Psoralen mediates BMSCs osteogenic differentiation via cross-talk between Wnt/ β -catenin and BMP/smad signaling pathways

Purpose To explore the mechanism of psoralen mediated Wnt/ β -catenin and bone morphogenetic protein (BMP) signaling pathway to induce osteogenesis of BMSC. Methods Bone marrow mesenchymal stem cells (BMSCs) were treated with psoralen to detect its osteogenic differentiation. In addition, lentivirus and siRNA were used to construct cell models of β -catenin or BMP2 overexpression and knockdown, separately. They may help to clarify the role of β -catenin and BMP2 crosstalk in osteogenic differentiation of BMSCs. What’s more, C57BL/6 mice were selected to be treated psoralen with psoralen to further verify the osteogenic effect. Results Various in vitro studies on BMSCs showed that psoralen could promote the osteogenic differentiation of BMSCs. Overexpression of β -catenin could promote the expression of BMP2 in BMSCs, and psoralen can enhance the effect of bone differentiation. Knockdown β -catenin decreased the expression of BMP2 and inhibited psoralen in promoting bone differentiation. In addition, it was found that the effect of psoralen on β -catenin level did not change signi�cantly after overexpression or knockdown of BMP2, but the effect of psoralen on promoting bone differentiation was inhibited by knockdown of BMP2. In mice, psoralen intervention regulated the crosstalk of Wnt/ β -catenin and BMP signaling pathway to reached to promote osteogenic differentiation of bone tissue.


Introduction
Bone defects due to the causes such as bone trauma, bone tumor, and infection are relatively common in the clinic.Because of the low cure rate of traditional treatment, bone defect is a thorny problem in orthopedics, which seriously affects the life of patients and brings a heavy burden to families, communities and countries.An effective therapy is urgently needed since the incidence of bone defects after fracture has been shown to be as high as 0.068 [1].Currently, stem cell-based tissue engineering and regenerative medicine hold new promise for the treatment of bone defects [2].The directed differentiation of stem cells into osteoblasts is a central di culty in tissue engineering and regenerative medicine for the treatment of bone defects, while osteogenic inducers play a central guiding role as stimulators of bone promoting bioactivity [3].However, conventional osteogenic inducers have disadvantages such as high cost, low differentiation e ciency, complex preparation and many side effects [4].Therefore, the search for simple, safe and effective osteogenic inducers is of great signi cance for tissue engineering and regenerative medicine.
Psoralen (Pso) is a naturally occurring compound.Chemically, it possesses the furocoumarin entity which consists of coumarin moiety fused furan ring [5].Medicinal plants are limitless sources of secondary metabolites.Therefore, these plants have been endowed as important sources of phytomedicines [6].Psoralen is produced in certain plants like in the fruits and seeds of Psoralea corylifolia, celery, parsley and in all citrus fruits.Furthermore, Pso is the major and the most active furocoumarin present in Psoralea corylifolia, which is a traditional Chinese medicine that has long been used to treat diseases such as, osteoporosis and bone defects [7].Modern pharmacological studies have also shown that Pso has the role of anti-tumor, anti-in ammatory, estrogen-like activities, bone promotion and regulation of bone metabolism, so it could be used in the treatment of vitiligo, leukemia, osteoporosis, bone defects and other diseases [8][9][10].
The bone morphogenetic protein (BMP) signaling pathway has an important regulatory role in the osteogenesis of BMSCs, whereas there are studies showing that Pso can activate the BMP signaling pathway to promote osteogenic differentiation of BMSCs [9,10].Bae SJ [11]found that BMP/Smad and Wnt/β-catenin signaling pathway has a synergistic effect, and upregulation of BMP2 expression can activate Smad and β-catenin, thereby promoting osteogenic differentiation of BMSCs.Coincidentally, Pso is also capable of mediating Wnt/β-catenin signaling pathway [12], which seems to be a further step from our understanding of the mechanism of action of Pso.Thus, we hypothesized that Pso may act through Wnt/β-Catenin and BMP2/Smad signaling pathways crosstalk to induce osteogenic differentiation of BMSCs.We hope to apply transgenic technology to elucidate the molecular biological mechanisms by which Pso promotes bone regeneration, resolve bottlenecks in the eld of bone tissue engineering, and promote clinical repair of bone defects.
