Danggui Buxue Tang promotes the adhesion and migration of bone marrow stromal cells via the focal adhesion pathway in vitro

Author’s Accepted Manuscript Danggui Buxue Tang promotes the adhesion and migration of bone marrow stromal cells via the focal adhesion pathway in vitro Huaben Bo, Junhua He, Xiao Wang, Ruilan Du, Haikang Bei, Jun Chen, Jinquan Wang, Fenglin Wu, Wenfeng Zhang, Qizhu Chen www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(18)32057-9 https://doi.org/10.1016/j.jep.2018.11.018 JEP11599

To appear in: Journal of Ethnopharmacology Received date: 13 June 2018 Revised date: 31 October 2018 Accepted date: 9 November 2018 Cite this article as: Huaben Bo, Junhua He, Xiao Wang, Ruilan Du, Haikang Bei, Jun Chen, Jinquan Wang, Fenglin Wu, Wenfeng Zhang and Qizhu Chen, Danggui Buxue Tang promotes the adhesion and migration of bone marrow stromal cells via the focal adhesion pathway in vitro, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.11.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Danggui Buxue Tang promotes the adhesion and migration of bone marrow stromal cells via the focal adhesion pathway in vitro Huaben Boa*, Junhua Hea, Xiao Wanga, Ruilan Dua, Haikang Beia, Jun Chenb, Jinquan Wanga, Fenglin Wua, Wenfeng Zhanga , Qizhu Chena*

a

School of Bioscience and Biopharmaceutics, Guangdong Province Key Laboratory

for Biotechnology Drug Candidates, Guangdong Pharmaceutical University, 510006 Guangzhou, Guangdong, China b

College of Pharmacy, Guangdong Pharmaceutical University, 510006 Guangzhou,

Guangdong, China *

Corresponding author: School of Bioscience and Biopharmaceutics, Guangdong

Province Key Laboratory for Biotechnology Drug Candidates, Guangdong Pharmaceutical

University,

510006

Guangzhou,

Guangdong,

China.

Tel:

86-20-39352199. Fax: 86-20-39352201. E-mail: [email protected]

Abstract

Ethnopharmacology relevance: Danggui Buxue Tang has been used in China to treat clinical anemia for more than 800 years. However, there is no scientific report on its effect on bone marrow stromal cells.

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Aim of the study: Here, we aimed to explore the effect of Danggui Buxue Tang on bone marrow stromal cell adhesion and migration. Materials and Methods: Bone marrow stromal cells were used as a model to evaluate the effect of Danggui Buxue Tang on the adhesion and migration of bone marrow stromal cells. RNA-sequencing, quantitative polymerase chain reaction, and western blotting were used to detect and confirm the expression of genes related to the focal adhesion pathway before and after drug delivery. Results: Danggui Buxue Tang significantly increased the number of bone marrow stromal cells. After 12 days of 16 mg/mL Danggui Buxue Tang treatment, bone marrow stromal cells were significantly increased (by 0.527 ± 0.008 fold; p < 0.001) as compared to the control group (0.180 ± 0.019). The effect was not due to enhanced cell proliferation, as there was no difference in the cell cycle (p > 0.05). The adhesion area of a single cell was doubled by Danggui Buxue Tang treatment (p < 0.001), and the time required for cell adhesion to a Petri dish was shortened. Thus, Danggui Buxue Tang increases the number of bone marrow stromal cells by promoting adhesion. Danggui Buxue Tang also significantly promoted bone marrow stromal cell migration (p < 0.001). Transcript analysis revealed that the focal adhesion and PI3K-Akt signaling pathways were activated. Expression analysis confirmed that the gene and

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protein expression of focal adhesion-related factors were upregulated. Conclusion: Danggui Buxue Tangaffects bone marrow stromal cell adhesion and migration by enhancing the focal adhesion pathway in vitro, and bone marrow stromal cells are a target of DBT-regulated hematopoiesis, and the active ingredients of DBT involved in the effects require further investigation. Graphical Abstract:

DBT can induce gene expression of Intergrin-α. The Intergrin-α can result in the phosphorylation of FAK, and sequential of PAXILLIN. PAXILLIN regulates the activity of VINCULIN. VINCULIN regulates focal adhesions assembly. The assembly of focal adhesions can cause the recruitment of F-ACTIN and direct the cytoskeletal organization. The cytoskeletal eventually lead to BMSCs adhesion and migration

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List of abbreviations DBT, Danggui Buxue Tang; BMSCs, Bone marrow stromal cells; HPLC, High-performance liquid chromatography; ECM, Extracellular matrix; TCM, Traditional Chinese medicine; IMDM, Iscove’s modified Dulbecco’s medium; DEGs, Differentially expressed genes; qPCR, Quantitative polymerase chain reaction Keywords: Danggui Buxue Tang; adhesion; migration; focal adhesion pathway

1.

