Effect of polygonimitin C on bone formation and resorption in human osteoblast-like MG 63 cells

Purpose: To investigate the effect of polygonimitin C (PC) on bone formation and resorption in human osteoblast-like MG63 cells. Methods: MG63 cells were treated with PC at doses of 0, 20, 40 or 80 μg/mL for 48 h, with an untreated group as control. The effect of PC on alkaline phosphatase (ALP) activity in MG63 cells was investigated by p-nitrophenyl phosphate disodium hexahydrate assay. Western blot assay was used to evaluate the effect of PC on the expressions of osterix (OSX), bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), fibronectin (FN), type I collagen (COL I), osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) proteins in MG63 cells. Results: ALP relative activity in MG63 cells treated with PC at 20, 40 or 80 μg/mL (123.58, 137.74 or 159.62 %, respectively) was significantly (p < 0.05 or 0.01) higher than that in control group (99.37 %). Expressions of OSX, BMP-2, RUNX-2, OC, FN, COL I and OPG proteins in MG63 cells treated with PC at 20, 40 or 80 μg/mL were significantly (p < 0.01) higher than those in control group. However, there were no statistically significant differences in RANKL protein expression between PC-treated MG63 cells and control group. Conclusion: These results show that PC exerts protective effects against osteoporosis by promoting bone formation and inhibiting bone resorption. Thus, PC may be useful in the development of new antiosteoporosis drugs.


INTRODUCTION
Osteoporosis (OP) is characterized by weak bone, which manifests clinically in bone fractures [1].It is a major public health problem which affects numerous people worldwide, and imposes significant mental stress and economic burden on family and society [2].The etiology of OP may be primary, secondary or idiopathic [3].Typical primary OP is sub-divided into postmenopausal and senile OP, while secondary OP is sub-divided into endocrine, metabolic, drug-induced and deficiency OP [3,4].The pathogenesis of idiopathic OP is related to genetic factors [5].Generally, OP results from imbalance between bone formation and bone resorption, which is controlled by bone-forming osteoblasts and bone-resorbing osteoclasts [6].In OP patients, the bone-forming function of osteoblasts is lower than the bone-resorbing function of osteoclasts [7].Therefore, the fundamental principle involved in the treatment of OP is to regulate the imbalance between the bone-forming function of osteoblasts and boneresorbing function of osteoclasts.
Studies have shown that the roots of Polygonum multiflorum Thunb.(Polygonaceae), also named as Polygoni multiflori Radix, have protective effects against OP [8][9][10].It has been reported that stilbene glucoside, the main active ingredient of Polygoni multiflori Radix, increased bone mineral density and bone strength in rats, suggesting that stilbene glucoside may exhibit a protective effect against OP [11].The chemical structure of polygonimitin C (PC) from Polygoni multiflori Radix is similar to that of stilbene glucoside [12].This suggests that PC may also exhibit some effects on OP.
Therefore, the aim of this work was to investigate the effect of PC on the bone formation and resorption functions of human osteoblast-like MG63 cells.

Isolation of PC
Air-dried and ground Polygoni multiflori Radix (30 kg) was extracted by refluxing 95 % ethanol three times.The combined ethanol solvent was evaporated under reduced pressure to yield the ethanol extract (1964 g), which was dissolved in water and then successively partitioned with chloroform, ethyl acetate and n-butyl alcohol.The n-butyl alcohol solvent was evaporated under reduced pressure to yield the n-butyl alcohol extract (206 g), which was dissolved in water for further separation.The water-soluble fraction was subjected to macroporous resin D101 column, eluting with a gradient solvent system of ethanol-water to yield 30 % ethanol fraction.
The 30 % ethanol fraction was chromatographed on Sephadex LH-20 column, eluting with 30 % methanol to yield fractions 1 -7.Fraction 5 (14 g) was chromatographed over silica gel column chromatography, eluting with chloroform-ethyl acetate-methanol (volume ratio = 5: 3: 2) solvent system to yield PC (56 mg).The purity and chemical structure of PC were determined by area normalization method of high performance liquid chromatography (HPLC), mass spectrum (MS) data and nuclear magnetic resonance (NMR) data.PC was dissolved in 0.5 % DMSO to obtain different concentrations used in subsequent assays.

Cell culture
MG63 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).The cells were cultured in MEM medium supplemented with 1 % penicillin, 1 % streptomycin and 10 % FBS at 37 ºC in a humidified atmosphere containing 5 % CO 2 .

Grouping and treatment
MG63 cells were seeded on 96-well culture plates at a density of 5 × 10 5 cells/well and divided into control and three doses of PC groups.After culture for 24 h, cells in control group were treated with PC at a dose of 0 μg/mL for 48 h, and cells in three doses of PC groups were treated with PC at doses of 20, 40 or 80 μg/mL for 48 h, respectively.All treatments were carried out in triplicate.

Assay of alkaline phosphatase (ALP) activity
After treatment with PC, MG63 cells were washed three times with PBS.Then the supernatant of MG63 cells were obtained with the aid of cell lysis buffer, ultrasound and centrifugation.According to the existing method in the literature [13], ALP activity in the supernatant was determined by pNPP assay.After reactions were completed, the absorbance (A) of each sample was read at 405 nm in a Multiskan MK3 microplate reader (Thermo, USA).Meanwhile, enhanced BCA protein assay kit was used to determine the protein concentration in the supernatant, and the absorbance value of each sample adjusted based on relatively stable protein concentration.The effect of PC on ALP activity in MG63 cells was evaluated by ALP relative activity, calculated as in Eq 1.
ALP relative activity (%) = (At/Ac)100 …… (1) Where At and Ac are the absorbance of the test and control samples, respectively.

