is a need to identify better and safer treatment options. For this, 63 plant extracts were screened and among them, six extracts showed high anti-osteoclastic activity with low cytotoxicity. Of these six extracts, three extracts, Cudrania tricuspidata (P371), Ulmus davidiana var. japonica (P401), and Torilis japonica (P411), showed more than 50 percent osteoclast inhibition. While the remaining, Stewartia pseudocamellia extracts I and II (P370, P397) and Cuscuta chinensis (P418), showed moderate or between 40-50 percent osteoclast inhibition. Among all the extracts, Torilis japonica (P411) showed the highest inhibitory action against osteoclast development. Torilis japonica (P411) primary components include Kaempferol, Quercetin, and Luteolin, all proven to inhibit osteoclastogenesis. Stewartia pseudocamellia extracts I and II (P370 and P397) showed moderate or 44% osteoclast inhibition. Stewartia pseudocamellia extract II (P397) enhanced the growth of RAW 264.7 cells by 19%. Torilis japonica (P411) and Stewartia pseudocamellia extract II (P397) suppressed the expression of osteoclast-specific genes in RANKL-induced osteoclastogenesis in RAW 246.7 cells. Torilis japonica (P411) extracts even increased osteoblast-specific RUNX2 gene expression. This results provide that six extracts could be used as a potential treatment option for osteoporosis disease with the extracts of Torilis japonica (P411) and Stewartia pseudocamellia (P397) as an ideal candidates. However, the combination of the extract with higher osteoclastic inhibition and less toxic effects with further analysis should be recommended.

Keyword

표/그림 (4)

그림 Figure 1. Cytotoxicity of plant extracts in RAW 264.7 cells. 1×10⁴ RAW 264.7 cells/well in a 96-well were treated with 100 μg/ml plant extracts. MTT assay was performed 24 h after the plant extracts treatment. Absorbance was measured at 570 nm. All 63 plant extracts were tested simultaneously for this experiment, and the experiments were repeated thrice. DMSO was used for vehicle control. *p < 0.05, **p < 0.01 compared with the control.
그림 Figure 2. Effects of plant extracts on RANKL-stimulated osteoclasts differentiation in RAW 246.7 cells. RAW 246.7 cells were stimulated with 100 ng/ml RANKL with or without 100 μg/ml plant extracts. The osteoclast cell-like morphology was analyzed using an inverted microscope. All the cells were fixed with 10% buffered formalin before imaging osteoclasts. Scale bar 1 millimeter. (a) Representative photographs of the morphological changes are presented, and cells were counted to determine osteoclast numbers. Negative Control: No RANKL treatment; Positive Control: RANKL (100 ng/ml) + 1% DMSO. (b) Percentage of osteoclast inhibited. Values are mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 compared with the positive control, ^##p < 0.01 compared with the negative control.
그림 Figure 3. RANKL-mediated osteoclastogenesis suppressed by treatment of extract of C. tricuspidata (P371), U. davidiana var. japonica (P401), T. japonica (P411) and S. pseudocamellia (P397). (a-e) Changes in the mRNA expression level of osteoclast-related genes (TRAP, RANKL, CTSK, OCSTAMP, and MMP9) and (f) osteoblast-related gene (RUNX2). RAW 264.7 cells were treated with 100 μg/ ml plant extracts 24 h incubation after seeding along with 100 ng/ml RANKL treatment. RANKL treatment was used as a positive control (PC), and the group without RANKL treatment was used as a negative control (NC). Ordinary one-way ANOVA analysis was used to compare the groups with the positive control (PC). ***p < 0.001; ****p < 0.0001.
표 Table 1. Osteoclast-specific gene primers

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데이터처리

Using Prism software (version 9.0; GraphPad, CA, USA), one-way ANOVA was performed to compare between groups, with a significance of <0.05, 0.01, 0.001, 0.0001.
Student’s t-test was used within groups using Microsoft Excel software

성능/효과

1, 5 of them showed severe toxicity (80-50%), 21 of them showed moderate toxicity (50-30%), 34 of them showed less toxicity (30-1%), and 3 of them increased the viability of RAW 264.7 cells, compared to the DMSO treated control at 100 μg/ml of a plant extract.
In order to test whether 63 plant extracts inhibit osteoclastogenesis, RAW 264.7 were stimulated with 100 ng/ml RANKL with or without 100 μg/ml of plant extracts
To find out if the plant extracts have any toxic effects, the cytotoxicity of 63 plant extracts against RAW 264.7 cells using the MTT reagent was tested. As shown in Fig.
As shown in Fig. 2a, osteoclast-like morphological changes in which many cells aggregated and bundled were observed in the RANKL treatment only. This suggested that RANKL-stimulated RAW 246.
2a, osteoclast-like morphological changes in which many cells aggregated and bundled were observed in the RANKL treatment only. This suggested that RANKL-stimulated RAW 246.7 cells were completely differentiated into osteoclast. However, among the 63 plants tested, extracts of Cudrania tricuspidata (P371), Ulmus davidiana var.
RUNX2 is a central transcription factor regulating osteoblast differentiation and promoting bone mineralization. TRAP, RANKL, CTSK, OCSTAMP, and MMP9 are known to be predominantly expressed in active osteoclasts. Therefore, to further examine the anti-osteoclastogenic activity of plant extracts, the expression of osteoclast-specific genes (TRAP, RANKL, CTSK, OCSTAMP, and MMP9) were investigated.
TRAP, RANKL, CTSK, OCSTAMP, and MMP9 are known to be predominantly expressed in active osteoclasts. Therefore, to further examine the anti-osteoclastogenic activity of plant extracts, the expression of osteoclast-specific genes (TRAP, RANKL, CTSK, OCSTAMP, and MMP9) were investigated. The mRNA expression of TRAP, RANKL, and CTSK were decreased by treatment of extract of C.
Note: Areas <0.5 mm in diameter were all ignored.
However, among the 63 plants tested, extracts of Cudrania tricuspidata (P371), Ulmus davidiana var. japonica (P401), Torilis japonica (P411), Stewartia pseudocamellia extracts I and II (P370 and P397), and Cuscuta chinensis (P418) decreased the osteoclast area by more than 40% when compared to the control. Among these 6 candidates, T.
chinensis (P418) and S. pseudocamellia extracts I and II (P370 and P397) showed moderate osteoclast inhibition (45-40%) (Fig. 2a, b).

후속연구

Hence, the observed osteoclast inhibition might be caused by one of these or the combination of these known compounds. Further experiments are necessary to investigate the unknown component or the mechanism of these known compounds in the inhibition of osteoclasts.
As osteoporosis is a disease that affects almost everyone, mainly in the later part of their life with the increase in their age, new treatment methods must be developed which are safer and more effective than the current treatment methods. We have found six potential candidates from natural plant sources that showed effective inhibitory action against RANKL-stimulated osteoclasts in RAW 264.
japonica extracts were highly efficient in controlling osteoclast proliferation. Further studies are required to confirm their activity in vivo systems.

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