Introduction

Hematopoietic dysfunction can have various clinical causes, such as bone marrow injury during bone marrow transplantation, chemotherapy, toxic side effects of certain drugs, and various autoimmune diseases (Lee and Kim, 2014; Reagan and Rosen, 2016). Hematopoietic dysfunction usually induces severe bone marrow inhibition and hematopoietic insufficiency and further leads to decreased immunity, thus forming a vicious cycle. There are various approaches to treating hematopoietic disorders, but they all have drawbacks. Intravenous iron supplementation for iron deficiency anemia can cause nausea and allergic reactions (Macdougall et al., 2016). Blood transfusion for the treatment of erythrocyte reduction can cause viral and bacterial infections (Farzad et al., 2016; Kurihara et al., 2016). Erythropoietin injection, which is used for the treatment of hematopoietic insufficiency, is associated with a risk of autoimmune erythropoietin-neutralizing antibody production and

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hypertension (Alves et al., 2014; Badve et al., 2013). Therefore, it is necessary to develop new drugs for the treatment of hematopoietic dysfunction. Hematopoiesis is a complex biological process that generally occurs in the bone marrow microenvironment. Bone marrow stromal cells (BMSCs) are an important part of the hematopoietic microenvironment (Yu and Scadden, 2016). BMSCs regulate hematopoiesis by secreting hematopoietic growth factor for hematopoietic cells, by providing a stable environment for hematopoietic cells, and by conveying hematopoietic cell information through cell-cell contact (Gordon et al., 1987; Long et al., 1992; Roberts et al., 1988). Thus, BMSCs may be a potential target cell of drug-regulated hematopoiesis. Traditional Chinese medicines have the advantages of being multi-component medicines, having active-target diversity and limited side effects. The traditional Chinese medicine used in this study, Danggui Buxue Tang (DBT), has been used to treat clinical anemia for more than 800 years in China (Lin, H.Q. et al., 2017). It is widely prescribed as a dietary supplement for menopausal women in China. A vast body of literature shows that DBT can promote blood production and improve angiogenesis (Gao et al., 2006; Zheng et al., 2010; Zhou et al., 2017). DBT remarkably increases the quantity of red and white blood cells, reticulocytes, and bone marrow nucleated cells. DBT can also affect hematopoietic function through immune-mediated aplastic anemia, bone tissue regeneration, and induction of ECM secretion (Ke et al., 2012; Wang et al., 2015; Yang et al., 2014). However, the effect of

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DBT treatment on BMSCs has not been elucidated. Therefore, we studied the effect of DBT on the adhesion and migration of BMSCs in vitro and elucidated whether BMSCs are a potential target cell of DBT.

2.

Materials and Methods

2.1. Plant materials and DBT preparation

Astragali radix (roots of Astragalus membranaceus (Fisch.) Bunge or Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao, Huangqi in Chinese) and Angelica Sinensis Radix (roots of Angelica sinensis (Oliv.) Diels, Danggui in Chinese), were purchased from Guangzhou Zhixin Herbal Medicine (Guangdong, China). Plant materials were authenticated morphologically in our lab by Dr. Chen. A voucher specimen (TCM-HQ-2016040513, TCM-DG-2016040711) has been deposited in the School of Bioscience and Biopharmaceutics, Guandong Pharmaceutical University, Guangzhou, China. DBT was prepared using the method described previously (Wang et al., 2014). Crude drugs were extracted under strict quality control. Briefly, RA and RAS were mixed at a ratio of 5:1 and boiled in 8 volumes of water for 2 h. The residue was re-extracted under the same conditions. The aqueous extracts were combined, filtered, and condensed to 1 g/mL (w/v, crude drugs/water) in a rotavapor, and stored at −80°C.

