Rhizoma Drynariae promotes the Osteogenic differentiation of Bone Mesenchymal Stem Cells by activating the Wnt/β-catenin Signaling Pathway

Background: Rhizoma Drynariae (RD), a traditional Chinese medicine with pleiotropic biological activities, exerts a protective effect against age-related osteoporosis. Osteoporosis is a serious clinical problem and characterized by the deterioration in bone volume and strength, mainly due to the dysfunction of bone marrow stromal cells (BMSCs). However, it remains unclear whether RD-containing serum regulates the osteogenic differentiation of BMSCs via Wnt/β-catenin signaling pathway. Methods: The effects of RD-containing serum on the osteogenesis of BMSCs were detected via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), western blotting, alkaline phosphatase (ALP) activity assay and alizarin red staining. Using Dickkopf-related protein-1 (DKK1), an inhibitor of the Wnt/β-catenin signaling pathway, we examined whether RD-containing serum regulates osteoblast differentiation of BMSCs via the Wnt/b-catenin signalling pathway. Results: The results showed that RD-containing serum promoted the BMSC proliferation and ALP activities, as well as up-regulated the expression of osteogenic markers and Wnt/β-catenin pathway-related genes, i.e., runt-related transcription factor 2, Sp7, osteocalcin, β-catenin and Wnt3a, in BMSCs. Moreover, we found that RD-containing serum activated the Wnt/β-catenin pathway. Adding DKK1 to the RD-containing serum could decrease the promotion of osteogenic differentiation of RD-containing serum on BMSCs. Conclusions: Collectively, RD-containing serum could promote the osteogenic differentiation of BMSCs, and the potential mechanism may involve regulation of Wnt/β-catenin signaling.


Introduction
Osteoporosis (OP) is a metabolic bone disease involving low bone mineral density (BMD), which usually leads to lower bone resistance to load and fragility fracture, and its development is affected by imbalance of bone homeostasis, with excessive bone resorption compared with bone formation [1][2][3][4] .OP is characterized by decreased bone mass and microarchitectural deterioration with a subsequent increased risk of fragility fracture effects.Multiple agents exist that are effective for the treatment of osteoporosis, such as teriparatide, bisphosphonates and denosumab 3,5 .Teriparatide has proven effective in reducing both vertebral and non-vertebral fractures and is currently the only bone formation-accelerating drug, whereas bisphosphonates act mainly through inhibition of bone resorption.However, teriparatide has been recently listed and its safety has not been fully veri ed 6 .Studies have shown that the oral bisphosphonate route is associated with a high incidence of osteonecrosis of the jaw and other complications 4,7 .Phytochemicals, such as avonoids, with therapeutic and preventive effects against OP have recently received more and more attention because they are potentially more suitable for long-term use than traditional therapeutic chemical compounds, such as denosumab, teriparatide, abaloparatide, romosozumab, and selective estrogen receptor modulators 1 .
The Wnt/β-catenin signaling pathway, which regulates the expression of osteogenic differentiation marker genes and the dissociation of physiological bone remodeling stages, has a bene cial role in bone formation and thus has been identi ed as a potential target for OP treatment 8 .So far, three different WNT signaling pathways have been described, all of which are activated by binding of the WNT ligands to the Frizzled receptors.Phytochemicals, mechanical stimuli and extracellular Wnt proteins could activate the Wnt/β-catenin signaling pathway and promote bone formation 9 .DKK1, an inhibitor of the Wnt/β-catenin signaling pathway, can competitively bind to LRP5/6 and thus has been identi ed as an inhibitor of the Wnt/β-catenin signaling pathway 10 .
Rhizoma Drynariae (RD), the dried rhizome of Drynaria fortune (Kunze) J. Sm., has been widespread and been proved to be effective in treating OP, bone fractures and defects [11][12][13][14][15][16] .Due to the minimum side effects and favorable cost-effective therapeutic effects, RD has been widely used in clinical practices for more than 2,100 years in China.RD, as one of the most common clinically used folk medicines, has the effects of tonifying kidney, promoting blood circulation, stopping bleeding, and prolonging damage.its main chemical components are avonoids, triterpenes, phenylpropanoids, etc. 16,17 .RD has been proved to exert multifaceted pharmacological effects on the bone homeostasis, such as up-regulating the expression levels of BMP-2 and Runx2 18, 19 , promoting osteoblast proliferation and differentiation 20,21 , inhibiting osteoclast activity and preventing bone loss 17,22,23 .In recent years, there have been a lot of reports on RD's anti-osteoporosis treatment 14,17,23 , but the mechanism of its anti-osteoporosis action from the perspective of Wnt/β-catenin signaling pathway is still very few.
In the current study, we used RD-containing serum to stimulate the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and used DKK1 as the inhibitor of Wnt/β-catenin to explore the osteogenesis of BMSCs and the potential mechanisms of RD.

