Taxifolin Protects Dental Pulp Stem Cells under Hypoxia and Inflammation Conditions

Cell Culture

DPSCs from human dental pulp tissue was purchased from Lonza (cat. no. PT-5025. Basel, Switzerland)) and identified by DPSC’s biomarkers. Cryopreserved human DPSCs were recovered from liquid nitrogen using an AccuVital cell recovery kit (AccuRef Scientific, Goshen, NY, USA) and cultured as previously reported. Single-cell suspensions (0.01 to 1 × 10⁵/well) of dental pulp were seeded into 6-well plates (Costar, USA) with alpha modification of Eagle’s medium (Gibco, BRL, USA) supplemented with 20% FBS, 1% Pen-Strept, 10 nM dexamethasone, 50 μg/ml L-ascorbic acid and 10 mM β-glycerophosphate,10 nM calcitriol (Sigma, USA) for 14 d to induce osteogenic differentiation and then incubated at 37°C in 5% CO₂. For the induction of adipogenesis, alpha-minimum essential medium (MEM) supplemented with 10% FBS, 10% horse serum, 1% Pen-Strept, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine, 3 μg/ml insulin (all purchased from Sigma, USA), and 1 μM rosiglitazone (BRL49653; Novo Nordisk, Denmark) was added to culture DPSCs for 14 d. For hypoxia treatment, DPSCs were cultured under 5% O₂ in a hypoxia chamber (STEMCELL Technologies, Vancouver, USA) for 24 h at 37°C. For TNF-α treatment, cells were treated with 20 ng/ml TNF-α for 24 h before collection. The addition of Taxifolin is associated with hypoxia and TNF-α treatment.

Flow Cytometry

Cells were dissociated into single cells and then fixed with 1% (vol/vol) paraformaldehyde for 20 min at room temperature and stained with primary and secondary antibodies in PBS plus 0.1% (vol/vol) Triton X-100 and 0.5% (wt/vol) bovine serum albumin (Sangon, Shanghai, China). Flow cytometry sorting was performed to determine the expression levels of the cell surface markers CD44, CD146, and CD105. DPSCs were harvested from T75 flasks, washed and aliquoted equally into tubes. Cells were first incubated with a blocking solution containing 10% FBS at room temperature for 10 min followed by washing. Cells were then incubated with the specific antibodies conjugated with fluorochromes (Biolegends, San Diego, CA) at 4°C. After washing these cells, data were collected on a FACSCaliber flow cytometer (Beckton Dickinson, Sunnyvale, USA) and analyzed using FlowJo (Version 10.5.4, FlowJo, LLC).

DPSCs Characterization and Differentiation

Adipogenic and Osteogenic differentiation were induced as recommended by the manufacturer (R&D systems). DPSCs were confirmed using flow cytometry sorting and measuring gene expression of DPSCs markers, such as CD44, CD105, and CD146. And cells were grown in 10% FBS containing alpha minimum essential medium (α-MEM). The differentiation medium of adipocytes was changed every 3–4 days. The differentiation medium of osteoblasts was changed twice per week. DPSCs were differentiated into chondrocytes, osteoblasts and adipocytes in vitro following the protocol of differentiation. Osteoblasts and adipocytes should be observed at around 2 weeks after differentiation.

Alizarin Red S (ARS) Staining

DPSCs were seeded in 6-wells plates (1 × 10⁵ cells/well) and cultured in osteogenic medium. After fixation with 4% PFA, the calcium deposits in the mineralization nodules formed were stained by ARS solution (Solarbio, China). To quantify ARS staining, the mineral stained was solubilized in 5% SDS in 0.5 ml 0.5 N HCl for 30 min, and the absorbance were further measured at 405 nm using a microplate reader.

Oil Red O Staining

DPSCs were seeded in 12-wells plates (1 × 10⁵ cells/well) and cultured in adipose medium for 14 d to induce adipose differentiation. After fixation with 4% PFA, the samples were stained with Oil Red O. The staining procedure required ∼18 min for Oil Red O. To perform the Oil Red O staining, the slices were immersed in ready-to-use formalin for 1 minute, washed in running tap water, covered with the working solution for 12 min, washed in source water for 1 minute, covered with Mayer’s Hematoxylin for 1 minute, washed a second time in running tap water for 3 min, and mounted with watery media. A working solution composed of 8 mL of Oil Red O diluted with 5 mL of deionized water was prepared and left to rest for 10 min (Diapath, Bergamo, Italy). Lipids on adipose differentiation cells were highlighted in flesh red.