2.2. Quality control of DBT by high-performance liquid chromatography (HPLC)

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DBT was subjected to quality control as reported previously (Yang et al., 2009), with minor modifications. Standards including ferulic acid (95.6%) and formononetin (99%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ferulic acid and formononetin were dissolved in MeOH. Ethanol was added to the extract at a final concentration of 70%. The mixtures were centrifuged (12,000 ×g, 10 min). The supernatant was dried in a rotavapor. The residues were dissolved in 1 mL of methanol and filtered through a Pall 25-mm syringe filter (0.2 μm) for HPLC analysis. HPLC was performed on a 100 × 4.6 mm, 2.7 μm, EC C-18 column using an HPLC system consisting of a controller (Waters 2420) and photodiode array detector (Waters 2996). Solvent A (methanol: water, 5:95) and Solvent B (methanol: water, 95:5) were used as mobile phases at a flow rate of 0.5 mL/min at room temperature. The gradient profile was 0% B isocratic for 10 min, 22% B linear for 15 min, 45% B isocratic for 20 min, 65% B linear for 15 min, and 100% B isocratic for 10 min. The signals were detected by the photodiode array detector.

2.3. Preparation of bone marrow single-cell suspension

KM mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangdong, China). All animals were kept under standard lighting conditions (12-h alternate day and night cycles) and were given free access to food and water. The animals were treated according to the Guidelines of Animal Care and Use

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Committee of Guangdong Pharmaceutical University, and the study was approved by the local Animal Research Welfare Committee. BMSCs were isolated and cultured according to reported methods (Soleimani and Nadri, 2009). Briefly, an 8-week-old male mouse was sacrificed by cervical dislocation. The tibias and femurs were collected in a sterile environment and the ends of the long bones were trimmed to expose the interior marrow shaft. Bone marrow cells were rinsed with Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS. To make a single-cell suspension, the cells were gently drawn up and down with a 3-cc syringe with 21-g needle. Live bone marrow mononuclear cells were counted with a Luna Fluorescence Cell Counter (Logos Biosystems, South Korea) after acridine orange/propidium iodide fluorescent staining.

2.4. Effect of DBT on BMSC viability

DBT was diluted in complete medium to 32 mg/mL, 16 mg/mL 8 mg/mL, 2 mg/mL, and 0.5 mg/mL. Bone marrow single cells were seeded in a 96-well plate (Corning, NY, USA) at a density of 4 × 105 cells/well in IMDM supplemented with 10% FBS and the various concentrations of DBT and incubated at 37°C in a humidified 5% CO2 incubator. After incubation for 3 days, cell viability was determined by using MTT according to the manufacturer’s instruction. Briefly, 20 μL MTT (5 mg/mL) was added to each well and the cells were incubated for another 4 h at 37°C. The supernatant was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to

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each well. The absorbance at a wavelength of 490 nm was measured with Varioskan Flash Multifunction reading instrument (Thermo Scientific, USA). All experiments were performed in triplicate.

2.5. Detection of cell cycle by flow cytometry

Bone marrow single cells were seeded in a 6-well plate (Corning) at a density of 1.2 × 107 cells/well in IMDM supplemented with 10% FBS and 2 mg/mL DBT and incubated at 37°C in a humidified 5% CO2 incubator. After incubation for 3 days and 6 days, flow cytometry analysis was carried out as described previously (Jafarnejad et al., 2008). Briefly, cells were fixed overnight in 70% ethanol, rinsed with PBS, and centrifuged to remove the ethanol thoroughly. The cell pellet was suspended in 1 mL of propidium iodide (PI)/Triton X-100 staining solution with RNase A and incubated for 30 min at room temperature. Then, the cells were analyzed in a Beckman Gallios flow cytometer.

2.6. Cell spreading assay

Bone marrow single cells were seeded in a 96-well plate (Corning) at 4 × 105 cells/well in IMDM supplemented with 10% FBS and various concentrations of DBT (16 mg/mL, 8 mg/mL, 2 mg/mL, 0.5 mg/mL) and incubated at 37°C in a humidified 5% CO2 incubator. After incubation for 48 h and 72 h, the cells were washed three times with pre-warmed PBS to remove non-adherent cells. Micrographs of the adherent

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cells were captured using an inverted microscope (Olympus IX51). Cell spreading was quantified by measuring the area of each cell using ImageJ software (National Institutes of Health, USA) (Nabiuni et al., 2012). To acquire more quantitative and real-time information on cell adhesion and spreading, we employed changes in impedance as a measure of the degree of cell attachment and spreading using the xCELLigence RTCA Control Unit (Obr et al., 2013). Bone marrow single cells were seeded in a CIM-plate xCELLigence assay plate at 4 × 105 cells/well in IMDM supplemented with 10% FBS and various concentrations of DBT (16 mg/mL, 2 mg/mL, 0.5 mg/mL), and placed on the Real-time xCELLigence Cell Analyzer (RTCA DP) platform at 37°C to measure the Cell Index every 15 min. The slope of Cell Index variation, calculated by the RTCA software, represented the efficiency of cell binding to the wells.

