Effects of Processed Polygonum multiflorum with KIOM Patent on Bone Remodeling-Related Protein Expression in Human Osteoblast-Like SaOS-2 Cells

This present study evaluated the effects of processed P. multiflorum on osteogenesis using Sarcoma osteogenic (SaOS-2) cell lines and osteoclastogenesis of bone marrow-derived macrophage cells (BMM) and to elucidate differences in effect on the expression of bone-related proteins between commercially sold P. multiflorum and patented, in vitro-propagated Korea Institute of Oriental Medicine (KIOM) P. multiflorum. Raw P. multiflorum and P. multiflorum that were stir-baked and steamed in black bean juice were compared, and western blotting analysis was performed to investigate the expression of bone remodeling-related proteins in SaOS-2 cells. In the cells treated with P. multiflorum steamed in black bean juice, the expression of RANKL was decreased, whereas that of osteoprotegerin, alkaline phosphatase, Runx2, and osterix was increased. Owing to these results, we conclude that processed P. multiflorum can be used as an alternative treatment for bone diseases such as osteoporosis, osteopenia, periodontitis, and Paget's disease.


Introduction
In bone metabolism, bones are constantly remodeled by balancing osteoblasts and osteoclasts [1]. In fact, an imbalance between osteoblasts and osteoclasts causes bone loss that can result in various bone diseases such as osteoporosis, osteopenia, periodontitis, and Paget's disease [2,3]. In recent years, herbal medicines have been used to increase osteoblast differentiation and decrease osteoclast differentiation for treating bone diseases, including osteoporosis [4][5][6]. e root tuber of Polygonum multiflorum unb. which belongs to the Polygonaceae family, is a medicinal herb that has been used in Traditional Korean Medicine (TKM) as a blood-tonifying medicine [7]. In Dong-Eue-Bo-Gam, which is the TKM book, P. multiflorum is regarded as a medicinal plant with many therapeutic effects, including bone-strengthening [8], potent antiaging, and cognitive-enhancing effects [9], as well as the ability to protect human foreskin melanocytes from oxidative stress and improve pigmentation in hair follicles [10]. Polygonum multiflorum also exerts beneficial effects on hippocampal neurons [11].
In TKM, P. multiflorum is used after processing because processed P. multiflorum exerts better effects than the raw plant [12,13]. e processing of herbal medicines, one of the core theories in TKM, is aimed at reducing their toxicity and increasing their beneficial effects [14]. To optimize the utilization of P. multiflorum in TKM, the Korea Institute of Oriental Medicine (KIOM) developed and patented a standard protocol for rapid in vitro production of its seedlings and enlarged root tubers [15]. erefore, the current study aimed to evaluate the potential effects of P. multiflorum produced using the patented protocol Korean Patent submission (no: 10-2019-0120751, September 30, 2019) of KIOM and commercially sold P. multiflorum on osteogenesis using osteoblast-like cells Saos-2 and osteoclastogenesis using BMM; this study will serve as a foundation for the use of P. multiflorum as a possible treatment of osteoporosis.

Preparation of Commercially Obtained P. multiflorum.
e 2 kg of the roots of P. multiflorum were purchased from Jirisan Hasuo Farming Co. (Sancheong, South Korea) and authenticated by Dr. Kang at Korea Institute of Oriental Medicine (Figure 1(a)) as Commercial Raw P. multiflorum (C-RPM), and 1 kg dried roots of raw P. multiflorum was baked in a pan with constant stirring at 160°C for 40 min and then maintained in dryer at 45°C for 4 hours (Figure 1(b)) as Commercial Stir-Baked to Yellow P. multiflorum (C-SBYPM). e 1 kg of black bean was purchased from Kwangmyongdang Co. (Ulsan, South Korea) and prepared using the method previously suggested [16] in which the obtained beans were boiled in 5 L of water at 100°C for 4 hours to obtain the black beans liquid extract. en, 4 litres of water was added to the cooked beans and again boiled at 100°C for 3 hours to obtain the second extract. e first and second extracts were then mixed to make the black bean juice. 250 g of P. multiflorum and the black bean extract were mixed together in a pot and stirred constantly for 2 hours, then steamed at 60°C for 1 hour, and maintained in an oven at 45°C for 8 hours (Figure 1(c)) as commercial Steamed with Black Bean Juice P. multiflorum (C-SBBJPM).  (Figure 1(f )).

Extraction of P. multiflorum.
e 10 g of each of the six P. multiflorum mentioned above was extracted using 100 mL of distilled water for 2 h at 20°C at 200 rpm. After filtration, the obtained extracts were concentrated in a vacuum evaporator and powdered by using a freeze-drying machine for 72 h at −80°C. e dried powder weights of C-RPM, C-SBYPM, C-SBBJPM, K-RPM, K-SBYPM, and K-SBBJPM were 2.56 g, 2.33 g, 1.55 g, 1.37 g, 1.95 g, and 2.37 g (yield: 25.6%, 23.3%, 15.5%, 13.7%, 19.5%, and 23.7%, respectively). e sample was stored at −20°C prior to further studies.

