Polydeoxyribonucleotide ameliorates IL-1β-induced impairment of chondrogenic differentiation in human bone marrow-derived mesenchymal stem cells

Abstract

Osteoarthritis (OA) is a degenerative disease of the joints, prevalent worldwide. Polydeoxyribonucleotide (PDRN) is used for treating knee OA. However, the role of PDRN in IL-1β-induced inflammatory responses in human bone marrow-derived mesenchymal stem cells (hBMSCs) remains unknown. Here, we investigated the role of PDRN in IL-1β-induced impairment of chondrogenic differentiation in hBMSCs. hBMSCs treated with PDRN showed a large micromass, enhanced safranin O and alcian blue staining intensity, and increased expression of chondrogenic genes in IL-1β-induced inflammatory responses, in addition to regulation of catabolic and anabolic genes. In addition, PDRN treatment suppressed the expression of inflammatory cytokines and mitigated IL-1β-induced apoptosis in hBMSCs. Mechanistically, PDRN treatment increased the formation of cyclic adenosine monophosphate (cAMP) and upregulated the phosphorylation of cAMP-dependent protein kinase A (PKA)/cAMP response element binding protein (CREB) through the adenosine A2A receptor in hBMSCs and thus blocked the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway. Thus, IL-1β-induced expression of inflammatory cytokines in hBMSCs was directly reduced by adenosine A2A receptor activation. Based on our results, we suggest that PDRN may be a promising MSC-based therapeutic agent for OA.

Keywords: Chondrogenic differentiation; Interleukin-1β; Mesenchymal stem cell; Osteoarthritis; Polydeoxyribonucleotide.

Fig. 1

Polydeoxyribonucleotide (PDRN) rescued IL-1β-induced inhibition of chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). (A) hBMSCs were treated with different doses (50 and 100 µg/mL) of PDRN or left untreated, and cell viability was determined using the CCK-8 reagent. (B) hBMSCs were treated with vehicle, IL-1β (10 ng/mL) and different doses (50 and 100 µg/mL) of PDRN, and cell viability was determined using the CCK-8 reagent. (C) hBMSCs were treated with different doses (50 and 100 µg/mL) of PDRN or left untreated, and then cultured with chondrogenic differentiation medium for 14 days. Thereafter, protein levels of SOX9 and COL2A1 were investigated by western blot analysis. (D) The western blot images were quantified using ImageJ. (E) hBMSCs were treated with vehicle, IL-1β (10 ng/mL), or IL-1β (10 ng/mL) with PDRN (100 µg/mL) and then subjected to chondrogenic differentiation for 14 days. Thereafter, alcian blue and safranin O staining of hBMSCs was performed. (F) Absorbance quantification of alcian blue and safranin O staining. (G) mRNA levels of chondrogenesis-related genes were analyzed using qRT-PCR; GAPDH used for normalization. (H) Expression levels of SOX9 and COL2A1 were determined using western blot analysis. (I) Densitometry of the protein bands shown in (H), using the ImageJ software. Results are means ± SDs. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001. (A, B, D, F, G, and I) P-values were determined using a one-way analysis of variance with Bonferroni post hoc tests.

Fig. 2

Polydeoxyribonucleotide (PDRN) downregulates the expression of catabolic markers and upregulates that of an anabolic marker in an IL-1β-induced inflammatory microenvironment. (A) mRNA expression of catabolic and anabolic genes was determined using qRT-PCR; GAPDH used for normalization. (B) Expression of catabolic and anabolic proteins was measured using western blot analysis. (C) Densitometry analysis of the protein bands shown in (B) using the ImageJ software. Results are means ± SDs. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001. (A,C) P-values were determined using a one-way analysis of variance with Bonferroni post hoc tests.

Fig. 3

Polydeoxyribonucleotide (PDRN) decreased the expression of inflammatory cytokines in chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. (A) mRNA expression of proinflammatory cytokine genes was evaluated using qRT-PCR; GAPDH used for normalization. (B) Expression of proinflammatory cytokines was determined using western blot analysis. (C) Densitometry analysis of protein bands shown in (B) using the ImageJ software. Results are means ± SDs. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001. (A,C) P-values were determined using a one-way analysis of variance with Bonferroni post hoc tests.