2.7. Immunofluorescence staining of the cell skeleton and focal adhesion protein

Immunofluorescence analysis was carried out as described previously (Rauh et al., 2015). After 48-h culture, BMSCs were washed with PBS, fixed with 4% paraformaldehyde solution for 10 min, and washed with PBS twice. The cells were permeabilized with 0.2% Triton X-100 in PBS for 30 min. After washing in PBS, the cells were blocked with 1% BSA for 30 min. Then, the cells were stained with 5 µg/mL fluorescent phalloidin conjugate solution in PBS (containing 1% BSA) for 20 min at 37°C. After washing in PBS, the cells were incubated with DAPI

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(Sigma-Aldrich, Castle Hill, NSW, and Australia) for 5 min to stain the nuclei. For VINCULIN staining, after 24-h culture, BMSCs were washed with PBS, fixed with 4% paraformaldehyde solution for 10 min, and washed with PBS twice. The cells were then incubated with mouse anti-VINCULIN monoclonal antibody (Proteintech, 1:250) at room temperature for 2 h followed by washing with PBS (containing 1% BSA) three times. Finally, the samples were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Proteintech, 1:250) at room temperature for 1 h. Images were captured with a confocal laser scanning microscope (FV1000).

2.8. Cell migration assay

Cell migration was assayed as described previously (Gao et al., 2016). Bone marrow single cells were seeded in a 6-well plate at 1.2 × 107 cells/well in IMDM supplemented with 10% FBS and grown to 80–90% confluency. The monolayer of cells was scraped using a sterile micropipette tip and washed with PBS. Then, the medium was replaced with low-serum IMDM (2% FBS, 0.1% BSA) with different concentrations of DBT (0.5 mg/mL, 2 mg/mL, 16 mg/mL). The migration capability was monitored and photographed after 24 h and 48 h of incubation using an inverted microscope (Olympus IX51). The migration rate of BMSCs was calculated using ImageJ software. The proliferation of BMSCs in low-serum IMDM was examined by MTT assay.

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2.9. RNA-sequencing-based transcriptome analysis

Bone marrow single cells were seeded in a 6-well plate (Corning) at a density of 1.2 × 107 cells/well in IMDM supplemented with 10% FBS and 2 mg/mL DBT, and were incubated at 37°C in a humidified 5% CO2 incubator. After 3 days and 6 days of culture, total RNA was extracted using TRIzol reagent. Total RNA from each sample was used for Illumina sequencing at Biomarker Technologies (Beijing, China). Fold change ≥2 and false discovery rate <0.01 were used as criteria for the identification of differentially expressed genes (DEGs). DEGs were classified based on KEGG pathway analysis. The most relevant pathways were sorted according to false discovery rate and p values.

2.10. Reverse-transcription qPCR analysis

After 6 days of culture, total RNA was extracted using TRIzol reagent (Bateman et al., 2017) and was used as template for first-strand cDNA synthesis. qPCR was conducted on a LightCycler 480 II (Roche) using RealStar Green Power Mixture (GenStar) according to the manufacturers’ instructions. qPCR primers (Supplemental Table S1) were designed using Primer 5.0 (Boston, USA). Thermal cycles consisted of denaturation at 95°C for 3 min, followed by 50 cycles of 10 s at 95°C, 15 s at 55°C, and 30 s at 72°C. Fluorescence was quantified at the end of the 55°C annealing step and product identity was confirmed by melting curve analysis. The relative expression level was calculated using the following the equation: relative gene expression

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 2(treated(GENE GAPDH)control(GENE GAPDH)) . Mouse GAPDH was used as an internal control to normalize mRNA content. Samples were tested in triplicate.

