Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model

Asperosaponin VI (ASA VI), a natural compound isolated from the well-known traditional Chinese herb Radix Dipsaci, has an important role in promoting osteoblast formation. However, its effects on osteoblasts in the context of osteoporosis is unknown. This study aimed to investigate the effects and mechanism of ASA VI action on the proliferation and osteogenic differentiation of bone marrow stromal cells isolated from the ovariectomized rats (OVX rBMSCs). The toxicity of ASA VI and its effects on the proliferation of OVX rBMSCs were measured using a CCK-8 assay. Various osteogenic differentiation markers were also analyzed, such as ALP activity, calcified nodule formation, and the expression of osteogenic genes, i.e., ALP, OCN, COL 1 and RUNX2. The results indicated that ASA VI promoted the proliferation of OVX rBMSCs and enhanced ALP activity and calcified nodule formation. In addition, while ASA VI enhanced the expression of ALP, OCN, Col 1 and RUNX2, treatment with LY294002 reduced all of these osteogenic effects and reduced the p-AKT levels induced by ASA VI. These results suggest that ASA VI promotes the osteogenic differentiation of OVX rBMSCs by acting on the phosphatidylinositol—3 kinase/AKT signaling pathway.

The ALP expression of the two groups of BMSCs were observed on 7d and 14d. The OVX group was significantly lower than that of the SHAM group. (E) The calcium deposit formation of the two groups were determined by Alizarin red staining. The OVX group was significantly lower than that of the SHAM group. (F) The data are represented as the mean ± SD. *P < 0.5 vs the SHAM group.

Materials and Methods
Reagents. ASA VI (purity > 99%, National Institute for the Control of Pharmaceuticals and Biological Products, Beijing, China) was dissolved in phosphate-buffered saline (PBS) and stored at − 20 °C. Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from Sigma (Sigma-Aldrich Co., USA). A CCK-8 kit was obtained from Dojindo (Kumamoto, Japan). Bicinchoninic acid (BCA) protein assay kit was obtained from Jiancheng (Nanjing, China)and alkaline phosphatase (ALP) activity assay kit was obtained from Wako (Japan). LY294002 was obtained from the Biyuntian Bioengineering Institute (Shanghai, China). Ascorbic acid phosphate, β -glycerophosphate, dexamethasone and alizarin red S were also purchased from Sigma (Sigma-Aldrich Co, USA).
Animals and OVX models. Three-month-old, female Sprague-Dawley rats weighing between 240 and 260 grams were obtained from the Animal Experiment Center of Zhejiang University (Hangzhou, China). All animal experiments were performed in accordance with the Animal Care and Use Committee guidelines of Zhejiang province. The experiments of this study were approved by the Institutional Animal Care and Use Committee of Zhejiang University, Hangzhou, China. 40 rats were divided into two groups randomly (20 OVX group, 20 Sham group). After receiving an intraperitoneal injection of 300 mg of chloral hydrate per kg of body weight, the rats underwent ovariectomy. The sham operation were used as controls. All rats were then housed in cages at room temperature (23 °C) with controlled humidity (55%) and a 12-hour light/dark cycle 24 . After 12 weeks, the femur of 10 rats (5 per group) were dissected and scanned using micro-computer tomography (Bruken, Belgium) to acpuire three-dimensional imagery of their cancellous bone micro structure and for analysis. Sample parameter included Bone mineral density (BMD), Bone volume/Total volume (BV/TV), The trabecular spacing (Tb. Sp).
Cell culture. 30 rats (15 per group) were sacrificed by an overdose of chloral hydrate to obtain rBMSCs for culture. These rBMSCs cultures were prepared according to the protocol developed in the Caplan laboratory 25 . In brief, the tibia and femur were excised, and all attached tissues were carefully removed under sterile conditions. The  . ALP activity was assessed using a commercial ALP kit after cells were cultured with various concentrations of ASA VI for 5 or 10 days. The data are represented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs the controls; # P < 0.05 and ## P < 0.01 vs ASA VI 10 −5 M. bone marrow was extracted with an injection of DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μ g/ml streptomycin. Cells were plated on plates 60 mm in diameter and incubated at 37 °C in a humidified atmosphere with 5% CO 2 for 24 hours before the first medium change. At 85% confluence, the cells were trypsinized (0.25% trypsin-EDTA, Sigma) and passaged into flasks at a ratio of 1:3. rBMSCs from the third passage were used in all experiments. For all subsequent experiments except the cell proliferation assay, the culture medium was replaced with an osteogenic medium supplemented with 10 mM β -glycerophosphate, 50 μ g/ml ascorbic acid and 10 -8 M dexamethasone 26 .
Detection of osteogenic capability. The osteogenic capability of OVX rBMSCs and SH rBMSCs was tested. After osteogenic induction, expression of alkaline phosphatase (ALP) were examined on day 7 and day 14 separately. The formation of calciumnodes was analyzed by alizarin red staining on day 21. The procedures were the same as that for the assay described following.
ALP activity assay. OVX rBMSCs were seeded in 24-well plates (5 × 10 3 cells/well) together with various concentrations of ASA VI (0, 10 −5 , 10 −6 , 10 −7 , and 10 −8 M), with 3 replicate wells for each concentration. After 5 or 10 days of osteogenic induction, the cells were lysed with 0.2% Triton X-100 on ice and then centrifuged at 14,000 rpm at 4 °C for 15 minutes. The supernatant was collected to measure ALP activity using an ALP activity kit, and protein concentrations were measured using a BCA protein assay kit 26 . Alizarin red staining and mineraNlization assay. To investigate the mineralization of the OVX rBMSCs, cells were seeded in 24-well plates (5 × 10 3 cell/well) in osteogenic medium with LY294002 (50 μ M) and/or ASA VI (10 −5 M), with 3 replicate wells per concentration. The cultures were maintained for 21 days, and the medium was changed every 3 days. The cells were washed twice with PBS and fixed in 98% ethanol for 20 minutes. Then, the cells were washed with PBS again, and 200 μ l of alizarin red solution was added to each well and incubated for 15 minutes at 37 °C. After the nodules were imaged, 10% cetylpyridinium chloride was used to dissolve the nodules, and absorbance at 562 nm was measured 27 . Western blot analysis. The obtained OVX rBMSCs were cultured for 2 days in osteogenic induction conditions, as previously described. Then, they were washed 3 times with PBS and lysed with RIPA buffer (Wuhan Guge, China) on ice before being centrifuged at 12,000 g for 5 minutes. The supernatant was then collected, and protein concentrations were determined using a BCA protein assay kit; 50 μ g of protein for each sample was boiled for 5 minutes and was then separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Germany). The membranes were blocked with 5% BSA in TBST buffer and were subsequently incubated with primary antibodies against p-AKT overnight at 4 °C. Then, the membranes were washed 3 times in TBST for 15 minutes and incubated with secondary antibodies (1:3,000) for 30 minutes at room temperature. After visualization using enhanced chemiluminescence, the experimental band absorbance values were determined relative to the absorbance of β -actin. Statistical Analysis. All experiments were repeated at least in triplicate, and one-way ANOVA was used to compare differences among groups. P values < 0.05 were considered statistically significant.