SaOS-2 Cells
Culture. Human osteoblast-like SaOS-2 cells, which have similar properties to primary osteoblasts, were obtained from Seoul National University cell bank (Seoul, South Korea). ey were derived from an osteosarcoma and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin at 37°C, 95% humidity, and 5% CO 2 . SaOS-2 cells were seeded at a density of 1 × 10 6 cells/ well in 24-well culture plates. After 24 h of cultivation, the medium was replaced with 3 mL medium supplemented with samples. e sample was diluted with the cell culture medium to obtain concentrations of 100 μg/mL. BMM were cultured in an α-MEM medium having 10% fetal bovine serum and 1% penicillin/streptomycin. BMM were seeded at a density of 1 × 10 4 cells/well in 96-well culture plates with M-CSF (60 ng/mL). After 2 hour preincubation with each sample (50 μg/mL) on BMM, RANKL (100 ng/mL) were treated for 6 days.

Western Blot Assay.
Western blot analysis was performed to investigate the effects of bone metabolism in SaOS-2 cells, osteoclast differentiation factor; RANKL and OPG, osteoblastogenesis factors; alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and Osterix expressions. Each sample of SaOS-2 cells was vortexed in RIPA buffer ( ermo scientific, Rockford, USA). 20 μg cell protein from SaOS-2 was denatured with 5% SDS buffer. e prepared protein samples were loaded on 10% polyacrylamide gels, separated by electrophoresis, and then electrotransferred to activated polyvinylidene fluoride (PVDF) membranes. Membranes were blocked by 3% bovine serum albumin (BSA) in tris-buffered saline (TBS) containing 1% Tween 20 (TBS-T) and incubated with the specific antibodies at 4°C for 12 h (β-actin, OPG, RANKL, Osterix, ALP, RUNX2; Santa Cruz Biotechnology, Inc., CA), 1 : 1000 dilutions in TBS-T). After washing of the membranes for 10 min 3 times, membranes were incubated with anti-rabbit and anti-mouse alkaline phosphataseconjugated secondary antibody (1 : 2000 dilution in TBS-T) for 1 h at 24°C. e membranes were washed three times in TBS-T for 3 min and then reacted with HRP-polymerized secondary antibody for 60 minutes, after completion of the reaction, using a chemiluminescent detection system (Phototope ® -HRP western blot assay Detection kit, New England Biolab). e cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution. For a short time, 1 × 10 5 cells/well were seeded in 96-well plates and allowed to adhere for 12 h. e cells were treated with 100 μL of 100 μg/mL samples each for 24 hours, and MTT (2 mg/mL) was added 50 μL/ well and incubated for 2 h, followed by solubilization of the formazan crystals by adding dimethyl sulfoxide (Sigma-Aldrich, Seoul, South Korea) 50 μL/well for 30 min. e color developed was read as optical density using a Gene5 (Biotek, Seoul, South Korea) at 570 nm.

Statistical
Analysis. All data were expressed as a means ± standard error of the mean. One-way ANOVA followed by the Tukey test (compare all pairs of columns) was used for the statistical analysis. In all analyses, p < 0.05 was taken to indicate statistical significance.

Effects of samples on osteoclast differentiation mediators,
RANKL, and OPG in SaOS-2 cells. C-RPM, K-RPM, C-SBYPM, and K-SBYPM were not effective on the decrease of the RANKL expression. On the other hand, C-SBBJPM and K-SBBJSPM treated with black bean juice decreased the expression level of RANKL by 62.3% and 60.2%, respectively, compared to the control group. C-SBBJPM significantly decreased RANKL expression compared to C-RPM and C-SBYPM. K-SBBJPM significantly decreased RANKL expression compared to K-RPM and K-SBYPM (Figure 2(a)).
C-RPM and K-RPM were not effective on the increase of OPG expression in SaOS-2 cells. On the other hand, C-SBYPM and K-SBYPM and C-SBBJPM and K-SBBJPM increased the level of OPG by 62.7%, 90.6%, 131.2%, and 153.1%, respectively, compared to the control group. C-SBYPM and C-SBBJPM increased OPG expression compared to C-RPM. C-SBBJPM increased the OPG expression compared to C-SBYPM. Likewise, K-SBYPM and K-SBBJPM increased the OPG expression compared to K-RPM. K-SBBJPM increased the OPG expression in SaOS-2 cells compared to K-SBYPM (Figure 2(b)).   by 81.9% and 74.6%, respectively, compared to the control group. C-SBBJPM significantly increased the ALP expression compared to C-RPM and C-SBYPM. K-SBBJPM significantly increased the ALP expression compared to K-RPM and K-SBYPM (Figure 3(a)).