2.11. Western blotting

After 6 days of culture, treated cells were washed twice with ice-cold PBS and collected in lysis buffer. Protein concentrations were determined using a bicinchoninic acid assay kit (Beyotime Biotechnology, Wuhan, China). Equal amounts of proteins (45 µg) were separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Specific antibodies for were used to detect the levels of F-actin (66009-1-Ig), FAK (AF6397), phospho-FAK (Tyr397), paxillin (AF6331), and phospho-paxillin (Tyr118) respectively.

Immunoreactive

proteins

were

visualized

using

general

chemiluminescence detection reagent (Beyotime Biotechnology, Wuhan, China), and GADPH was used as an internal control.

2.12. Statistical analysis

Statistical charts were drawn with the statistical software GraphPad Prism 5.0. Data were analyzed with statistical software SPSS 18.0 (Chicago, USA). All data were first tested for normality and homogeneity of variances. All values were expressed as the mean ± standard deviation (SD). Two groups were compared using a t-test, multiple groups were compared using one-way ANOVA. In case of multiple comparisons, least significant difference (LSD) analysis was applied. A value of p <

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0.05 was considered significant.

3.

Results

3.1. Quality control of DBT by HPLC

Representative liquid chromatograms are shown in Figure 1. The retention times of ferulic acid and fermononetin standards were 24.5 min and 59.5 min, respectively (Figure 1B and D). The maximal absorption peaks of ferulic acid and fermononetin were 323 nm and 249 nm, respectively (Figure 1A and C). The peaks of ferulic acid and fermononetin in DBT were determined by the retention time and the ultraviolet absorption spectrum. DBT peaks appeared in the corresponding positions and ultraviolet spectra were similar to those of the standards (Figure 1E, inset). The chromatographic fingerprints were further analyzed with the similarity evaluation system for chromatographic fingerprints of TCM (2012 edition). Compared with previous laboratory-constructed fingerprint maps, the similarity reached 97.4 % (Supplemental Figure S1, Table S2).

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Figure 1 DBT quality control by HPLC. (A) UV spectra for ferulic acid, (B) retention time of ferulic acid, (C) UV spectra for fermononetin, (D) retention time of fermononetin, (E) peak diagram of DBT at 249 nm.

3.2. Effects of DBT on BMSC viability in vitro

At concentrations between 0.5 mg/mL and 16 mg/mL, the number of BMSCs increased with increasing drug concentration, showing a significant dose–effect relationship (Figure 2A). When the drug concentration reached 16 mg/mL, the effect was the strongest. After 12 days of 16 mg/mL DBT treatment, BMSCs had increased significantly (by 0.527 ± 0.008 p < 0.001) as compared to the control group (0.180

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± 0.019). Interestingly, the morphology of BMSCs showed a conspicuous change in the DBT-treated group. With the increase in DBT concentration, BMSCs became larger, and the cell contour became more obvious (Figure 2B). In the meantime, the number of BMSCs in the control group and the groups treated with 0.5, 2, 8, and 16 mg/mL DBT increased with the prolongation of culture, showing an obvious time– effect relationship. However, when the concentration of DBT reached 32 mg/mL, the number of cells decreased obviously. These findings indicated that at low concentrations, DBT positively affects BMSC viability, whereas high concentrations of DBT may produce drug toxicity, possibly via changes in pH or osmotic pressure.

Figure 2 Effect of DBT on BMSC viability in vitro. (A) Cell viability of BMSCs under the indicated concentrations of DBT was assessed from day 4 to day 12. (B) The cell morphology of BMSCs under the indicated concentrations of DBT was observed over 12 days by light microscopy (magnification 100×).

3.3. Effect of DBT on the cell cycle in BMSCs

To determine whether DBT increases the number of BMSCs by promoting their proliferation, the cell cycle was examined in suspended cells and adherent cells

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treated with 2 mg/mL DBT for 3 days and 6 days in vitro. There was no significant difference in the number of cells in G0/G1 phase after treatment for suspended (p = 0.101) and adherent cells (p = 0.521) at day 3 (Figure 3A). However, after 6 days of 2 mg/mL DBT treatment, the fraction of adherent BMSCs in G0/G1 phase (p = 0.005) had increased significantly as compared to the control group (Figure 3B). The higher the proportion of cells in G0/G1 phase, the lower the rate of cell proliferation. The results indicated that the increased cell number was not due to normal cell proliferation.

Figure 3 Cell cycle in suspended cells and adherent cells treated with 2 mg/mL DBT for 3 days (A) or 6 days (B). Values are the mean ± SD. (**p < 0.01 compared to the control group, t-test).