Cell Counting Kit-8 (CCK-8) Assay

Cells were seeded in 96-well flat-bottom microplates (Costar; Painted Post, NY, USA) with a density of 2 × 10⁴ cells/well. After overnight incubation, the medium was replaced by DMEM containing 10% FBS. The cells were incubated for additional 24 hours, 48 hours, 72 hours, separately. At each time point, 100 µl CCK-8 reagent (EXINNO, Goshen, NY, USA) was added into each well. Cells were incubated at 37°C subsequently for 2 hours. Against a background control, the sample absorbance was tested at 450 nm via a microplate reader (Bio-Rad Laboratories, Inc.; Hercules, CA, USA).

Apoptosis Assay

DPSCs seeded in 6-well plates were cultured in vitro under conditions of hypoxia (5% O₂, 24 h) and/or inflammation TNF-α (20 ng/ml, 24 h) after 24 hours. Then cells were collected after digestion with 0.25% typsin-EDTA solution (Cell biologics; Beijing, China), and subjected to apoptosis analysis using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Kit (BD Biosciences; San Jose, CA, USA) following the manufacturer’s instructions. Briefly, after washing with PBS (phosphate-buffered saline) and the binding buffer for one time each, cells were stained with Annexin V-FITC/PI for 20 min at room temperature in a dark. After washing with the binding buffer once, the labeled cells were detected immediately by a flow cytometer (CytoFLEX; Beckman Coulter, Brea, CA, USA). Flow cytometry data were analyzed using expo32 software (Beckman Coulter).

Real-Time Quantitative PCR

Total RNA and miRNA were extracted with the TRIzol reagent (EXINNO) and the miRNeasy mini kit (Qiagen, Hilden, Germany), respectively, per the manufacturer’s protocols. Total RNA (1 µg each sample) was used to synthesize cDNA utilizing the PrimeScript^® RT Master Mix Perfect Real Time Reagent Kit (Takara Bio Inc.). For miRNA reverse transcription, cDNA was synthesized using a universal tag by using a miScript II RT kit (Qiagen). Quantitative reverse transcription PCR (qRT-PCR) for miRNA and mRNA was performed using a standard protocol from the SYBR Green PCR kit (Toyobo, Osaka, Japan) on an AB7500 RT-PCR instrument (Applied Biosystems, Foster City, CA, USA). Relative quantification was determined by normalization to GAPDH. The primers for qRT-PCR analysis were as follows: ALP, forward 5′-CCATACAGGATGGCAGTGAAGG-3′, reverse 5′-TTGACCTCCTCGGAAGACACTC-3′; RUNX2, forward 5′-TGCTTTGGTCTTGAAATCACA-3′, reverse 5′-TCTTAGAACAAATTCTGCCCTTT-3′; OCN, forward 5′-TGCCTGGAGAGGAGCAGAACT-3′, reverse 5′-GGCGCTACCTGTATCAATGGC-3′; GAPDH, forward 5′-GGCATCCACTGTGGTCATGAG -3′, reverse 5′-TGCACCACCAACTGCTTAGC-3′; WNT3a, forward 5′-GTGAGGACATTGAATTTGGAGG-3′, reverse 5′-ACTTGAGGTGCATGTGACTG-3′; β-catenin, forward 5′-GCTACTGTTGGATTGATTCGAAATC-3′, reverse 5′-CCCTGCTCACGCAAAGGT-3′;The PCR reaction protocol consisted of two steps: step one, initial denaturation for 30 s at 95°C step two, denaturation for 5 s at 95°C, annealing and extension for 60 s at 60°C and fluorescence signal acquisition. The experiments were repeated for 3 times and each sample was run in triplicates. PCR product specificity was confirmed by melting curve analysis. Gene expression levels were calculated with the 2^-ΔΔCT method.