Cell culture and interventions P3 BMScs were collected and seeded into 96-well plates at a density of 1x10 4 cell/ml and were cocultured with varying concentrations of Pso (0, 5, 10, 15, 20, 25, and 30 μM).Next, 10 µl Cell Counting Kit-8 assay (CCK-8, E-CK-A362, Elabscience Biotechnology Co, China) solution was added into each well after cell culture for 24, 48 and 72 h.The cells were incubated at 37˚C for 2 h in the dark.The absorbance values at a wavelength of 490 nm were detected using a microplate reader (Bio-rad laboratories, inc., USA) to determine the optimal intervention concentration for Pso on cell viability.BMSCs that were not co-cultured with Pso were considered the control group.Each experiment was repeated three times.
Cells were cultured in complete medium and osteogenic induction medium (OIM, β-glycerol-phosphate 10 mM, L-Ascorbic acid 50 mg/L, dexamethasone 0.1 μM add into complete medium) with or without Pso for 3, 7, 14 and 21 d.Alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, real time-PCR (RT-PCR) and western blotting were performed to determine the osteogenic effect of Pso on BMSCs and the most suitable time.BMSCs with BMP2 or β-catenin gene overexpression or knockdown were cultured in osteogenic induction medium, and Pso was added to intervene for the suitable time determined by the previous experiment.BMSCs induced by empty virus served as blank control group, while BMSCs without Pso served as vehicle group.ALP staining, ARS staining, real time-PCR and Western blotting were performed to evaluate the mechanism of Pso effects on BMSCs.
Animals and treatments 60 C57BL/6 mice (6-8 weeks old, weighing 18 ± 2 g) were purchased from the Animal Experimental Center of Xi'an Jiaotong University.All mice were housed in microisolator in the barrier facility of Shaanxi University of Chinese Medicine.After adaptively housed for 1 week, mice were randomly allocated, and injected daily (i.p.) with β-catenin agonist (WAY-262611, 2.5 mg/kg/d, HY-11035), β-catenin inhibitor (XAV939, 2.5 mg/kg/d, HY-15147), BMP2 agonist (LDN-193189, 3 mg/kg/d, HY-12071), and BMP2 inhibitor (SJ000291942, 3 mg/kg/d, HY-112331), separately, purchased from MedChemExpress and orally co-administrated with Pso (40 mg/kg/d) for 4 weeks.A group of age-matched mice without any treatment was designed as control.All experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of Shaanxi University of Chinese Medicine.

Collection of animal samples
After 4 weeks of administration and intervention, the mice were sacri ced by overdose anesthesia (pentobarbital, i.p., 70 mg/kg) and blood was collected by cardiac puncture.The femur and tibia of the mice were harvested for a variety of biochemical, histological and molecular analyses.
Alkaline Phosphatase staining, Alizarin Red S staining and H&E staining After completing the experiment, cells or bone biopsy were stained with ALP staining (BCIP/NBT Chromogen Kit, PR1100, Solarbio, China), ARS staining (Alizarin Red S Solution, G1450, Solarbio, China) and to H&E staining (Hematoxylin -Eosin Stain kit, D006-1-1, Nanjing Jiancheng Bioengineering Institute, China) evaluate the osteogenic differentiation of BMSCs in in vitro and in vivo.Brie y, cells or bone biopsy were xed with a 4% formaldehyde for 15 min and washed in PBS 3 times.Then, the specimens were stained with ALP staining solution for 60 min or ARS for 30 min according to the operating instructions.Samples were observed using a phase-contrast microscope (CKX41, Olympus, Japan).Each sample was assessed in triplicate.