3.4. Effect of DBT on the adhesion of BMSCs

To evaluate whether DBT increased the number of BMSCs through adhesion, we counted the average area of single BMSCs after culture in the presence of DBT for 48 h. With increasing DBT concentration, the number of adherent cells increased, and the

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cells became larger (Figure 4A). However, at a DBT concentration of 16 mg/ml, BMSCs were larger but less numerous. The single-cell area of the control group (1.000 ± 0.139) was significantly lower than that of DBT group, including 0.5 mg/mL, 2 mg/mL, 8 mg/mL, and 16 mg/mL (1.364 ± 0.104, 1.842 ± 0.124, 2.178 ±

0.167, 2.598 ±

0.720, respectively) (Figure 4B), consistent with the

morphological alterations. To quantitatively monitor the effect of DBT on the adherence ability of BMSCs in real time, we employed the xCELLigence RTCA DP analyzer. This system provides a cell index that increases as a function of cell adhesion. As shown in Figure. 4C, the cell index increased within 100 min and further increased under DBT treatment, whereas it stabilized after 100 min in non-treated control cells. When the DBT concentration reached 16 mg/mL, the cell index dropped after 200 min. Collectively, these results indicated that low concentrations of DBT positively promote BMSC adhesion.

Figure 4 Effect of DBT on the adhesion of BMSCs. (A) After DBT treatment for 48 h

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and 72 h, cell morphology of BMSCs was observed under a light microscope (magnification, 200×). (B) Single-cell areas in BMSCs treated or not with DBT for 48 h. Single cell area = total cell area/total cell count. Values are the mean ± SD. (*p < 0.05; **p < 0.01, ***p < 0.001 compared with the control group, LSD). (C) Adhesion of BMSCs in response to DBT as measured in a CIM-plate 16 xCELLigence RTCA DP instrument.

3.5. Effect of DBT on cell skeleton and focal adhesion

We observed the cell skeleton (f-actin) after 48-h DBT treatment and focal adhesion protein (vinculin) after 24-h treatment of BMSCs by confocal laser microscopy after fluorescent labeling of the target proteins. The results showed a significant difference in cytoskeletal filament between the control and DBT-treated cells at 48 h. The cytoskeletal filament of DBT-treated cells stretched longer, and pseudopodia extended outside the cells, suggesting the cells might be capable of migrating (Figure 5A, right panels). There were no obvious pseudopodia in the control group. Meanwhile, the expression of the vinculin was significantly increased after 24-h DBT treatment (Figure 5A, left panels). To explain this phenomenon, migration was assessed to verify the cellular motility. The results showed that the migration ability of the control group (5.937 ± 0.469) was significantly lower than that of DBT group, including 2 mg/mL, 8 mg/mL and 16 mg/mL (12.523 ± 0.942, 23.453 ± 0.821, 9.427 ± 0.574, respectively)

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after 48 h (Figure 5B). However, when the concentration of DBT reached 16 mg/mL, this effect was inhibited (Figure 5C). These results suggested that DBT affects the BMSCs cytoskeleton, prompting BMSC adhesion and migration.

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Figure 5 Effect of DBT on the cell skeleton and cell migration. (A) Immunofluorescence staining of vinculin in BMSCs treated with 2 mg/mL DBT for 24 h and of actin (cell skeleton) in BMSCs treated with 2 mg/mL DBT for 48 h. (B) The migration of BMSCs was photographed at 0 h, 24 h, and 48 h after treatment with DBT at the indicated concentrations. (magnification, 200×) (C) The migration rate of BMSCs was quantified using ImageJ software. Values are the mean ± SD. (*p < 0.05; **p < 0.01, ***p < 0.001 compared with the control group, LSD).

3.6. Transcriptome analysis of BMSCs treated with DBT

The numbers of genes up- or downregulated in BMSCs after treatment for 3 days and 6 days in comparison with the control group are shown in Figure 6A. In total, there were 128 DEGs between the DBT and control groups after 3 days. There were 911 DEGs between the DBT and control groups after 6 days. Forty-eight DEGs were in common between the 3 days versus 6 days groups (Figure 6A). Forty-eight Unigenes were significantly differentially expressed between the DBT and control groups (Figure 6B). Functional annotation was adopted to study the functions of the DEGs. KEGG pathway analysis showed that 531 DEGs were enriched mainly in 20 pathways (Supplemental Table S3). The PI3K-Akt signaling and focal adhesion pathways were the two most enriched pathways. Forty-seven DEGs were significantly enriched in the focal adhesion pathway. Fifty-one DEGs were significantly enriched in PI3K-Akt

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signaling pathway (Figure 6C). The expression of genes related to focal adhesion was the most obvious (Figure 6D). Further analysis indicated that the 47 DEGs enriched in the focal adhesion pathway included Itga11, Itga1, Itga2b, Itga3, Pik3r3, Tln2, Rtk, Parva, paxillin, Fak, and vinculin.