Results
Micro-CT evaluation. Micro-CT three-dimensional imagery showed that in OVX group the bone microstructure of femur were damaged badly with the bone trabecula being sparser when compared to the Sham group (Fig. 1A). Meanwhile, OVX group showed a significant decrease in the BMD (Fig. 1B) and BV/TV (Fig. 1C), but Tb. Sp (Fig. 1D) was significantly higher (P < 0.05).
Detection of osteogenic capability. The results showed that the OVX group was significantly lower than that of the Sham group, including the expression of ALP (P < 0.05) (Fig. 1E) and calcium deposit formation ( Cell proliferation. OVX rBMSC proliferation was analyzed using a CCK-8 assay on days 1, 4, and 7. ASA VI did not affect cell proliferation on day 1. On day 4 and 7, ASA VI (10 −5 , 10 −6 , 10 −7 , and 10 −8 M) promoted the proliferation of OVX BMSCs in a dose-dependent manner, while ASA VI 10 −4 M inhibited cell proliferation (Fig. 2). ALP activity. ALP activity is an early marker of osteoblast differentiation. After 5 days of cultivation, compared with the control groups, the 10 −5 M or 10 −6 M ASA VI groups showed significantly greater ALP activity (P < 0.05). On day 10, we observed an increase in ALP activity in all four ASA VI groups and the 10 −5 M ASA VI groups showed the greatest ALP activity among them, These findings indicated that 10 −5 M ASA VI was the optimal concentration for stimulating osteogenic differentiation in OVX rBMSCs. Thus, we adopted this concentration for use in subsequent experiments. (Fig. 3).

Matrix mineralization.
To assess matrix mineralization, we measured the area of calcium deposition and the absorbance index values (Fig. 4). The ASA VI treatment resulted in a significantly larger calcium deposition area compared with the controls (P < 0.01). In addition, cells treated with ASA VI and the PI3K inhibitor LY294002 exhibited a much smaller area of calcium deposition than did cells treated with ASA VI alone (P < 0.05); this value was similar to that of the control groups. These data indicated that the PI3K pathway inhibitor LY294002 interfered with the osteogenic differentiation of OVX rBMSCs induced by ASA VI.