Quantitative real-time PCR (RT-PCR) Total RNA was extracted using standard TRIZOL method.Extracted RNA was used as a template for cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scienti c) according to manufacturer's protocol.After the cDNA was synthesized, RT-PCR was carried out using Lightcycler® 96 kit (Roche, Switzerland).Primer sequences are shown in Table 1.β-actin was used as an internal control for normalization.Reverse CACCCTTCAACTATCTCCTCC

Statistics
The data were expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8.0 statistical software (GraphPad Inc, San Diego, CA, USA).One-way analysis of variance was used to determine the statistical signi cance, and post-hoc comparisons were performed using Tukey's multiple comparisons test.P < 0.05 was considered as statistically signi cant.

Results
Morphology and phenotype of BMSCs Passage1(P1)-BMSCs showed a typical adherent growth state (Fig. 1A).As the passage number increased, small quantities of epithelial and dead cells rapidly disappeared.Until P3, the cultured cells presented high purity with a uniformly typical broblast-like fusiform morphology in a radial or swirling arrangement (Fig. 1B).The immuno uorescence staining indicated that BMSCs highly expressed MSC surface markers such as CD90 and CD44 (Fig. 1C), but did not express hematopoietic stem cells surface markers such as CD34 and CD45 (Fig. 1D).These typical characteristics were in accordance with the minimum criteria for de ning multipotent mesenchymal stem/stromal cells proposed by The International Society for Cellular Therapy [14].
Effects of Pso on osteogenic differentiation and viability of BMSCs Firstly, BMSCs were incubated in complete medium with or without different concentrations of Pso (0, 5, 10, 15, 20 25 or 30 μM) for 24-72 h to ascertain the effect of Pso on cell proliferation.According to the results of the CCK-8 assay, different concentrations of Pso had signi cant effect on cell proliferation at different time points (Fig. 2A-2D).The results showed that the concentration of Pso between 0 and 30 μM promoted the proliferation of rat BMSCs.At 24h, the proliferation of BMSCs increased with the concentration of 0-20 μM, and decreased with the concentration of 20-30 μM (Fig. 2A).At 48h and 72h, the pro-proliferation effect of BMSCs increased with concentration at 0-15 μM, and decreased with concentration at 15-30 μM (Fig. 2B-2C).Among them, the trend of drug action was the most obvious after 48 hours of intervention (Fig. 2D).Comprehensive analysis showed that 15 μM was the best intervention concentration of Pso, so 15μM concentration of Pso was used in subsequent experiments.
To clarify the effect of Pso on osteoblastic differentiation, BMSCs were cultured in complete medium with or without OIM and Pso (15 μM ) for 3, 7, 14 and 21 days (Fig. 2E-2F).The analysis of the ALP activity assay evidenced that ALP staining was positively correlated with the time of OIM and Pso except for the Con group, and the number of stained cells was the highest at 21th days.At the third day, there was no signi cant difference between the experimental groups.On the 7th and 14th day, the number of stained cells in OIM + Pso group was the highest, while the Pso group and OIM group was slightly lower.The cell ALP activity of the above three groups were increased, and the differences were statistically signi cant (Fig. 2E).This result was also consistent with the outcome of ARS staining (Fig. 2F).All these data implied that Pso accelerated osteoblastic differentiation of BMSCs.

Effects of Pso on the expression of osteogenesis-related genes and proteins
The P3 BMSCs were cultured with 15 μM Pso, OIM, and Pso + OIM for 14 d and 21 d (Fig. 3A-3N).As shown in Fig. 3A-3F, the mRNA expression of osteogenic markers osteocalcin (OC), type I collagen (Col1) and BMP2 were signi cantly elevated compared to that of the Con group on 14th and 21th days.