Western Blot

Protein samples from DPSCs culture in vitro were obtained after cell lysis with the radioimmunoprecipitation assay lysis buffer (RIPA; EXINNO, Xi'an, China) and subsequent centrifugation at 13 000 g for 20 min at 4°C. Equal amount of protein samples (20 µg each) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes in an AccuRef fast transferring buffer (AccuRef Scientific), and incubated with indicated primary antibodies overnight. Antibody including anti-ALP (cat. no. ab83259), anti-RUNX2 (cat. no. ab236639), OCN (cat. no. ab133612), WNT3a (cat. no. ab219412), β-catenin (cat. no. ab32572), pro-casp3 (cat. no. ab32499), cleaved-casp3 (cat. no. ab32042), cleaved-casp-9 (cat. no. ab2324), anti-CA9 (cat. no. ab243660), anti-HIF-1a (cat. no. ab51608), GLUT1 (cat. no. ab115730), anti-pro-casp9 (cat. no ab138412), BAX (cat. no. ab32503) and the antibody for the loading control GAPDH (cat. No. ab8245) and β-actin (cat. no. ab8226) were all purchased from Abcam Company (Cambridge, UK). After binding with the secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (cat. no. ab7090, Abcam) or the secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG (cat. no. ab97040, Abcam), the membranes were processed with the enhanced chemiluminescence kit (EMD Millipore, Burlington, MA, USA). The expressions of _targeted proteins were quantified by densitometry using the ImageJ software (version 1.49; National Institutes of Health, Bethesda, MD, USA).

RNA Interference

CA9-specific small interfering RNAs (siRNA) (ON-_target plus Human CA9 siRNAs J-005244-05) were purchased from Dharmacon (GE Healthcare, Japan). 20 hours after DPSCs plated as monolayer in 2 × 104/cm², DPSCs was incubated with siRNAs at 37°C for 24 hours. Cells were transfected with the CA9 siRNA oligos with Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. At 48 hours after transfection, cells were subjected to further analyses.

Statistics

All of the experiments in this study were repeated for at least 3 times, quantitative results are expressed as mean ± standard deviation (SD), and groups were compared using unpaired, two-tailed Student’s t-tests. Statistical analyses were performed using SPSS v. 19.0 statistical software (IBM Corporation; Armonk, NY, USA), and a P-value <0.05 was considered to be statistically significant.

Differentiation of DPSCs into Osteoblasts and Adipocytes

DPSCs were expanded in a complete isolation/proliferation culture medium added with 1.25% HS. Following expansion, cell surface marker expression levels of CD44, CD105, and CD146 were evaluated for DPSCs in that the presence or absence (CD45, CD31, and CD34) of these markers are important determinants in defining “stemness” associated with DPSCs. Expansion conditions resulted in yielding DPSCs which expressed high levels of CD44 (>99.8%), CD105 (>99.9%), and CD146 (>99.7%), while expressed low levels of CD31 (<0.9%), CD34 (<1.1%), and CD45 (<1.2%) (Fig. 1A). Additionally, all these data confirmed the successful isolation of DPSCs. Isolated DPSCs from all individuals were differentiated into osteoblasts and adipocytes according toosteoblasts and adipocytes’ differentiation protocol as previously described. 0.1% ARS staining yielded a strong mineral staining at 2 wk after osteogenic differentiation (Fig. 1B). Oil red staining yielded bright-red droplets in differentiated adipocytes (Fig. 1C).

Hypoxia and TNF-α Synergistically Inhibit the Survival and Osteogenesis of DPSCs

It has been reported by other groups that DPSCs tend to apoptosis under abnormal culture conditions in vitro and in vivo. To determine what the specific phenotype of DPSC in abnormal culture conditions, we cultured and differentiated DPSCs under hypoxic and inflammatory conditions. CCK-8 analysis for the determination of cell viability in cell proliferation assays in day 30 post cultures and differentiation under normal conditions indicated 100% cell viability, while only 80% and 60% cell viability respectively under hypoxic and inflammatory conditions. Further, the cell viability is <50% under hypoxia and inflammation (Fig. 2A). The corresponding cell apoptosis results are match with the above cell viability respectively (Hypoxia+TNF-α >TNF-α >Hypoxia >Control) (Fig. 2B). Real time PCR data, expressed as differentiated DPSCs, showed an increased and comparable expression, 2 weeks of osteoblastic induction, for all the tested markers with significant differences deriving from the different proliferating conditions, as evidenced in Fig. 2C. In order to evaluate the effect of hypoxia and inflammation on osteogenic differentiation of DPSCs, proliferating DPSCs cultured in 1.25% HS were osteo-induced for 2 weeks. During the osteoblastic inductive period cells changed their osteoblasts’ morphology, 0.1% ARS staining showed that the efficiency of osteogenic differentiation under normal conditions is much greater than that of hypoxia and inflammation groups (Fig. 2D).