Materials And Methods
Preparation of ethanol extracts of RD Rhizoma Drynariae (collection in Hunan, China) was purchased from pharmacy department of Second A liated Hospital of Heilongjiang University of Chinese Medicine (Heilongjiang, Harbin, China), and authenticated as the dried rhizome of Drynaria fortunei (Kunze) J. Sm. by Professor Bingyou Yang (School of Chinese Medicine, Heilongjiang University of Chinese Medicine, China).Content of naringin (pharmacological active component in Rhizoma Drynariae, 0.84%) was determined referring to the authentic method (pharmacopeia of the People's Republic of China 2010, volume I, page 239).Powdered RD (500g) was extracted with 75% ethanol (6000 mL, 3×) under thermal re ux for 2.5 h.After ltration, the ethanol extract was concentrated under reduced pressure.The residue was dissolved in 0.5% sodium carboxyl methyl cellulose to give an extract with a concentration of 2 g/mL (expressed as the weight of raw materials).

Animal experiments
Ten female Sprague-Dawley (SD) rats were purchased from the Animal Experiment Center of Heilongjiang University of Chinese Medicine (permit number: SYXK-2016004) and were housed at Heilongjiang University of Chinese Medicine (Heilongjiang, Harbin, China).This experiment was approved by the Animal Ethics Committee of the Second A liated Hospital of Heilongjiang University of Chinese Medicine.All animal procedures were carried out in accordance with the UK Animals (Scienti c Procedures) Act 1986.The rats were maintained under standard laboratory conditions (temperature of 21-23 ℃, relative humidity of 45-65%, and 12 h/12 h light/dark cycle) with food and water freely available.
The 32 rats were randomly divided into three groups: high RD group (n = 10) in which RD extract was administered orally at dose of 40 mg/kg body weight once daily between 8:00 and 10:00 a.m. for the following 7 days; low RD group (n = 10) RD extract was administered orally at dose of 20 mg/kg; control group (n = 12) which was fed naturally without any handle for following 7 days.

Preparation of RD-containing serum
At 1 h after the last gavage, SD rats were anesthetized with chloral hydrate.With a sterilized technique, blood was collected through their abdominal aorta and centrifuged at 1,610 × g for 10 min to separate the drug-containing serum.The same group of serum was mixed with each other.Then the serum was inactivated at 56°C for 30 min in water bath, ltered through a 0.22 mm pore size lter, dispensed into a cryotube, and stored in a -80°C refrigerator.Mixing, ltration, and dispensing were all performed in a biosafety cabinet.

Isolation and culture of BMSCs
BMSCs were obtained from the bone marrow of the femurs and tibias of 3-week-old rats according to our previously reported study 24 .Brie y, the bone marrow was ushed out with 2 ml of α-MEM containing 10% FBS using a 5-ml syringe.The bone marrow was layered on top of the separating medium.After cell centrifugation at a speed of 3500 r/min, the buffy coat was removed and seeded in a culture ask.The isolated BMSCs were cultured in α-Minimal Essential Medium with 10% (v/v) FBS and 1% (v/v) penicillin and streptomycin in a 5% CO 2 incubator at 37 °C.The BMSCs from 3 to 5 passages were used for subsequent experiments.Osteogenic induction medium (OIM) comprised the basal culture medium supplemented with 5 × 10 -5 M L-ascorbic acid, 10 -2 M β-sodium glycerophosphate, and 10 -7 M dexamethasone.