Compared with the OIM group, the increased levels of genes in the Pso group were decreased.However, the combination of Pso and OIM increased the genes content of OC, Col1 and BMP2 signi cantly, especially the BMP2 levels.These results indicate that Pso promoted osteogenic marker expression at the transcriptional level.
Analogously, we found that Pso could also signi cantly promote protein expression of the OC, Col1 and BPM2, and the effect of the Pso + OIM group was more obvious (Fig. 3G-3N).Therefore, these results show that Pso may lead to high expression of osteogenesis-speci c proteins in BMSCs during induction differentiation.
BMSCs models with overexpression or knockdown of β-catenin or BMP2 were constructed Three BMP2 siRNA were designed according to the principles of siRNA design, including BMP2-rat-444 BMP2-rat-859 and BMP2-rat-1096.BMSCs were cultured in serum-free medium for 1 h and transfected with BMP2 siRNA or control siRNA.After transfection, there was no difference in siRNA transfection e ciency among the three groups (Fig. 4A).RT-PCR showed that the relative expression of BMP2 mRNA in the BMP2-rat-444 group was only 1/50 of that in the vehicle group, and the interference effect was the best.Therefore, the BMP2-rat-444 gene fragment was selected for the subsequent experiments (Fig. 4B).
Similarly, we found that the relative expression of β-catenin mRNA in the β-catenin-rat-1231 group was only 1/14 of that in the vehicle group, and the interference effect was the best (Fig. 4C).Therefore, the βcatenin-rat-1231 gene fragment was selected the subsequent experiments (Fig. 4C-4D).In addition, shRNA combined with lentiviral vector was used to overexpress BMP2 (Fig. 4E) and β-catenin (Fig. 4F) gene in BMSCS, respectively.RT-PCR results showed that compared with vehicle group, BMP2 and βcatenin gene expression were signi cantly increased after transfection.These results suggested that BMSCs overexpressed BMP2 or β-catenin gene were successfully constructed, respectively.
Pso increases the expression of BMP2/Smad and Wnt/β-catenin signaling in BMSCs Previous studies reported that BMP2/Smad and Wnt/β-catenin signaling pathways cooperatively regulate cytoskeletal dynamics and osteogenesis.Therefore, we tested the activation effect and crosstalk of Pso treatment on the BMP2/Smad and Wnt/β-catenin signal pathways in BMSCs.As showed in Fig. 5A-K, βcatenin and BMP2 knockdown or overexpressed were performed in BMSCs with Pso treatment.The mRNA expression of Wnt (Wnt1 and Wnt3a), β-catenin, LEF1, BMP2, and Smad (Smad 1, 5 and 9) were markedly reduced by β-catenin or BMP2 knockdown, as compared to Pso group, while the mRNA expression of GSK3β and Smurf2 showed the opposite trend.As expected, when overexpressed the βcatenin or BMP2 gene, the mRNA expression of Wnt (Wnt1 and Wnt3a), β-catenin, LEF1, BMP2, and Smad (Smad 1, 5 and 9) were markedly increased in comparison to Pso group, but the GSK3β and Smurf2 were lower.These results indicate that Pso promoted osteogenic marker expression at the transcriptional level.
At the translational level, as shown in Fig. 5L-U, the relative expression levels of BMP2/Smad and Wnt/βcatenin signal pathways such as Wnt (Wnt1 and Wnt3a), β-catenin, LEF1, Runx2, BMP2, and Smad (Smad 1, 5 and 9) were lower than that in the Pso group when knockdown of β-catenin or BMP2, and the GSK3β was on the contrary.In addition, it was con rmed that the Pso-induced increase in the expression of Wnt (Wnt1 and Wnt3a), β-catenin, LEF1, Runx2, BMP2, and Smad (Smad 1, 5 and 9) was promoted by overexpression of β-catenin or BMP2.These results indicate that the effect of Pso on osteoblastogenesis is exerted through the promotion of crosstalk between BMP2/Smad and Wnt/β-catenin signal pathways.