Taxifolin Protects DPSCs Against TNF-α and Hypoxia-Induced Apoptosis

To examine protective effects of taxifolin on apoptosis, DPSCs were incubated with different concentrations of taxifolin for 24 h and subsequently treated with CCK-8 for the determination of cell viability. Pretreatment with taxifolin showed a final concentration of 10 μM taxifolin can significantly improve the viability of DPSCs under inflammation- and hypoxia-induced conditions (Fig. 3A, B). Next, flow cytometric analysis showed that 10 μM taxifolin can significantly inhibit DPSCs apoptosis under hypoxic and inflammatory conditions (Fig. 3C, D). We then examined cleaved-Casp3, cleaved-Casp9and BAX protein levels by Western blotting to further analyze the potential influence of the taxifolin on inhibit DPSCs apoptosis activities. Hypoxia and TNF-α group exhibited increased ratio of these proteins, which were much higher than the taxifolin group (Fig. 3E).

Taxifolin Promotes Osteogenic Differentiation of DPSCs Under Hypoxia and Inflammation Conditions

After we demonstrated that taxifolin inhibited the TNF-α and hypoxia decrease in DPSCs viability, we investigated whether taxifolin promotes ostegenic differentiation of DPSCs under hypoxia and inflammation conditions. We divided the DPSCs into seven groups: a control group, hypoxia group, TNF-α group, hypoxia and TNF-α group, hypoxia plus taxifolin (10 μM)-treated group, TNF-αplus taxifolin (10μM)-treated group, and hypoxia and TNF-α plus taxifolin (10 μM)-treated group. After incubation for 24 h, the seven groups were stained with ARS, and ARS staining was used to determine the osteogenic rate. control group, hypoxia plus taxifolin (10 μM)-treated group and TNF-αplus taxifolin (10 μM)-treated group were equivalent on ostegenic differentiation of DPSCs, which were much higher than the other groups (Fig. 4A, B). Moreover, the mRNA expression of osteogenesis differentiation markers such as ALP, OCN, Runx2, WNT3a, and β-catenin, were determined by quantitative real-time PCR, and the protein expression levels of ALP, OCN, Runx2, WNT3a, and β-catenin were determined by western blot. The results revealed that ALP, OCN, Runx2, WNT3a, and β-catenin mRNA levels were significantly elevated in DPSCs in the presence of taxifolin in a concentration dependent manner (Fig. 4C). Similarly, protein expression of ALP, OCN, Runx2, WNT3a, and β-catenin increased in the presence of taxifolin, especially at 10 μM taxifolin-treated group (Fig. 4D).

Molecular Mechanisms Involved in Taxifolin Protects DPSC

Carbonic anhydrase IX (CA9), as well as a membrane associated zinc metallo-enzyme, are reported to enhance hypoxic tumor cells to survive in the acidic microenvironment ^20,21 . Therefore, we examined whether taxifolin could increase CA9 expression in a dose-dependent manner. We first determined protects of taxifolin in the DPSCs under difference conditions, the results of protects of taxifolin were significant (Fig. 5A). Western blotting results from our study showed that taxifolin significantly increased protein CA9 expression but not HIF1a, the corresponding mRNA suck as CA9, HIF-1a, and GLUT1, were determined by quantitative real-time PCR, match with Western blotting results (Fig. 5B).

Further, to determine whether taxifolin and CA9 synergistically protect the survival and ostegenesis of DPSCs, in vitro RNAi inhibits CA9 protein expression were performed.

The results from our study demonstrated that inhibit CA9 protein expression cell viability cell survival decreased significantly, RNAi groups only 60%, while control groups have two-fold to RNAi groups (Fig. 6A, B). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Kit assay the apoptotic rate for difference groups. The results match with cell viability, which apoptosis rate of RNAi group is much higher than other groups (Fig. 6C, D).