Cell culture and treatments
The experiment was performed as follows: (1) The BMSCs were treated with α-MEM supplemented with 10% FBS and 1% high or low RD-containing serum.The control BMSCs were treated with α-MEM supplemented with 10% FBS and 1% serum without RD-containing.(2) The BMSCs were treated with OIM supplemented with 10% FBS and 1% high or low RD-containing serum.The control BMSCs were treated with OIM with 10% FBS and 1% serum without RD-containing.To determine the potential effects of the Wnt/β-catenin signaling pathway on the osteogenic differentiation of BMSCs, we used DKK1 (0.6 mg/mL) to inhibit the Wnt/β-catenin signaling.

Cell proliferation assay
The MTT assay was performed to evaluate the effect of RD-containing serum on the proliferation of BMSCs.BMSCs were plated at a density of 1 × 10 4 per well in 96-well plates and incubated in α-MEM for 20 h.When BMSCs covered the 96-well plates, the RD-containing serum was added for 1, 2, 4, and 8 days.
At days 1, 2, 4, and 8, MTT (5mg/mL) was added to the α-MEM medium.Then, 100μl of dimethyl sulfoxide (DMSO) added to the 96-well to dissolve the formazan crystals and and the absorbance at 570 nm was measured using a microplate reader (SpectraMax Plus384, Molecular Devices, Sunnyvale, CA, USA).

ALP activity assay
For quantitative ALP activity determination, the cells were lysed in 0.5% (v/v) Triton X-100 in PBS for 30 min.ALP activity was measured by the conversion of the p-nitrophenyl phosphate liquid substrate system (Sigma-Aldrich).The absorbance was measured at 405 nm (Bio-Rad, Hercules, CA, USA).The same cell lysate was used to determine the protein content with the BCA kit according to the manufacturer's instructions.

Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA was extracted using the TRIzol® reagent (Invitrogen), and RNA concentrations were measured with a NanoDrop® 8000 spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA).RNA was reverse-transcribed to cDNA using a GoScript TM Reverse Transcription System.The level of mRNA expression was quanti ed via qRT-PCR using a GoTaq® 2-Step RT-qPCR System and a CFX96TM realtime system (Bio-Rad, Hercules, CA, USA).The primer sequences are shown in Table 1.We used β-actin as the reference control.The relative mRNA levels of Runx2, Sp7, β-catenin, Wnt3a, Wnt7a, c-myc, cyclin-D, Tcf7, c-jun, and Lef-1 were expressed as fold changes normalized to GAPDH mRNA and were analyzed according to the 2 −ΔΔCt comparative method.
Western blot analysis BMSCs were lysed using the RIPA buffer containing phenylmethane sulfonyl uoride (PMSF).Protein concentrations were quanti ed by the BCA assay.Proteins from individual groups (100μg) were separated in a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA, USA).The membrane was blocked with 5% nonfat dry milk in PBST for 2.5 h at room temperature and then incubated with rabbit polyclonal anti-rat β-catenin, Runx2, Sp7, Wnt4a and Wnt7a antibodies for 2 h at room temperature.After washing three times for 10 min each with PBST, the PVDF membranes were incubated with the corresponding secondary antibodies at room temperature for 2.5 h and washed three times with PBST.The immunoreactive bands from triplicates were visualized and quanti ed using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA).

Immuno uorescence staining
At day 8 of cell culture, cells were xed with 4% paraformaldehyde for ten minutes and washed with PBS for three times.Then, 0.15% Triton X-100 was added for 15min to break the cell membranes.Next, the BMSCs were blocked with 1% bovine serum albumin (BSA) to prevent nonspeci c reactions.BMSCs were then incubated with anti-β-catenin antibodies (Abcam, USA, 1:500) overnight in a 4 °C refrigerator.

