Thymoquinone-Mediated Modulation of Toll-like Receptors and Pluripotency Factors in Gingival Mesenchymal Stem/Progenitor Cells

Abstract

Thymoquinone (TQ), the key active component of Nigella sativa (NS), demonstrates very promising biomedical anti-inflammatory, antioxidant, antimicrobial and anticancer properties. Several investigations have inspected the modulative activities of TQ on different stem/progenitor cell types, but its possible role in the regulation of gingival mesenchymal stem/progenitor cells (G-MSCs) has not yet been characterized. For the first time, this study investigates the effects of TQ on G-MSCs’ stemness and Toll-like receptor expression profiles. G-MSCs (n = 5) were isolated, sorted via anti-STRO-1 antibodies and then disseminated on cell culture dishes to create colony-forming units (CFUs), and their stem/progenitor cell attributes were characterized. TQ stimulation of the G-MSCs was performed, followed by an examination of the expression of pluripotency-related factors using RT-PCR and the expression profiles of TLRs 1−10 using flowcytometry, and they were compared to a non-stimulated control group. The G-MSCs presented all the predefined stem/progenitor cells’ features. The TQ-activated G-MSCs displayed significantly higher expressions of TLR3 and NANOG with a significantly reduced expression of TLR1 (p < 0.05, Wilcoxon signed-rank test). TQ-mediated stimulation preserves G-MSCs’ pluripotency and facilitates a cellular shift into an immunocompetent-differentiating phenotype through increased TLR3 expression. This characteristic modulation might impact the potential therapeutic applications of G-MSCs.

Keywords: Toll-like receptors; gingiva; pluripotency; stem cells; stemness; thymoquinone.

Figure 1

Chemical structure of thymoquinone.

Figure 2

Microscopic appearance, colony formation, surface marker expression and differentiation potential of G-MSCs. (A) Crystal violet staining of G-MSCs’ colonies formed after 12 days and (B) microscopic appearance of the G-MSCs. (C) Flowcytometric expression profile of G-MSCs’ surface marker expression. Multilineage differentiation ability of G-MSCs. G-MSCs after stimulation with osteogenic inductive medium stained with Alizarin Red (D) and their controls (E) with the analysis of RUNX (F) and ALP (G) expressions. G-MSCs after stimulation with adipogenic differentiation medium stained with Oil Red O (H) and their controls (I) with the analysis of PPARɣ (J) and LPL (K) expressions. G-MSCs after stimulation with chondrogenic differentiation medium stained with Alcian Blue and Nuclear Fast Red (L) and their controls (M) with the analysis of ACAN (N). (n = 5; box-and-whisker plots with medians and quartiles; Wilcoxon signed-rank test, statistical significance marked with asterisk, *: p < 0.05). ACAN, aggrecan; ALP, alkaline phosphatase; LPL, lipoproteinlipase; PPARɣ, peroxisome proliferator-activated receptor-gamma; RUNX, runt-related transcription factor-2. Statistical outliers have been marked by blue dots.

Figure 3

Flowcytometric percentage expression of TLRs 1–10 in G-MSCs with and without TQ stimulation. Expressed TLRs in G-MSCs (blue and red curves) and their isotype controls (grey curves) after incubation in a basic control medium and basic medium with TQ (n = 5; box-and-whisker plots with medians and quartiles). Significance at p < 0.05 marked with an asterisk * (Wilcoxon signed-rank test). Statistical outliers have been marked by blue and red dots.

Figure 4

mRNA expression of pluripotency factors after TQ stimulation. Changes in mRNA expression of the stemness-associated factors (A) homeobox protein Nanog (NANOG), (B) sex-determining region Y (SRY)-box 2 (SOX2), (C) octamer-binding transcription factor 4 (OCT4A), (D) Krüppel-like factor 4 (KLF4), (E) REX1 and (F) muscle segment homeobox (MSX-1) after G-MSC stimulation with TQ compared to the unstimulated control group. Significance at p < 0.05 marked with an asterisk * (Wilcoxon signed-rank test). Statistical outliers have been marked by blue and red dots.

Figure 5

Potential thymoquinone-mediated modulation of gingival mesenchymal stem/progenitor cells (G-MSCs) during periodontal inflammation (created with BioRender.com; accessed on 9 April 2022).