Real-time PCR.
We explored the expression of the osteogenic genes ALP, OCN, COL 1 and RUNX2 after 48 hours of culture with ASA VI and/or LY294002, as described above. Compared with the controls, the ASA VI treatment significantly enhanced the expression of osteogenic genes in OVX rBMSCs (ALP, P < 0.001; OCN, P < 0.05; COL 1, P < 0.05; and RUNX2, P < 0.001). In comparison, the treatment with both LY294002 and ASA VI yielded normal expression levels (Fig. 5). Western blot analysis. The Western blot analysis indicated that ASA VI induced a significant increase in the expression of p-AKT (P < 0.01), while LY294002 decreased this expression (P < 0.01) (Fig. 6). These results demonstrate that ASA VI stimulates osteogenic differentiation in OVX rBMSCs through PI3K/AKT pathway activation.

Discussion
ASA VI is a major active component that can be extracted from Radix Dipsaci, which is used in traditional Chinese medicine to enhance bone formation 19 . Previous reports have shown that Radix Dipsaci can alleviate OP [16][17][18] . Although Radix Dipsaci is a natural, stable and widely available drug, there are few reports on this compound in the literature. ASA VI has shown the ability to stimulate the proliferation and osteogenic differentiation of MC3T3-E1 cells and calvarial osteoblasts in healthy mice 20 . However, the mechanism by which ASA VI promotes ossification is unknown. Moreover, to the best of our knowledge, there are no reports on the role of ASA VI in bone formation during OP. In this study, we showed that ASA VI was involved in proliferation, osteogenic differentiation and calcification of rBMSC isolated from osteoporotic rats.
The PI3K pathway plays a major role in cell proliferation, differentiation, adhesion and apoptosis 28 , and recent research has demonstrated a close relationship between PI3K/AKT signaling and bone tissue metabolism 22,29 . In osteoblasts, PI3K/AKT pathway activation has been shown to stimulate proliferation and differentiation while also inhibiting apoptosis 30,31 . Additionally, the PI3K/AKT pathway can affect osteoclast formation 29 . There are also reports that mice deficient in AKT1 or AKT2 (two of the three mammalian AKTs) 32,33 displayed serious bone defects, as well as other major developmental defects. In contrast, the activation of AKT increases bone mass by blocking osteoblastic phosphatidylinositol-3,4,5-trisphosphate 3-phosphataseexpression in mice 34 . Recent studies of PI3K signaling have underscored the involvement of the PI3K/AKT signaling pathway in OP 22 . Therefore, this pathway is critical for maintaining bone stability under both normal and pathological conditions. Accordingly, it has enormous potential as a therapeutic target for OP. Our findings show that at a concentration of 10 −5 M, ASA VI enhanced ALP activity in OVX rBMSCs, improved mineralization, and promoted the expression of the osteogenic genes ALP, OCN, COL 1, and RUNX2. ASA VI also enhanced the expression of p-AKT in OVX rBMSCs. These effects were inhibited by the PI3K/AKT inhibitor LY294002, indicating that the ability of ASA VI to stimulate osteogenic differentiation in OVX rBMSCs is dependent on the PI3K/AKT signaling pathway.
As a natural medicine that can promote osteogenesis, Icariin has been extensively studied, and it has been linked to the promotion of osteogenesis through different pathways, such as the BMP-2, SMAD, RUNX2 and osterix 35 , estrogen 36 , MAPK 37 and PI3K/AKT 26 signaling pathways. These observations indicate that a single drug can function synergistically via multiple signaling pathways, with the added benefit of some crosstalk occurring among them. Previous studies have demonstrated the ability of ASA VI to promote the expression of BMP-2 and the activation of p38 and ERK1/2, confirming that its role in promoting osteoblast differentiation is dependent on the BMP-2 pathway 20 . In this study, we showed that ASA VI promoted the osteogenic differentiation of OVX rBMSCs and that the increased expression of RUNX2 is dependent on PI3K/AKT. We believe that these two results are not contradictory. The two pathways are related to each other in terms of osteoblast differentiation. RUNX2 is one of the most important transcription factors for osteoblast differentiation and bone formation 38 . BMPs are best known for their actions as signals for bone formation 39 , and BMP-2 is capable of promoting the expression of the osteogenic gene RUNX2 40 . In turn, RUNX2 is capable of improving the activity of the PI3K/AKT signaling pathway by increasing the protein levels of PI3K p85, p110β and AKT. Furthermore, PI3K/AKT signaling enhances the DNA binding of RUNX2 and RUNX2-dependent transcription 41 . This positive feedback loop further enhances RUNX2 activity during osteoblast differentiation. Moreover, crosstalk exists between the BMP-specific SMAD and PI3K/AKT signaling pathways in the regulation of BMP-2 transcription 42 . However, although we have demonstrated that ASA VI is a natural drug that can enhance osteogenesis in vitro, how this compound regulates several signaling pathways remains to be elucidated.

Conclusion
ASA VI has the ability to promote the proliferation, osteogenic differentiation and mineralization of OVX rBMSCs. Its optimal concentration is 10 −5 M, and its action is dependent on the PI3K/AKT signaling pathway. Therefore, ASA VI has potential as a new drug for treating women who suffer from post-menopausal OP.