Anti-inflammatory effect of polydeoxyribonucleotide on zoledronic acid-pretreated and lipopolysaccharide-stimulated RAW 264.7 cells

Abstract

Bisphosphonates are generally used as therapeutic agents for bone diseases. However, previous reports on bisphosphonates-related osteonecrosis of the jaw (BRONJ) demonstrated that inflammation triggers and worsens the disease. Recently, polydeoxynucleotide (PDRN), an A_2A receptor agonist, has been suggested for the treatment of various diseases and broadly studied for its anti-inflammatory effect. The present study aimed to measure the effect of PDRN on macrophage cells treated with zoledronic acid (ZA) and lipopolysaccharide (LPS). Macrophage cells were cultured with ZA for 24 h, following which they were stimulated with LPS in the presence or absence of varying concentrations of PDRN for 24 h. The cell viability and nitric oxide (NO) production of the cells were analyzed. In addition, protein expression levels were quantified by western blotting. Cell viability was compromised and NO was overexpressed by ZA and LPS stimulation. However, under ZA and LPS stimulation cell viability was enhanced, and NO production, and inducible nitric oxide synthase, interleukin (IL)-1β, -6, and tumor necrosis factor-α overexpression were suppressed on exposure to PDRN. A_2A receptor and vascular endothelial growth factor (VEGF) expression levels increased following PDRN treatment. These results indicate that PDRN treatment of macrophages inhibits the inflammatory cytokines induced by ZA and LPS stimulation. It was hypothesized that the inflammatory cytokines were inhibited through A_2A activation by PDRN. In addition, increased VEGF expression may contribute to increased vascularization and subsequently improve the pathological condition in BRONJ. As inflammation and LPS may stimulate the occurrence of BRONJ, the present study postulated that PDRN is possibly a candidate for the therapeutic management of BRONJ by decreasing inflammation and increasing vascularization.

Keywords: adenosine A2 receptor; bisphosphonate-related osteonecrosis of the jaw; lipopolysaccharide; polydeoxynucleotide; zoledronic acid.

Figure 1.

Cell viability is decreased by LPS and ZA+LPS stimulation. Relative cell viabilities were compared with the control group, which was set to 100%. Cell viability was measured in RAW 264.7 cells cultured with (A) ZA for 48 h, (B) LPS for 24 h and (C) ZA for 24 h, followed by LPS for 24 h. (D) Cell viability was measured in RAW 264.7 cells pretreated with 10 µM ZA and stimulated 0.1 µg/ml LPS compared with 0.1 µg/ml LPS-stimulated cells. The experiments were conducted in triplicate. Data are presented as the mean + standard error of the mean. *P<0.05 compared with the control group. ^#P<0.05 compared with the LPS group. ZA, zoledronic acid; LPS, lipopolysaccharide.

Figure 2.

PDRN increases cell viability of RAW 264.7 treated with ZA+LPS. Relative cell viabilities were compared with the control group, which was set to 100%. Cell viability was measured in RAW 264.7 cells treated with 10 µM ZA for 24 h, and then with 0.1 µg/ml LPS and PDRN (1, 10, and 100 µg/ml) for 24 h. The experiments were conducted in triplicate. Data are presented as mean ± standard error of the mean. *P<0.05 compared with the control group. ^#P<0.05 compared with the ZA+LPS group. ZA, zoledronic acid; LPS, lipopolysaccharide; PDRN, polydeoxynucleotide.

Figure 3.

PDRN decreases ZA+LPS-induced NO production. NO production in the culture medium was analyzed with an NO detection kit. RAW 264.7 cells were cultured with 10 µM ZA for 24 h, and then with 0.1 µg/ml LPS and PDRN (1, 10, and 100 µg/ml) for 24 h. The experiments were conducted in triplicate. Data are presented as mean ± standard error of the mean. *P<0.05 compared with the control group. ^#P<0.05 compared with the ZA+LPS group. ZA, zoledronic acid; LPS, lipopolysaccharide; PDRN, polydeoxynucleotide.

Figure 4.

PDRN decreased inflammatory cytokine expression and increased A_2A and VEGF expression. The expression levels of iNOS, IL-1β, IL-6, TNF-α, A_2A and VEGF were measured in RAW 264.7 cells treated with 10 µM ZA for 24 h, and then with 0.1 µg/ml LPS and PDRN (1, 10, and 100 µg/ml) for 24 h. Data are presented as mean ± standard error of the mean. *P<0.05 compared with the control group. ^#P<0.05 compared with the ZA+LPS group. ZA, zoledronic acid; LPS, lipopolysaccharide; PDRN, polydeoxynucleotide; iNOS, inducible nitric oxide synthase; IL, interleukin; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.