To verify this, ARS staining was performed on the BMSCs with β-catenin and BMP2 knockdown or overexpression.The result showed that overexpression β-catenin or BMP2 gene could promoted the formation of calci ed nodules, while knockdown of β-catenin or BMP2 gene does the opposite (Fig. 5V).It was consistent with the overall expression levels of mRNA and protein.

Pso increases the expression of BMP2/Smad and Wnt/β-catenin signaling in in vivo
To further demonstrate that Pso can also promote the cross-talk of BMP2/Smad and Wnt/β-catenin signaling pathways in in vivo, we treated mice with Pso and added inhibitors and agonists of β-catenin and BMP2, respectively.It turned out that Pso signi cantly promoted the mRNA and protein expressions of BMP2/Smad and Wnt/β-catenin signaling pathways.When β-catenin was activated, the mRNA and protein expressions of Wnt3a, β-catenin, Runx2, Smad5 and SmAD8 were increased (Fig. 6A-R).When βcatenin was inhibited by XAV939, the mRNA and protein expressions of Wnt1, Wnt3a, β-catenin, BMP2, Runx2, Smad5 and Smad8 were decreased (Fig. 6A-R).Similarly, activation of BMP2 promoted the expression of Wnt1, Wnt3a, β-catenin, BMP2, Runx2, Smad1, Smad5 and Smad8 mRNA and proteins (Fig. 6A-R), while inhibition of BMP2 showed the opposite trend (Fig. 6A-R).In addition, the distal femoral trabeculae were observed by H&E staining.The results of staining showed that compared with the Con group, the number of femur trabecular bone in the Pso group was increased, and the trabecular mesh structure was increased.Agonists of β-catenin and BMP2 intervention further improved trabecular bone number and mesh structure, while the inhibitors treatment signi cantly restrained the effect of Pso in promoting trabecular bone increase (Fig. 6S).Further in vivo experiments in mice con rmed that Pso could indeed promote the cross-talk of BMP2/Smad and Wnt/β-catenin signaling pathways to promote the osteogenic differentiation of cells.

Discussion
The repair of bone defects is complicated.At present, the application of bone tissue engineering scaffolds is more and more widespread, but the biological scaffolds must have good bone conductivity, which is the key to inducing bone repair [15].With the deepening of the research on BMSCs, their application in bone tissue engineering is gradually widespread [16].Through many studies, it has been found that BMSCs have strong advantages in the treatment of bone defect fracture reconstruction and other metabolic bone diseases.The balance of bone metabolism between osteoblast bone formation and osteoclast bone resorption plays an important role in maintaining normal bone mass and strength and promoting bone repair and reconstruction.Traditional Chinese medicine (TCM) can regulate the balance in bone metabolism and have been used to treat bone diseases, such as osteoporosis and bone defects, and promote fractured bone healing for centuries among the Chinese population.
TCM believes that the kidney dominates bone and generates marrow.Only when the kidney essence is su cient, can bone marrow be nourished, bones be strong, and fractures be repaired and regenerated normally [17].For example, in clinical practice, kidney-toning herbs, such as Epimedium, psoralea and Eucommia ulmoides, are commonly used individually or in combination to enhance osteogenesis and inhibit bone resorption, and they show a positive effect on the treatment of bone defects [18].In addition, many natural small-molecule compounds derived from these TCM have also been found to promote the osteogenic differentiation of BMSCs [19][20][21][22][23][24][25].Recently, a number of small molecules have been identi ed that can govern stem cell fate, such as self-renewal, pluripotency, and differentiation [26].