Western blot assay
MG63 cells were harvested after treatment with PC, and then their total proteins were extracted with the aid of cell lysis buffer, ultrasound and centrifugation.Enhanced BCA protein assay kit was used to determine the concentration of total proteins.Equal amounts of total proteins (about 30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane.After blocking with 5 % non-fat milk, the PVDF membranes were incubated with primary antibodies for β-actin, OSX, BMP-2, RUNX-2, OC, FN, COL I, OPG and RANKL overnight at 4ºC.Subsequently, the PVDF membranes were washed with Tris-buffer saline-Tween (TBS-T) and incubated with HRP-conjugated goat antirabbit secondary antibody in TBS-T at room temperature for 2 h.Also, after being washed with TBS-T, the proteins on the PVDF membranes were detected on X-ray film by using enhanced chemiluminescence detection kit for HRP.β-actin was used as internal reference protein for equalizing protein loading.The expression level of target protein was expressed relative to β-actin i.e. protein level/β-actin level.

Statistical analysis
All data are presented as mean ± standard deviation (SD).Differences among different groups were analyzed by one-way ANOVA on SPSS 21.0 software (Chicago, IL, USA).Differences were considered statistically significant at p < 0.05 or 0.01.

Purity and identification of PC
Results of area normalization method in HPLC indicated that the purity of PC was 99.6 %.The chemical structure of PC (Figure 1) was identified by comparing its MS and NMR data with existing literature [12].

Effects of PC on expressions of bone formation-related proteins in MG63 cells
As shown in Figure 3, after treatment with PC (20, 40 or 80 μg/mL), expressions of OSX, BMP-2, RUNX-2, OC, FN and COL I proteins in MG63 cells were significantly (p < 0.01) higher than those in the control group.

Effects of PC on expressions of bone resorption-related proteins in MG63 cells
As shown in Figure 4, after treatment with PC (20, 40 or 80 μg/mL), the expression of OPG protein in MG63 cells was significantly (p < 0.01) higher than that in the control group, while the expression of RANKL protein in MG63 cells was not significantly different from that in the control group.

DISCUSSION
MG63 cells are classic osteoblast-like cells for investigation of the effects of agents on bone formation and bone resorption [14,15].In the present study, MG63 cells were used to assess the effect of PC on bone formation and bone resorption with the aid of pNNP and Western blot assays.
The differentiation of pre-osteoblasts into mature osteoblasts is promoted by OSX and RUNX-2 [16,17].RUNX-2 plays an important role in early osteoblasts differentiation and promotes the formation of bone matrix proteins, such as ALP and COL I [16].OSX is an important transcription factor in terminal osteoblasts differentiation and regulates the expressions of bone formationrelated proteins, such as OC and COL I [18,19].The differentiation of pre-osteoblasts into mature osteoblasts is enhanced by BMP-2 through upregulation of the expression of OSX [20].OC, a key protein involved in osteoblasts differentiation and maturity, is positively related to the rate of bone formation and exhibits a regulative effect on extracellular matrix mineralization [21].FN, an important cell adhesion factor, has a positive influence on osteoblasts differentiation and expression of COL I [22,23].ALP and COL I are the main protein of reflecting osteoblasts functions [15].Thus, ALP activity and the expressions of OSX, BMP-2, RUNX-2, OC, FN and COL I have positively influences on the differentiation of pre-osteoblasts into mature osteoblasts and on the osteoblasts functions.These indicate that ALP activity and the expression levels of these proteins are positively related to bone formation.In this study, PC significantly increased ALP activity and the expressions of OSX, BMP-2, RUNX-2, OC, FN and COL I in MG63 cells relative to those in the control group, suggesting that PC promoted the bone formation function of MG63 cells.
Osteoclasts differentiation and maturity are enhanced by the combination of RANKL produced by osteoblasts and RANK [24].The OPG produced by osteoblasts is a decoy receptor for RANKL and inhibits combination of RANKL and RANK [25].Briefly, RANKL promotes osteoclasts differentiation and maturity, and OPG inhibits RANKL-induced osteoclasts differentiation and maturity.Thus, RANKL is positively related to bone resorption, and OPG is negative related to bone resorption.In this study, PC significantly increased the expression of OPG protein in MG63 cells without any effect on the expression of RANKL protein in MG63 cells relative to those in the control group, indicating that PC inhibited the bone resorption function of MG63 cells.

CONCLUSION
The findings of this work indicate that PC promotes the bone formation function of MG63 cells by up-regulating ALP activity and the expressions of bone formation-related proteins (OSX, BMP-2, RUNX-2, OC, FN and COL I).In addition, PC inhibits bone resorption function of MG63 cells by up-regulating the expression of bone resorption-related protein (OPG) without significant effect on the expression of bone resorption-related protein (RANKL).Thus, PC may have a potential application in the treatment of OP.
institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.
Radix was purchased from Tongrentang Drugstore (Beijing, China) in 2013.It was authenticated by Xiao-Feng Yang, a taxonomist in the Department of Medicine, Hangzhou Medical College.A voucher specimen (no.HZMC 2013DM08) was deposited in the herbarium of Department of Medicine, Hangzhou Medical College for future reference.

Figure 1 :
Figure 1: Chemical structure of PC

Figure 2 :
Figure 2: Up-regulative effect of PC on ALP activity in MG63 cells; *p < 0.05, **p < 0.01, compared with that in the control group

Figure 3 :
Figure 3: Up-regulative effect of PC on expressions of bone formation-related proteins (OSX, BMP-2, RUNX-2, OC, FN and COL I) in MG63 cells; **p < 0.01, compared with those in the control group

Figure 4 :
Figure 4: Effect of PC on expressions of bone resorption-related proteins (OPG and RANKL) in MG63 cells; **p < 0.01, compared with that in the control group