Asperosaponin VI promotes human mesenchymal stem cell differentiation into nucleus pulposus like-cells via up-regulation of ERK1/2 and Smad2/3 signaling pathways.

At present, the regeneration of nucleus pulposus cells is an effective method to prevent intervertebral disc degeneration (IVDD). In this study, we investigated the role of Asperosaponin VI (ASA VI), isolated from a traditional Chinese medicine (TCM), the root of Dipsacus asper Wall, in promoting human mesenchymal stem cell (HMSC) proliferation and differentiation into nucleus pulposus like-cells and explored its possible mechanism of action. the effects of on vitality and proliferation were determined by the and staining. Then, HMSCs were cultured with VI. Real-time PCR, immunocytochemistry and immunouorescence were used to measure the expression of extracellular matrix (ECM) components in nucleus pulposus cells, such as type II collagen(COL2A1), aggrecan, SOX9, KRT19, PAX1 and glycosaminoglycans (GAGs), and Western blot was used to investigate its potential mechanism.

The intervertebral disc(IVD) is mainly composed of cartilage tissue, including nucleus pulposus, annulus brous and endplates. Some studies have shown that the changes of ECM biosynthesis and the decreases in nucleus pulposus cell function and number are the primary factors of IVDD [6]. Therefore, increasing extracellular matrix and promoting the regeneration of NP cells have become an ideal treatment. Due to poor regeneration of NP cells, cell-based therapy may be a promising treatment for IVDD [7]. At present, increasing amounts of attention have been paid to using the multidirectional differentiation potential of stem cells to promote the regeneration of degenerative IVD [8].
Moreover, a large number of experimental studies have con rmed that stem cell applications are effective treatments with great clinical application potential [9,10]. There are many research methods to promote the differentiation of stem cells into nucleus pulposus for the treatment of degenerative IVD, including growth factor intervention, coculture of stem cells and nucleus pulposus cells, hypoxia induction, stem cells planted in three-dimensional scaffolds, and applied stress, which promote the differentiation of stem cells into nucleus pulposus-like cells to a certain extent [11][12][13][14][15]. However, it is rare to use traditional Chinese medicine to promote the differentiation of stem cells into nucleus pulposus-like cells to repair a degenerative IVD.
Asperosaponin VI (ASA VI), also known as Akebia Saponin D(ASD), is the main bioactive component of the traditional Chinese medicine (TCM) Radix Dipsaci. The Chinese Pharmacopoeia speci es ASA VI content as Dipsacus asper Wall quality standard.
Dipsacus asper Wall, as an herbal medicine, has the effect of tonifying the liver and kidney, with a long history of safe use for strengthening the tendons and bones. The study found that Radix Dipsaci functions by inhibiting osteoclast differentiation, preventing osteoporosis and promoting fracture healing [16][17][18]. In addition, Radix Dipsaci can upregulate the expression of TGF-β1, increase collagen formation and promote Achilles tendon healing [19]. It has not yet been reported that ASA VI can promote the differentiation of stem cells into nucleus pulposus-like cells.

Cell culture
Human mesenchymal stem cells (HMSCs) (Sciencell, 7500, USA) were purchased from Sciencell. Primary HMSCs were obtained by digestion. The cell line was cultured in mesenchymal stem cell medium (MSCm), supplemented with 10% FBS, 1% penicillin and 1% streptomycin at 37℃ in a humidi ed atmosphere of 5% CO 2 in a T-75 ask for 48 hours before the rst medium change. At 80% con uence,

Assessment of cellular vitality
Cell vitality was evaluated by cell counting XTT assays (Sigma-Aldrich, X4626, USA) according to the manufacturer's instructions. In brief, HMSCs were seeded on 96-well plates (2 × 10 3 cells/well) at 37℃ in a humidi ed atmosphere of 5% CO 2 for 24 hours. Then, 50 µl of XTT testing work uid was added to each well, and the plates were cultured at 37℃ in a humidi ed atmosphere of 5% CO 2 for 4 hours. After that, cells were treated with different concentrations of ASA VI (0, 0.01, 0.1, 1, 10, and 100 mg/l), the MSCm and ASA VI were changed every 72 hours, 50 µl of XTT test working solution was added to each well, and the plates were cultured at 37 °C in a humidi ed atmosphere of 5% CO 2 for 4 hours after 1, 3 and 5 days. The absorbance was measured at 450 nm using a microplate reader (Bio Tek, USA) after each addition of XTT test working solution.