Figure 6 Transcriptome analysis of BMSCs treated with 2 mg/mL DBT. (A) Venn diagram showing the DEGs between the DBT and control groups. DEGs that are common to multiple time points are shown by the overlap. (B) Heatmap analysis of 48 common DEGs. The gene expression level increased with color from green to red. (C) Annotation results of DEGs in the treatment groups classified according to KEGG pathways. (D) Signal pathways significantly enriched in DEGs on the sixth day of 2

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mg/mL DBT treatment.

3.7. Validation of gene expression by qPCR

To confirm the differential expression values obtained from the statistical comparison of sequencing data, qPCR was performed to validate genes involved in the focal adhesion pathway (Figure 7A). In total, 7 DEGs were selected, including Rtk, integrin-α, Fak, Pi3k, paxillin, vinculin, and actin. The results showed highly significant and positive correlations between the qPCR and transcriptome data. The relative expression of integrin-α (3.656 ± 0.950), Pi3k (3.908 ± 1.530), paxillin (1.950 ± 0.493) was significantly enhanced as compared to their expression in non-treated control cells (Figure 7B). The relative expression of Fak (3.203 ± 1.345) and vinculin (3.491 ± 1.510) was enhanced than control groups (p = 0.073, p = 0.051 respectively).

Figure 7 qPCR verification of RNA-sequencing-derived expression levels of the

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indicated target genes after 6 days of 2 mg/mL DBT treatment. Values are the mean ± SD. (*p < 0.05; **p < 0.01, compared to control group, t-test).

3.8. Focal adhesion pathway-related protein detection

Focal adhesion-related proteins including F-actin, FAK, phospho-FAK (Tyr397), paxillin, and phospho-paxillin (Tyr118) were detected by western blotting (Figure 8A, C). After 6 days of 4 mg/mL DBT treatment, the protein level of FAK (0.819 ± 0.042) and FAK phosphorylation (0.752 ± 0.070) was significant increased as compared to the control group (p < 0.05). There was no significant difference in the protein level of paxillin (0.810 ± 0.254) and paxillin phosphorylation (1.242 ± 0.233) (p = 0.0934). However, paxillin protein phosphorylation increased with increasing dose. Similarly, protein level of F-actin increased with increasing dose.

Figure 8 Western blot analysis of key proteins in the focal adhesion pathway after

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DBT treatment for 6 days. (A, C) Representatives blot from 3 independent experiments. (B, D) Densitometric quantification. GAPDH was used as an internal control. Values are the mean ± SEM, where n = 3. (*p < 0.05 as compared to the control, LSD).

Figure 9 Signaling routes in the key regulatory mechanisms of DBT promoting BMSC migration. Solid arrows represent direct activation, dotted arrows represent indirect activation, “+p” represents phosphorylation. Yellow arrows represent the direction of signal conduction predicted by experiments. Green background white

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fonts are key genes tested by qPCR. Abbreviations: FAK, focal adhesion kinase; RTK, epidermal growth factor receptor, PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase.

4.

Discussion

Quality control of DBT is a precondition for cell experimental stability and reliability. However, the quality of Chinese herbal medicines is difficult to control because they characteristically have multiple effective components and multiple targets. Ferulic acid and fermononetin are the main components of Astragali Radix and Radix Angelicae Sinensis and can promote cell adhesion and bone marrow hematopoietic function (Bouzaiene et al., 2015; de Andrade et al., 2012; Ma et al., 2011; Yu et al., 2013). Therefore, ferulic acid and fermononetin were used as standards in this study. Further, taking into account the characteristics of multiple components, fingerprinting was used for quality control. For centuries, DBT has been used as a medicine or nutritional supplement with very good effects in the treatment of women with menstruation problems and “QI” deficiency. Various reports have confirmed that DBT enhances the cloning ability of stem cells and significantly increases the proportions of stem and progenitor cells (Li et al., 2017; Lin, P.L. et al., 2017; Yang et al., 2009). Our previous studies have shown that DBT promotes the formation of erythrocytes and BMSC growth in vivo (He et al., 2017; Yang et al., 2009). Based on these previous studies, BMSCs may play an