MTT assay analysis of the BMSC proliferation
The MTT assay showed that high RD-containing serum signi cantly enhanced proliferation of BMSCs at days 2, 4, and 8 compared with the control group (P < 0.05).However, low RD-containing serum signi cantly enhanced proliferation of BMSCs at days 4, and 8 compared with the control group (P < 0.05).The effect of high RD-containing was more pronounced than that of low RD-containing serum (P < 0.05).Compared with the high RD-containing serum-treated group, BMSCs co-cultured with DKK1 (0.6 mg/mL) showed a signi cant decrease in proliferation (P < 0.05).Compared with control group, the proliferation of BMSCs in DKK1 (0.6 mg/mL) group was increased, however, the difference was not statistically signi cant (P > 0.05, Fig. 1).

ALP activity assay of the BMSC osteogenic differentiation
The ALP activity results shown that the high and low RD-containing serum-treated groups signi cantly increased the ALP activity at days 4, 8 and 16 compared with the control group (P < 0.05).The effect of high RD-containing was more pronounced than that of low RD-containing serum (P < 0.05).When DKK1 (0.6 mg/mL) was added in the high RD-containing serum-treated group, the ALP activity decreased with statistical signi cance (P < 0.05, Fig. 2).

Alizarin red staining (ARS) results
BMSCs were cultured in an OIM for 16 days, xed and stained with Alizarin red.Both low and high RDcontaining serum markedly increased the osteogenic differentiation of BMSCs, while DKK1 inhibited the promotion of high RD-containing serum on the osteogenic differentiation of BMSCs (Fig. 3).

qRT-PCR results
The PCR results indicated that, compared with the control group, both high and low RD-containing serum increase in the expression of the osteogenic genes (Runx2, Sp7 and Ocn) and the Wnt/β-catenin pathwayrelated genes (β-catenin, Wnt3a, Fig. 4).The effect of high RD-containing was more pronounced than that of low RD-containing serum (P < 0.05).When DKK1 (0.6 mg/mL) was added in the high RD-containing serum-treated group, the relative expression of these genes decreased signi cantly (P < 0.05, Fig. 4).However, no signi cant difference was observed for the reference gene Wnt7a (P > 0.05, Fig. 4).
Furthermore, the relative expression of c-myc, cyclin-D, Tcf7, c-jun, and Lef1 in the high and low RDcontaining serum-treated groups was statistically signi cantly higher than in the control group (P < 0.05, Fig. 5).When DKK1 (0.6 mg/mL) was added in the high RD-containing serum-treated group, the relative expression of these genes decreased signi cantly (P < 0.05, Fig. 5).

Western blot results
As shown in Fig. 6, the high RD-containing serum upregulated the expression of the osteogenic genes (Runx2, and Sp7) and the Wnt/β-catenin pathway-related genes (β-catenin, and Wnt3a).There was no signi cant difference between the expression of Wnt7a in the experimental and control groups.When DKK1 (0.6 mg/mL) was added in the high RD-containing serum-treated group, the relative expression of these genes decreased signi cantly (P < 0.05).However, no signi cant difference was observed for the reference gene Wnt7a (P > 0.05).

RD-containing serum induced β-catenin nuclear translocation in BMSCs
Next, we examined whether the RD-containing serum could activate Wnt/β-catenin signaling and enhance β-catenin translocation to the nucleus.As shown in Fig. 7, when high RD-containing serum was added to the BMSCs, we observed the β-catenin (green) transfer into the nucleus (blue).The added DKK1 (0.6 mg/mL) blocked the RD-containing serum induced the nuclear translocation of β-catenin.