Assessment of cellular proliferation
Cell proliferation was assessed by an EDU test using a BeyoClick EdU-488 cell proliferation kit combined with DAPI staining according to the manufacturer's instructions. The HMSCs were plated on 96-well plates (2 × 10 3 cells/well) at 37℃ in a humidi ed atmosphere of 5% CO 2 for 24 hours. Then, different doses of ASA VI (0, 0.01, 0.1, and 1 mg/L) were added to the wells, and the MSCm and ASA VI were changed every 72 hours. Five days later, the EDU reagent (0.5 µL, 50 µmol/L) was added into each well containing 200 µL of MSCm and incubated at 37℃ in a humidi ed atmosphere of 5% CO 2 . for 2 hours.
Then, cells were xed with 4% paraformaldehyde and incubated with amino acetic acid for 5 minutes.
After that, 100 µL of penetrant was added to each well and incubated for 5 minutes. Then, each well was supplemented with 100 µL of 1xEDU work uid, and 30 minutes later, DAPI was used to stain cell nuclei for 10 minutes [23]. The stained cells were observed under a uorescence microscope (OLYMPUS, USA).

Quantitatie Real-time Polymerase Chain Reaction
The HMSCs were plated on 6-well dishes. Then, cells were treated with different concentrations of ASA VI (0, 0.01, 0.1, 1, 10, and 100 mg/L). The mixture was changed every 72 hours. Total RNA was extracted from cells after 3 and 7 days using TRIzol reagent (INV, 15596026, USA) according to the manufacturer's instructions. Then, cDNA was synthesized by applying a cDNA reverse transcription kit according to the manufacturer's instructions. In brief, rst, an 8-µL reaction mixture containing 1 µg of total RNA, 2 µL of oligo d(T)23VN(50 µM) and RNase-free dH 2 O was incubated at 65 ℃ for 5 minutes. Subsequently, 10 µL of ProtoScript II Reaction Mix (2X) and 2 µL of Proto-Script II Enzyme Mix (10X) were added to a nal volume of 20 µL, and the mixture was incubated at 42 ℃ for 60 minutes. Finally, the mixture was incubated at 80 ℃ for 5 minutes. Gene expression was analyzed by quantitative real-time polymerase chain reaction( PCR, ABI Stepone Plus, USA). GAPDH was used to quantify PCR products and to con rm the use of equivalent RNA. Reactions were carried out in duplicate in a 96-well plate with a nal volume of 20 µL. The PCR included an initial enzyme activation stage at 95℃ for 10 minutes, followed by 40 cycles of 95℃ for 15 s and 60℃ for 60 s. Products were quanti ed using a melting curve analysis. Results were calculated using the 2 −ΔΔct method. The primers used in this study are shown in table 1.

Glycosaminoglycans (GAGs) assay
The HMSCs were plated on 6-well plates. After 24 hours, cells were treated with the appropriate concentration of ASA VI (1 mg/L), while cells in the control group were not treated with ASA VI. The medium was changed every 72 hours. To investigate the effect of ASA VI on the secreted ECM proteins, a dimethylmethylene blue assay (DMMB) was used to quantify the soluble GAGs in the cell culture media. After 7 and 14 days, the cell culture media were collected. A portion of the media was mixed with DMMB dye and incubated with moderate agitation at room temperature for 30 minutes. Upon incubation, the solution was centrifuged to form a pellet of GAGs that bound to the dye. The pellet was washed in icecold acid-salt solution, centrifuged and resuspended in 10% SDS for the DMMB assay. The DMMB assay was quanti ed at 525 nm using a microplate reader (Bio Tek, USA). Absorbance was converted to GAG concentrations using a calibration curve obtained using different concentrations of bovine chondroitin 4sulfate as the standard. The GAGs were normalized using the total protein in the media quanti ed at UV 280 nm using a microplate reader (Bio Tek, USA) [24].
The HMSCs were plated on 96-well plates (2 × 10 3 cells/well) at 37℃ in a humidi ed atmosphere of 5% CO 2 for 24 hours. Then, cells were treated with the appropriate concentration of ASA VI (1 mg/L), while control cells were not treated with ASA VI. The medium was changed every 72 hours. After 14 days, cells were xed with 4% paraformaldehyde for 5 minutes. After that, each well was treated with 0.2% Triton X-100 in 1X PBS for 5 minutes at room temperature. Cells were then blocked with 5% blocking serum from speci c species in 1X PBS at room temperature for 1 hour. Subsequently, cells were incubated with primary antibody (1:50) diluted in antibody dilution buffer for 1 hour at room temperature, followed by incubation with the corresponding uorochrome-labeled secondary antibodies diluted in antibody dilution buffer (1:200) for 1 hour at room temperature in the dark. Finally, DAPI was used to stain cell nuclei for 10 minutes [25]. The stained cells were observed under a uorescence microscope (OLYMPUS, USA).