Fig. 4

Polydeoxyribonucleotide (PDRN) rescued IL-1β-induced apoptosis in chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. (A) Cell apoptosis was determined using the TUNEL assay. Scale bar = 20 μm. (B) Percentage of TUNEL-positive cells. (C) mRNA expression of apoptosis-related genes (BAX and BCL2) was evaluated using qRT-PCR; GAPDH used for normalization. (D) Expression of apoptosis-related proteins was evaluated using western blot analysis. (E) Densitometry analysis of protein bands shown in (D) using the ImageJ software. Results means ± SDs. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001. (B-D) P-values were determined using a one-way analysis of variance with Bonferroni post hoc tests.

Fig. 5

Polydeoxyribonucleotide (PDRN) upregulates the phosphorylation of the cAMP/PKA/CREB pathway proteins under IL-1β-induced inflammation. (A) Intracellular cAMP levels of hBMSCs treated with vehicle, IL-1β (10 ng/mL), or IL-1β (10 ng/mL) with PDRN (100 µg/mL). (B) Expression of PKA/CREB signaling pathway mediators in hBMSCs treated with vehicle, IL-1β (10 ng/mL), PDRN (100 µg/mL), or DMPX (50 µg/mL) were evaluated using western blot analysis. (C) Densitometry analysis of protein bands shown in (B) using the ImageJ software. Results are means ± SDs. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001. (A,C) P-values were determined using a one-way analysis of variance with Bonferroni post hoc tests.

Fig. 6

Adenosine A2A receptor agonist suppressed the expression of inflammatory cytokines in chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. (A) hBMSCs were treated with PDRN (100 µg/mL), IL-1β (10 ng/mL) with PDRN (100 µg/mL), IL-1β (10 ng/mL) with CGS21680 (1 µM) and IL-1β (10 ng/mL) with PDRN (100 µg/mL), ZM241385 (1 µM) or left untreated in chondrogenic differentiation medium for 14 days. The Expression of proinflammatory cytokines was determined using western blot analysis. (B) Densitometry analysis of protein bands shown in (A) using the ImageJ software. Results are means ± SDs. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001. P-values were determined using a one-way analysis of variance with Bonferroni post hoc tests.

Fig. 7

Polydeoxyribonucleotide (PDRN) downregulates the NF-κB signaling pathway under IL-1β-induced inflammation. (A) Expression of NF-κB signaling pathway mediators in human bone marrow-derived mesenchymal stem cells (hBMSCs) treated with IL-1β (10 ng/mL) in the presence or absence of PDRN (100 µg/mL) was evaluated using western blot analysis. (B) Densitometry analysis of protein bands shown in (A) using the ImageJ software. P-values were determined using a one-way analysis of variance with Bonferroni post hoc tests. (C) Immunofluorescence images showing nuclear translocation of p65 in hBMSCs treated with IL-1β (10 ng/mL) in the presence or absence of PDRN (100 µg/mL). Nuclei were counterstained with DAPI. Scale bar = 20 μm. Results are means ± SDs. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 8

Schematic diagram of polydeoxyribonucleotide (PDRN) treatment in IL-1β-induced impairment of chondrogenic differentiation. PDRN protects against IL-1β-induced inflammation and promotes chondrogenic differentiation of hBMSCs by activating the cAMP/PKA/CREB pathway and inhibiting NF-κB activation. Binding of PDRN to the adenosine A2A purigenic receptor activates the downstream kinase cascade which ultimately results in CREB-mediated suppression of NF-kB. Inhibition of this pathway prevents suppression of growth factors necessary for chondrocyte development such as growth hormone (GH) and insulin-like growth factor 1 (IGF-1) as well as bone morphogenic protein 2 (BMP2) expression. Suppression of these factors normally leads to apoptosis and catabolism. Thus, PDRN action via the A2A receptor effectively prevents apoptosis and supports development and differentiation of hBMSCs.