27

important role in the promotion of erythrocyte production by DBT. Therefore, we further explored the effects of DBT on the proliferation, adhesion, and migration of BMSCs in vitro. In vitro, the number of BMSCs increased with increasing drug concentration, showing a significant dose–effect relationship. However, there were no significant differences in the numbers of cells in G0/G1 phase at 3 days after treatment for suspended and adherent cells. The higher the proportion of cells in G0/G1 phase, the lower the rate of cell proliferation. Thus, these results indicated that the increased cell number was not due to normal cell proliferation. We found that BMSCs became larger and their contours became more obvious with increasing DBT concentration. DBT significantly accelerated cell adhesion and increased the cell adhesive area. Therefore, we consider that DBT increases BMSC numbers by promoting their adhesion. Vinculin is a scaffolding protein that plays key roles in regulating focal adhesion assembly and disassembly. The assembly of focal adhesions can cause the recruitment of F-actin and direct cytoskeletal organization (Kuo, 2013). Thus, we observed the cell skeleton and vinculin involvement by staining assays. Vinculin expression was significantly increased after 24-h DBT treatment. The cytoskeletal filament of DBT-treated cells stretched longer, and pseudopodia extended outside of the cells. There were no obvious pseudopodia in the control group. This corroborated that DBT can promote the adhesion of BMSCs. The extension of cell pseudopodia often affects

28

the migration abilities of cells (Allena, 2013). Indeed, DBT significantly promoted the migration ability of BMSCs. Thus, BMSCs are a target of DBT-regulated hematopoiesis. The active ingredient(s) of DBT should be investigated in future studies using bone marrow stromal cells as a cell model. Many proteins are involved in cytoskeletal rearrangement and cell migration. By using RNA-sequencing, we identified candidate genes and pathways involved in the effect of DBT on BMSC adhesion and migration. The results showed that the focal adhesion signaling pathway was significantly affected by DBT treatment. Focal adhesion involves a classic cell-signaling pathway connecting the cytoskeleton with the ECM. Integrins exist on the cell membrane surface and transmit ECM signals to intracellular protein complexes that further regulate tension for cell motility. In this study, integrin-α expression was significantly enhanced by DBT treatment. FAK is a widely expressed cytoplasmic protein tyrosine kinase that is recruited by the integrin family to phosphorylate paxillin and PI3K. FAK protein and FAK phosphorylation were significantly increased by DBT treatment. Paxillin is a key component of integrin signaling and tyrosine-118 phosphorylation of paxillin is required for integrin-mediated cytoskeletal reorganization. There were no significant differences in the protein level and phosphorylation of paxillin. However, the phosphorylation of paxillin protein increased with increasing DBT dose.

29

Conclusions

DBT can promote BMSC adhesion and migration by via the focal adhesion pathway. Thus, BMSCs are a potential target of DBT. The active ingredients of DBT involved in the effects require further investigation. DBT

can

induce

gene

expression

of

integrin-α.

Integrin-α

induces

phosphorylation of FAK, and consequently, phosphorylation of paxillin. Paxillin regulates the activity of vinculin, which regulates the assembly of focal adhesions, which in turn can cause the recruitment of F-actin and direct cytoskeletal organization. The cytoskeletal re-organization eventually leads to BMSC adhesion and migration (Figure 9).

Acknowledgements

This work was supported by National Natural Science Foundation of China (Grant No. 81403317);

the

Guangzhou

Science

and

Technology

Project

(grant

no.

201707010441); the Guangzhou Science and Technology Project (grant no. 201607010087); the Natural Science Foundation of Guangdong Province (grant no. 2015A030310310); and the Science and Technology Planning Project of Guangdong Province (grant no. 2014A020212462)

Conflict of interest

The authors declare that they have no competing financial interests.

30

Authors’ contributions

HBB designed experiments, supervised the experiments, and revised and finalized the manuscript. JH, XW, and RD performed the experiments, analyzed the data, and prepared the figures and the manuscript. HKB performed the cell culture. JC, JW, and FW performed the quality control of DBT. WZ and QC contributed to the results discussion and paper writing. All authors reviewed and approved the final paper.

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