Western blotting analysis
The HMSCs were plated on 6-well plates. After 24 hours, cells were treated with the appropriate concentration of ASA VI (1 mg/L), while control cells were not treated with ASA VI. After 48 hours, cells were washed with PBS and lysed with lysis buffer mixed with PMSF for 30 minutes on the ice, followed by ultrasonic fragmentation (400W, Ultrasound 15 seconds, stop 15 seconds) for 10 minutes on an ice bath. After centrifuging for 10 minutes at 10,000xg and 4 ℃, the soluble was added with Loading Buffer(5:1) and Boiled in boiling water for 5 minutes. Protein samples were separated by 10% SDS-PAGE under 80 V for 30 minutes and 100 V for 90 minutes and then transferred to nitrocellulose membranes. The membranes were blocked with TBS buffer for 1 hour at room temperature. The primary antibodies (rabbit polyclonal anti-ERK1/2, anti-phosphospeci c ERK1/2, anti-Smad2/3, anti-phosphospeci c Smad2/3, 1/500 dilution; rabbit monoclonal anti-β-actin, 1/5000 dilution) were added to the nitrocellulose membranes and incubated overnight at 4 ℃.Subsequently, the membranes were washed thrice for 5 minutes each with TBS buffer and incubated with anti-rabbit secondary antibodies (1:5000) for 1 hour at room temperature. The membranes were washed thrice for 5 minutes each again, and detection was performed using a dual-color infrared imaging system (Odyssey, LI-COR,USA).

Statistical analysis
The results are expressed as the mean ± standard deviation. Statistical signi cance was determined using a one-way ANOVA test and a T-test to compare the different groups in SPSS 19.0 statistical software. For each test, at least three independent parallel experiments were performed. P < 0.05 was considered to be statistically signi cant.

Effect of ASA VI on HMSC proliferation
The effect of ASA VI on the cell proliferation of HMSCs was evaluated by the XTT and EDU assays. Cell numbers were increased after incubation with different concentrations of ASA VI (0.01, 0.1, 1, 10, and 100 mg/L) for 1, 3 and 5 days and peaked at 1 mg/mL (Fig. 1A ). The EDU assay results showed that the proliferation of HMSCs can be enhanced with the increase of ASA VI dose (Fig. 1B and 1C), which was similar to the XTT results.

Nucleus Pulposus Cells Gene Expression under Different Concentrations of ASA VI
To study the effects of different concentrations of ASA VI on the biosynthesis of HMSCs, ECM expression was analyzed by RT-PCR. The gene expression pro les were investigated after 3 and 7 days of ASA VI cultivation at 0 (control), 0.01, 0.1, 1, 10 and 100 mg/L. Figure 2 shows the relative gene expression of the nucleus pulposus markers (type II collagen, aggrecan, SOX9, KRT19, and PAX1) for the ASA VI and control groups. The results showed that the concentrations of 0.01, 0.1 and 1 mg/l ASA VI upregulated the gene expression levels of the markers, which peaked at 1 mg/L. However, when the concentration of ASA VI increased to 10 mg/l and 100 mg/l, the gene expression did not upregulate with the increased concentration and even appeared to be inhibited.
These ndings indicated that 1 mg/L was the optimal concentration of ASA VI for stimulating HMSCs differentiation into nucleus pulposus like-cells. Thus, we adopted this concentration for the subsequent experiments.
GAGs expression under ASA VI GAGs expression levels were investigated after 7 and 14 days of ASA VI cultivation at 0 (control) and 1 mg/l. Figure 3 shows that the GAGs contents in the cell supernatants signi cantly increased with time in the experimental and control groups. The increase in the rate of GAGs contents in the cell supernatant of the experimental group was higher than that of the control group at 7 and 14 days.

ASA VI accelerated aggrecan and PAX1 deposition
Aggrecan and PAX1 immuno uorescence staining revealed stronger green staining in ASA VI (1 mg/l)treated groups compared with the control groups after being cultured for 14 days, which suggests that there was more abundant regenerated aggrecan and PAX1 deposition (Fig. 4).
ASA VI upregulated P-ERK1/2 and P-Smad2/3 expression To investigate the mechanism of action of ASA VI in promoting the differentiation of HMSCs into nucleus pulposus like-cells, we explored the effect of ASA VI on P-Smad2/3 and P-ERK1/2 expression using Western blotting. The results indicated that ASA VI (1 mg/l) could better upregulate both P-Smad2/3 and P-ERK1/2 protein levels than in the control group (Fig. 5 ).

