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. 2021 Jan-Dec:30:9636897211034452.
doi: 10.1177/09636897211034452.

Taxifolin Protects Dental Pulp Stem Cells under Hypoxia and Inflammation Conditions

Xiaohui Fu  1 , Yimiao Feng  2 , Bingyi Shao  3 , Yanzhen Zhang  1
Affiliations

Taxifolin Protects Dental Pulp Stem Cells under Hypoxia and Inflammation Conditions

Xiaohui Fu et al. Cell Transplant. 2021 Jan-Dec.

Abstract

Background: Dental pulp stem cells (DPSCs) are a unique source for future clinical application in dentistry such as periodontology or endodontics. However, DPSCs are prone to apoptosis under abnormal conditions. Taxifolin is a natural flavonoid and possesses many pharmacological activities including anti-hypoxic and anti-inflammatory. We aimed to elucidate the mechanisms of taxifolin protects DPSC under hypoxia and inflammatory conditions.

Methods: DPSCs from human dental pulp tissue was purchased from Lonza (cat. no. PT-5025. Basel, Switzerland)) and identified by DPSC's biomarkers. DPSC differentiation in vitro following the manufacturers' instructions. ARS staining and Oil red staining verify the efficiency of differentiation in vitro after 2 weeks. The changes of various genes and proteins were identified by Q-PCR and western-blot, respectively. Cell viability was determined by the CCK-8 method, while apoptosis was determined by Annexin V/PI staining.

Results: DPSC differentiation in vitro shows that hypoxia and TNF-α synergistically inhibit the survival and osteogenesis of DPSCs. A final concentration of 10 μM Taxifolin can significantly reduce the apoptosis of DPSCs under inflammation and hypoxia conditions. Taxifolin substantially increases carbonic anhydrase IX (CA9) expression but not HIF1a, and inhibitions of CA9 expression nullify the protective role of taxifolin under hypoxia and inflammatory condition.

Conclusion: Taxifolin significantly increased the expression of CA9 when it inhibits DPSC apoptosis and taxifolin synergistically to protect DPSCs against apoptosis with CA9 under hypoxia and inflammatory conditions. Taxifolin can be used as a potential drug for clinical treatment of DPSC-related diseases.

Keywords: CA9; DPSCs; apoptosis; hypoxia and inflammation; taxifolin.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Isolation and characterization of DPSCs. (A) Representative flow cytometry data of undifferentiated DPSCs. Results indicating surface markers of DPSCs (n = 5). (B) ARS staining at 2 wk after osteogenic differentiation (n = 5). (C) Oil red staining at 2 wk after adipose differentiation (n = 5). Scale bars, 200 μm.
Figure 2.
Figure 2.
Hypoxia and TNF-α synergistically inhibit the survival and osteogenesis of DPSCs. (A) CCK-8 analysis of cell viability under different conditions (n = 5). (B) Flow cytometric analysis of cell apoptosis in different conditions (n = 5). (C) Relative expression level of ALP, RUNX2, and OCN at 2 wk after osteogenic differentiation was determined by qRT-PCR (n = 5 for each group). (D) ARS staining showed that hypoxia and TNF-α synergistically inhibited osteoclast formation in vitro. Scale bars, 200 μm.
Figure 3.
Figure 3.
Taxifolin protects DPSCs against TNF-α and hypoxia-induced apoptosis. CCK-8 analysis of cell viability with the addition of different concentrations of taxifolin under hypoxia conditions (n = 5). (B) CCK-8 analysis of cell viability with the addition of different concentrations of taxifolin under TNF-α treatment (n = 5). (C, D) DPSCs (cultured with TNF-α and hypoxia) were treated with taxifolin 10 μM concentration. Flow cytometric analysis cell apoptosis in different proliferating conditions (n = 5). (E) Western blot analysis of apoptosis-related proteins. *P < 0.05; **P < 0.01 compared with indicated groups.
Figure 4.
Figure 4.
Taxifolin promotes osteogenic differentiation of DPSCs under hypoxia and inflammation conditions. (A) ARS staining at 2 wk after osteogenic differentiation (n = 5). Scale bars = 200 μm. Taxifolin significantly promoted osteogenic differentiation of DPSCs under hypoxia and inflammation conditions. (B) Quantitative data showed Alizarin red-positive areas quantified by ImageJ software. (C) The relative expression levels of ALP, OCN, RUNX2, WNT3a, and β-catenin were determined by real-time quantitative PCR. (D) The protein expression levels of ALP, OCN, RUNX2, WNT3a, and β-catenin were determined by western blot. Grayscale was quantified by ImageJ software. Data were shown as the mean ± SD (n = 5); *P < 0.05 and **P < 0.01 compared with indicated groups.
Figure 5.
Figure 5.
Taxifolin increases CA9 expression but not HIF1a. (A) CCK-8 assay showed the viability of DPSCs under different culture conditions with or without Taxifolin treatment. (B) The protein expression levels of HIF-1a, CA9, and GLUT1 were determined by western blot. Grayscale was quantified by ImageJ software. Data were shown as the mean ± SD (n = 5). *P < 0.05 compared with indicated groups.
Figure 6.
Figure 6.
Inhibition of CA9 nullifies the protective role of Taxifolin under hypoxia and inflammation conditions. (A and B) CCK-8 assay showed the cell viability of DPSCs under hypoxia (A) or inflammation (B) conditions. Cells transfected with si-CA9 showed significant decrease of viability compared with cells transfected with scrambled siRNA (NC). (C, D) Flow cytometry analysis showed the apoptosis rate of DPSCs under hypoxia (C) or inflammation (D) conditions with or without downregulation of CA9. Data were shown as the mean ± SD (n = 5); *P < 0.05.
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