Effects of Samples on Osteoblast Differentiation Mediators
C-RPM and K-RPM did not change the expression of Runx2 in SaOS-2 cells. On the other hand, C-SBYPM and K-SBYPM and C-SBBJPM and K-SBBJPM increased the Runx2 expressions by 32.8%, 38.3%, 41.3%, and 49.0%, respectively, compared to the control group. C-SBYPM and C-SBBJPM increased the Runx2 expression compared to C-RPM. K-SBYPM and K-SBBJPM increased the Runx2 expression compared to K-RPM (Figure 3(b)).
C-RPM and K-RPM and C-SBYPM and K-SBYPM did not increase the expression of Osterix. On the other hand, C-SBBJPM and K-SBBJPM increased the expression of Osterix by 65.5% and 79.3%, respectively, compared to the control group. C-SBBJPM significantly increased the Osterix expression compared to C-RPM and C-SBYPM. K-SBBJPM significantly increased the Osterix expression compared to K-RPM and K-SBYPM (Figure 3(c)).

Difference between Commercial P. multiflorum Samples and KIOM Samples.
ere was no difference of RANKL and OPG expression between commercial P. multiflorum samples and KIOM samples. In addition, there was no difference of ALP, Runx2, and osterix expression between commercial P. multiflorum samples and KIOM samples.

Effect of P. multiflorum on SaOS-2 Cells Toxicity.
e effect of P. multiflorum on toxicity in SaOS-2 cells was examined by the MTT assay. e cells were treated with different doses of P. multiflorum, and no toxicity was found (Figure 4).

Discussion
e balance between osteoblasts and osteoclasts is one of the key components of bone metabolism [17]. Bone homeostasis cannot be maintained if there is excessive osteoclastic bone resorption or insufficient osteoblastic bone formation [18]. Attenuation of RANKL or activation of OPG (RANKL inhibitor) might be helpful to inhibit the differentiation of osteoclasts, resulting in alleviation of bone resorption [2]. In this study, the expression of RANKL was significantly decreased by C-SBBJPM and K-SBBJPM treatment, while C-RPM, C-SBYPM and K-RPM, K-SBYPM did not change the expression of RANKL in SaOS-2 cells. is result showed that steamed black bean juice of P. multiflorum apparently affected the production of RANKL in osteoblasts. In terms of OPG, all processed P. multiflorum samples including C-SBYPM, C-SBBJPM, K-SBYPM, and K-SBBJPM except C-RPM and K-RPM were effective on the expression of OPG in SaOS-2 cells. Nevertheless, steamed black bean juice of P. multiflorum was more effective than that of stir-baked to yellow P. multiflorum in both of commercial P. multiflorum samples and KIOM samples. Steamed black bean juice of P. multiflorum increases the inhibitory effects of P. multiflorum on bone resorption by osteoblasts by decreasing RANKL expression and increasing OPG expression.
At the stage of osteoblast differentiation, ALP, Runx2 and Osterix, and osteoblast-specific transcription factors are released from osteoblasts, leading to induction of bone formation [2]. is experiment showed that the levels of ALP and Osterix were significantly increased in C-SBBJPM and K-SBBJPM-treated cells. In part of Runx2 expression, C-SBYPM, C-SBBJPM, K-SBYPM, and K-SBBJPM increased the Runx2 compared to raw C-RPM and K-RPM. However, the level of Runx2 in cells treated with C-SBBJPM and K-SBBJPM was higher than C-SBYPM and K-SBYPM. e steamed with black bean juice method apparently upregulated the effects of P. multiflorum on osteoblast activation rather than the stir-baking method which is consistent with the result from osteoclast-related factors. e cultivation of P. multiflorum typically takes long time to propagate which leads to decreasing the yield and quality [19]. However, with the KIOM-patented root tuber enlargement protocol for P. multiflorum, rapid in vitro propagation can be carried out without compromising its chemical composition [15]. is experiment shows that the overall tendency of expressions of bone remodeling-related factors between commercial P. multiflorum samples and KIOM patented samples is very similar. To clarify the pharmacological potential of K-RPM and K-SBBJPM against bone disease, we are currently investigating the in vivo efficacy of the samples in preclinical animal models.

Conclusion
Processed P. multiflorum regulated osteoclast differentiation mediators, RANKL, and OPG and increased osteoblast differentiation mediators, ALP, Runx2, and osterix. In addition, there was no significant difference in expressions of RANKL, OPG, ALP, Runx2, and osterix between commercial P. multiflorum samples and KIOM samples. However, although this study has showed promising evidence that processed P. multiflorum might be considered as a possible treatment for bone diseases, there is need for further preclinical and clinical studies to enhance future drug development from it for the treatment of bone diseases. Additionally, future study should also consider comparing the phytochemical compositions in the different P. multiflorum roots.

Data Availability
e data for this current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.