Discussion
Due to the increasing aging of the population, the incidence of degenerative IVD is becoming higher and higher, and the treatment of degenerative IVD has received wide attention. Compared with traditional therapies, biotherapy may be more bene cial to relieve pain, repair degenerative nucleus pulposus and restore the biomechanical function of IVD [26]. Stem cells with high-e ciency self-renewal and pluripotency can be differentiated into various cell lines, including cartilage cell-like cells and nucleus pulposus-like cells. Therefore, inducing stem cells to differentiate into nucleus pulposus-like cells for IVDD has become the focus of biotherapy for IVDD. How to induce stem cells to differentiate into nucleus pulposus phenotype more effectively becomes a key problem in the treatment of IVDD [27]. Promoting the phenotypic differentiation of stem cells into nucleus pulposus cells is the basis of nucleus pulposus regeneration.
At present, there is no speci c cell phenotype to identify nucleus pulposus cells. By comparing the phenotypes of cartilage cells and nucleus pulposus cells, it was found that type II collagen, aggrecan and SOX9 expression was shared by nucleus pulposus cells and cartilage cells [28,29]. However, nucleus pulposus cells are signi cantly different from chondrocyte cells in terms of composition and biological function. Thus, to ensure the accumulation of proper ECM, it is necessary to identify the cell phenotype of differentiated cells. KRT19 as a speci c marker of human chordae was used to identify positive markers in nucleus pulposus cells [30]. Thorpe AA et al have found that KRT19 can be used as a unique gene for the identi cation of nucleus pulposus cells [31]. PAX1 is involved in the regulation of IVD formation in the embryonic stage and has been veri ed in human nucleus pulposus cells. Moreover, it is widely used as a new positive phenotype for the identi cation of nucleus pulposus cells in the study of stem cell differentiation into nucleus pulposus [32]. Thus, genes such as type II collagen, aggrecan, SOX9, KRT19 and PAX1 can be used as a genetic phenotype to identify nucleus pulposus cells.
As the main bioactive component of the Radix Dipsaci, previous studies have shown that ASA VI has the effects of protecting the nerves, heart, and liver and resisting osteoporosis [33]. In recent years, it has been reported that ASA VI has the effects of regulating intestinal micro ora, preventing atherosclerosis, resisting in ammation, reducing cortisol, and promoting angiogenesis and wound healing [20,[34][35][36][37]. In this study, we found that compared with the control group, ASA VI had more potential to promote differentiation of HMSCs into nucleus pulposus-like cells, especially when the concentration of ASA VI was 1 mg/L. To better understand the possible mechanism of ASA VI promoting differentiation of HMSCs into nucleus pulposus-like cells, we studied its effects on the ERK1/2 and Smad2/3 signaling pathways. Previous studies have found that the ERK and Smad signaling pathways are involved in the proliferation and differentiation of stem cells [38][39][40]. Moreover, in recent years, it has been found that the ERK1/2 and Smad2/3 signaling pathways regulate the differentiation of stem cells into nucleus pulposus-like cells and cartilage cell-like cells [41][42]. In this study, we found that ASA VI may promote the differentiation of HMSCs into nucleus pulposus cells by upregulation of P-ERK1/2 and P-smad2/3. However, the exact mechanism remains to be further veri ed.

Conclusions
We found that ASA VI promoted the differentiation of HMSCs into nucleus pulposus-like cells, probably by activating ERK1/2 and Smad2/3 signaling pathways. Our research increases our understanding of the potential mechanism of ASA VI promoting the differentiation of HMSCs into nucleus pulposus-like cells and suggests that ASA VI has therapeutic potential in the treatment of IVDD with stem cells.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was funded by the Foundation for leading talent in traditional Chinese medicine of Jiangsu province (2018SLJ0210) Authors' contributions YN conducted the study and drafted the manuscript. LX supervised the study and revised the manuscript. RD and XZ provided the technical support and advices for the study. all authors read and approved the nal manuscript.     The expression levels of aggrecan and PAX1 genes were veri ed by immuno-staining. The immuno uorescence signals were stronger (green staining) compared with the controls(scale bar: 200 µm).