As the fruit of the legume plant psoralea, psoralea is a TCM commonly used in the department of bone injury.It has the effects of warming the kidney and toning the Yang, strengthening the tendon and bones, and has been effectively used to treat orthopedic related diseases for thousands of years [27].Current studies have shown that Pso is one of the main pharmacological active components of psoralea.Modern pharmacological studies have shown that Pso has the role of anti-tumor, anti-in ammatory, estrogen-like activities, bone promotion and regulation of bone metabolism, so it could be used in the treatment of vitiligo, leukemia, osteoporosis, bone defects and other diseases[8].To our knowledge, however, the osteogenic effect of Pso was still unclear so far.In the present study, the potent osteogenic effect on BMSCs by Pso was reported.Further data showed that Pso induced the high expression of multiple osteoblast-speci c markers via crosstalk between Wnt/β-catenin and BMP2/Smad signaling pathways.
Our previous study found that the concentration of Pso less than 100 µM could promote the proliferation of BMSCs, and when the concentration reached 100 µM, toxic reactions would occur [28].Therefore, in order to determine the optimal concentration of Pso on BMSCs, different concentrations of 0, 5, 10, 15, 20, 25 and 30 µM Pso were used to intervene BMSCs for 24 h, 48 h and 72 h.The results of CCK-8 kit showed that Pso could promote the proliferation of rat BMSCs at the concentration of 0-30 µM, and the effect was most obvious at 15 µM.Furthermore, 15 µM Pso was used to intervene BMSCs for 3d, 7d, 14d and 21d. it was showed that Pso could signi cantly promote the ALP activity and the number of mineralized nodules of BMSCs, and enhance with the increase of time.Therefore, 15 µM Pso showed the highest cell viability and osteogenic activity in BMSCs, indicating that osteogenic BMSCs induction is sensitive to Pso.Meanwhile, naturally occurring small molecules were reported to have only synergistic osteoinductive activity in the presence of a routine osteogenic inducer in stem/progenitor cells [20][21][22][23][24][25][29][30][31].Moreover, the routine osteogenic inducer includes dexamethasone, a glucocorticoid that can lead to osteoporosis or bone losses.Therefore, the routine osteogenic inducer has a potential risk for clinical applications, and osteogenic Pso is more suitable for clinical development than the previously reported naturally occurring small molecules.
Osteogenic differentiation of BMSCs is a complex process involving a variety of growth transcription genes.BMP is a member of TGF-β1 family, which is mainly involved in regulating the proliferation and differentiation of BMSCs [32].BMP, as an important osteogenic factor, plays an important role in inducing the differentiation of BMSCs into osteoblasts.In the process of osteogenesis, the target of BMP is BMSCs and osteoblast precursor cells.At present, more than 20 kinds of BMP have been found, which can promote the differentiation of BMSCs, among which BMP2 is widely used in bone regeneration and other aspects [33].Col1 is one of the markers of BMSCs differentiation into osteoblasts.As a structural protein, Col1 is abundant in bone, especially as much as 90% in organic matter.As a bone metabolite, it can in turn regulate bone metabolism, enhance the osteogenic activity of osteoblasts, affect the expression of ALP and OC, and participate in bone tissue repair [34].In addition, as a non-collagen protein, the synthesis and secretion of OC depend on osteoblasts, and can re ect the degree of differentiation and maturation of osteoblasts, promote the deposition of hydroxyapatite, enrich osteoblasts, promote bone formation and bone calci cation, which is a speci c index of late differentiation of osteoblasts [35].In this study, BMP2, Col1 and OC were used to observe the effect of Pso on the osteogenesis differentiation of BMSCs.The expression of OC, Col1, and BMP2 gene and protein signi cantly increased on the 14d and 21d of BMSCs treated with Pso, indicating that Pso could induce BMSCs to differentiate into osteoblasts and promote bone repair.