Discussion
Osteoporosis is a serious clinical problem and characterized by the deterioration in bone volume and strength, partly due to the dysfunction of BMSCs.This study indicates that RD-containing serum could enhance the proliferation of BMSCs in a concentration-and time-dependent manner.Furthermore, RDcontaining serum increases the osteogenic differentiation of BMSCs, which is re ected in the upregulation of ALP activity and increased number of calci ed nodules in the RD-containing serum group.We have also found that the RD-containing serum promotes the expression of osteogenic genes (Runx2, Sp7, Ocn), and Wnt/β-catenin-related genes (Wnt3a and β-catenin).Previous studies have identi ed the Wnt/β-catenin signaling pathway as a mediator of BMSC differentiation to osteoblasts 25- 28 .When the Wnt/β-catenin inhibitor DKK1 is added (0.6 µg/mL) to the RD-containing serum, it negatively regulates osteogenic differentiation of BMSCs.
Research has shown that RD could enhance angiogenic-osteogenic coupling during distraction osteogenesis by promoting type H vessel formation through PDGF-BB/PDGFR-β signaling pathway 14 .RD could promote osteoblast differentiation and mineralization in MC3T3-E1 cells through regulation of bone morphogenetic protein-2, alkaline phosphatase, type I collagen and collagenase-1 29 .UPLC-MS metabolomics method can provide valuable insights into the effect and underlying mechanisms of RD protecting osteoporosis, and the results showed RD mainly plays a role in treating OP through lipid metabolic pathways, arachidonic acid metabolism and glycerol 3-phosphate ester metabolism 12 .Studies, based on network pharmacology, showed that RD may play an anti-osteoporotic role by directly or indirectly targeting PI3k-akt signaling pathway, osteoclast differentiation, Wnt signaling pathway, and estrogen signaling pathway 30 .Cathepsin K, which plays an important role in bone resorption and acts as a potential target in the treatment of osteoporosis, can be regulated by RD 31 .RD extract prevented the intracellular maturation of cathepsin K and thus, it was considered that RD is a pro-drug of a potent bone resorption inhibitor 29 .DR has a variety of effects on bone homeostasis through the promotion of osteoblastic differentiation and inhibition of osteoclast activity to prevent bone loss 17,22,23 .This study indicated that RD-containing serum could enhance the osteogenesis of BMSCs through the Wnt/βcatenin signaling pathway.
Further, RD-containing serum could enhance the BMSC proliferation and ALP activity, with the higher RD dose exerting a more pronounced effect than the lower RD dose.Next, we measured the mRNA and protein levels in the RD-containing serum-treated and control groups and showed that RD-containing serum could enhance the mRNA and protein levels of Runx2, Sp7, Wnt3a and β-catenin.Upon DKK1 addition to high RD-containing serum-treated medium, the relative levels of Runx2, Sp7, Wnt3a and β-catenin were decreased.
To further analyze the the mechanism of Wnt /β-catenin signaling pathway, we tested whether RDcontaining serum enhanced the osteogenesis of BMSCs via activating Wnt/β-catenin signaling.Wnt/βcatenin signaling is a key regulator of bone biology and participates in the differentiation of BMSCs into osteoblasts.However, whether the RD-containing serum could activate Wnt/β-catenin signaling and promote the osteogenic of BMSCs has been unknown.When BMSCs were cultured in the presence of both DKK1 and RD-containing serum, the relative expression of Runx2 and Sp7 was decreased.Thus, RDcontaining serum could enhance the osteogenesis of BMSCs into osteoblasts by regulating the Wnt/βcatenin signaling pathway.We further explored the relative expression of Wnt3a, β-catenin, and Wnt7a and showed that RD-containing serum mainly acts through the regulation of Wnt3a rather than Wnt7a.βcatenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression 32 .We used the immuno uorescence method to visualize the β-catenin translocation into the cell nucleus.Our results showed that RD-containing serum could enhance the β-catenin nuclear translocation, while DKK1 could inhibit the promotion of β-catenin nuclear translocation induced by RDcontaining serum.
In conclusion, RD-containing serum exerts a bene cial role in promoting the proliferation of BMSCs and osteogenic activity by increasing the expression of Runx2 and Sp7.

Supplementary Files
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Figures Figure 1 MTT
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Figure 5 Effects
Figure 5

Figure 7 Effects
Figure 7 Furthermore, RD-containing serum activates the Wnt/β-catenin signaling pathway and increases osteogenic activity.Therefore, our ndings indicate that RD-containing serum could enhance the osteogenesis of BMSCs through the Wnt/β-catenin signaling pathway.