Numerous signal transduction pathways and transcriptional factors, including the activation of Wnt/βcatenin, TGF-β/BMP, and MAPK signaling, have been widely reported to regulate osteoblastic MSC differentiation induced by the routine osteogenic inducer [36,37].Accordingly, the osteogenic effect of natural small molecules might be involved in these signaling pathways due to the use of the routine osteogenic inducer during osteogenic differentiation.Additional, Our previous study found that Pso signi cantly upregulated β-catenin and Runx2 gene expression [28,38], and multiple studies have shown that Pso has a strong osteogenic effect [39,40], Which may be relevant to Wnt/β-catenin and BMP/Smad signaling pathway [41][42][43].Therefore, Pso may be considered as an ideal bone tissue engineering seed cell differentiation inducer.To assess the possible mechanism of osteogenic Pso on Wnt/β-catenin and BMP2/Smad signaling pathway, we induced BMSCs models with BMP2 and β-catenin knockdown and overexpression, respectively.RT-PCR results con rmed that BMP2 and β-catenin overexpression and knockdown cell models were constructed successfully.
The Wnt/β-catenin pathway has been shown in various studies to increase the weight of bone and cortical bone through the activation of MSCs, inhibition of osteoblast death, and induction of osteoblast differentiation.For these reasons, this mechanism is an important strategic indicator of recent bonerelated diseases [44,45].The canonical Wnt signaling pathway is associated with the stabilization of βcatenin.In the normal cellular state, the proteasome complex of β-catenin is degraded by GSK-3β.When the Wnt mechanism is activated, it is attached to the membrane receptor to inhibit the interaction of GSK3β with β-catenin, resulting in β-catenin being phosphorylated and then transferred to the nucleus and attached to TCF/LEF, where it upregulates the expression of Runx2 [46,47].Moreover, the BMP pathway is one of the main signaling cascades that stimulate bone formation.The mechanism of receptor activation involves BMP-induced phosphorylation of two sequentially activated kinases, with the type I receptor acting as a substrate for the type II receptor kinase.The activated type I receptor relays the signal to the cytoplasm by phosphorylating its downstream target, Smad1/5/8 protein, which then interacts with Smad4 and translocates into the nucleus.A number of compounds have been found to affect this pathway by increasing the expression of BMPs and/or activating the downstream signaling pathway.Our results showed that Wnt/β-catenin and BMP2/Smad signaling-associated key factors, such as BMP2 Smad1 Runx2 Wnt1 β-catenin and LEF1, were signi cantly up-regulated intervened with Pso.
Furthermore, the effect of Pso was signi cantly inhibited when β-catenin and BMP2 were knocked down.
Therefore, osteoblastogenesis induced by Pso was maximized in the presence of both Wnt/β-catenin and BMP2/Smad signaling.
Furthermore, how does Pso induce osteoblastic BMSCs differentiation via cross-talk between Wnt/βcatenin and BMP2/Smad signaling pathways?How do they interplay both signals?How do they interplay both signals?Our results showed that blocking Wnt/β-catenin signaling exerted a more potent inhibitory effect on osteogenic differentiation and even on key factors of the BMP2/Smad signaling pathway(Fig.5A-U).It is known that the Wnt/β-catenin signaling pathway plays a crucial role in suppressing MSC commitment to chondrogenic and adipogenic differentiation and enhancing osteogenic differentiation and bone formation in vitro/in vivo [30,48,49].Previous studies showed that de cient or inactivated βcatenin could result in substantial osteogenesis inhibition with dramatic Runx2 down-regulation [48,50] Runx2 are the most important transcription factors in the osteogenic differentiation process as well as the principal downstream targets of canonical Wnt signaling [30].Furthermore, Runx2 is the downstream targets of β-catenin activity for the stimulation of osteoblast differentiation and bone formation [48,51].Similar to previous studies, Wnt1 p-β-catenin and LEF1 of Wnt/β-catenin signaling were distinctly upregulated in BMSCs after Pso treatment in the study.In addition, TGF-β/BMP signaling activates Runx2 gene expression in MSCs to control osteoblast differentiation and skeletal development [36,51].In our study, BMP 2, Smad1, Smad5 and smad9 were markedly up-regulated at the transcriptional and translation level in BMSCs after Pso addition.As described previously, when BMP2/Smad signaling was activated, pSmad1/5/8 was recruited to form a complex with Smad4, which was then transferred into the nucleus to regulate Runx2 transcription [52].Even BMP signaling is essential for almost every step during osteoblast differentiation and bone formation [53].Interestingly, many previous studies showed that BMPs could promote Wnt/β-catenin signal transduction in the process of osteogenic regulation.During the osteogenic differentiation of BMSCs induced by Pso, when BMP2 is overexpressed, Smad1 downstream of BMP2 is activated and forms a complex with other Smad proteins to enter the nucleus and up-regulate the expression of Runx2.Up-regulated Runx2 can promote the expression of Wnt1, βcatenin, LEF1 and other Wnt/β-catenin signaling related genes.When β-catenin is overexpressed, the protein level of β-catenin increases and binds to transcription factors such as LEF1 and Runx2 after entering the nucleus, which can promote the expression of BMP2 and Smad1 genes.Furthermore, when the expression of BMP2 or β-catenin was disrupted, the expression levels of Smad1, Runx2, Wnt1, βcatenin and LEF1 were down-regulated, but the expression levels were still higher than those in Con group without Pso.In other words, when the single signal of BMP2/Smad or Wnt/β-catenin is blocked, Pso can still compensate for the effect of the other pathway through the crosstalk of the two, and maintain the expression of BMP2/Smad and Wnt/β-catenin related genes at a relatively stable level.
To further con rm the ndings in in vivo, mice were treated with Pso combined with an agonist and inhibitor of BMP2 and β-catenin.The results showed that Pso could promote the expression of Wnt/βcatenin and BMP2/Smad related factor genes and proteins, increase the number of femoral trabecular bone and improve the trabecular meshwork structure, which was consistent with the results of in in vitro.
Overall, there are numerous osteogenic differentiation and bone formation processes that involve Wnt, βcatenin and BMP, yet there are con icting data on the role and interaction of these factors.However, Runx2 is regarded as a common downstream target gene or crucial mediator of Wnt/β-catenin and BMP2/Smad signaling during osteogenic differentiation.Based on the above analysis, a schematic diagram presenting the possible osteoinduction mechanism of Pso in BMSCs can be speculated as shown in Fig. 7.All in all, here we report osteogenic BMSCs differentiation induced by Pso through crosstalk between Wnt/β-catenin and BMP2/Smad signaling pathways.Due to the complexity of regulation of these signal transduction pathways, however, there remains a need for further investigation on the signaling transduction network of Pso on osteogenic BMSCs differentiation as well as its therapeutic effect on bone defect.
In summary, we have demonstrated that Pso is a safe, effective, and novel natural osteogenic inducer for BMSCs, and its action mechanism may be involved in cross-talk between the Wnt/β-catenin and BMP2/Smad signaling pathways.In addition, Pso might be used as a potential candidate compound for stem cell-based therapy of bone defects.BMSCs models with overexpression or knockdown of β-catenin or BMP2 were constructed.Three BMP2 siRNA were designed according to the principles of siRNA design, including BMP2-rat-44, BMP2-rat-859 and BMP2-rat-1096.After transfection, there was no difference in siRNA transfection e ciency among the three groups (Fig. 4A).RT-PCR showed that the relative expression of BMP2 mRNA in the BMP2-rat-444 group was only 1/50 of that in the vehicle group, and the interference effect was the best (Fig. 4B).
Similarly, the interference effect of the β-catenin-rat-1231 was the best and selected for subsequent experiments (Fig. 4C-4D).In addition, RT-PCR results showed that compared with vehicle group, BMP2 (Fig. 4E) and β-catenin (Fig. 4F) gene expression were signi cantly increased after transfection.

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Figure 2 Effect
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Figure 3 Effects
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Table 1
Primer sequences of target genes