Korean J Physiol Pharmacol.  2018 Mar;22(2):163-172. 10.4196/kjpp.2018.22.2.163.

Effects of salmon DNA fraction in vitro and in a monosodium iodoacetate-induced osteoarthritis rat model

• Ra HJ
• Oh MY
• Kim HJ
• Lee SY
• Eom DW
• Lee SK
• Kim SN
• Chung KS
• Jang HJ

• KMID: 2410106
• DOI: http://doi.org/10.4196/kjpp.2018.22.2.163

Abstract

PRF001 is a fragmented DNA polymer extracted from the testes of salmon. The purpose of this study was to assess the anti-inflammatory effect of PRF001 in vitro as well as the protective effect of PRF001 intake against arthritis in a rat model. In vitro, cell survival and inflammatory markers after H₂O₂ treatment to induce cell damage were investigated in CHON-001 cells treated with different concentrations of PRF001. In vivo, osteoarthritis was induced by intra-articular injection of monosodium iodoacetate (MIA) into the knee joints of rats. After consumption of PRF001 (10, 50, or 100 mg/kg) for 4 weeks, inflammatory mediators and cytokines in articular cartilage were investigated. In vitro, the levels of inflammatory markers, IL-1β, TNF-α, COX-2, iNOS, and PGE2, were significantly suppressed by PRF001 treatment. In vivo, the inflammatory mediators and cytokines, IL-1β, p-Erk1/2, NF-κB, TNF-α, COX-2, and PGE2, as well as MMP3 and MMP7, which have catabolic activity in chondrocytes, were decreased in the MIA-induced osteoarthritic rats following intake of PRF001. Histological analysis revealed that PRF001 had a protective effect on the articular cartilage. Altogether, these results demonstrated that the anti-inflammatory property of PRF001 contributes to its protective effects in osteoarthritis through deregulating IL-1β, TNF-α, and subsequent signals, such as p-Erk1/2, NF-κB, COX-2, PGE2, and MMPs.

Keyword

Chondrocyte; Cytokine; Inflammation; Osteoarthritis; Polydeoxyribonucleotide; PRF001

MeSH Terms

Animals
Arthritis
Cartilage, Articular
Cell Survival
Chondrocytes
Cytokines
Dinoprostone
DNA*
In Vitro Techniques*
Inflammation
Injections, Intra-Articular
Knee Joint
Matrix Metalloproteinases
Models, Animal*
Osteoarthritis*
Polymers
Rats*
Salmon*
Testis
Cytokines
DNA
Dinoprostone
Matrix Metalloproteinases
Polymers

Figure

• 

  Fig. 1 PRF001 is not toxic to CHON-001 cells and improves the viability of and H2O2-induced CHON-001 cells in a concentration-dependent manner.(A) Cytotoxicity of PRF001 on CHON-001 cells. PRF001 was added to CHON-001 cells at 6.3, 12.5, 25, 50, or 100 µg/ml for 24 h. Cell viability was measured in supernatants of treated CHON-001 cells and expressed as a percentage of untreated control cells in each group. (B) Cell viability in H2O2-induced CHON-001 cells. H2O2 was added to CHON-001 cells for 2 h followed by treatment with PRF001 for 24 h. Cell viability was measured in supernatants of treated CHON-001 cells and expressed as a percentage of untreated control cells in each group. (C) ROS level in H2O2-induced CHON-001 cells. H2DCFDA was added to CHON-001 cells after treatment with PRF001 for 24 h. ROS levels were measured as DCF fluorescence intensity in supernatants of 50 and 100 µg/ml-treated CHON-001 cells by flow cytometry. The data are expressed as the mean±SEM of triplicate experiments. *Statistically significant, p<0.05.

• 

  Fig. 2 PRF001 lowers inflammatory mediators and cytokines in H2O2-induced CHON-001 cells.(A–E) CHON-001 cells were treated with H2O2 and PRF001 at concentrations of 50 and 100 µg/ml for 24 h. Protein levels of IL-1β, TNF-α, COX-2, and iNOS were measured in cell lysates using western blot assay. (F, G) CHON-001 cells were pretreated with H2O2 for 2 h, followed by treatment with PRF001 at concentrations of 50 and 100 µg/ml for 24 h. Protein levels of PGE2 and TNF-α were measured in cell supernatants by ELISA. The data are expressed as the mean±SEM from triplicate experiments. *Statistically significant, p<0.05.

• 

  Fig. 3 PRF001 diminishes the production of inflammatory mediators and cytokines in MIA-induced OA rats.(A–E, G) MIA (60 mg/ml) was injected into the right knee joint of rats, and seven days later, PRF001 was administered orally at different concentrations (low, medium, and high; 10, 50, and 100 mg/kg/day) for four weeks. Protein levels of IL-1β, Erk, NF-κB, TNF-α, and COX-2 were measured in the cartilage tissue of all groups using western blot assay. (F, H) Protein levels of TNF-α and PGE2 were measured in sera of all groups using ELISA. The data are expressed as the mean±SEM of ten animals per group. *Statistically significant, p<0.05.

• 

  Fig. 4 PRF001 is involved in catabolic activity of chondrocytes by diminishing the MMP expression in MIA-induced OA rats.MIA (60 mg/ml) was injected into the right knee joint of rats, and seven days later, PRF001 was administered orally at different concentrations (low, medium, and high; 10, 50, and 100 mg/kg/day) for four weeks. Protein levels of MMP3 and MMP7 were measured in the cartilage tissue of all groups using western blot assay (A, C). The data are expressed as the mean±SEM for ten animals per group. *Statistically significant, p<0.05.

• 

  Fig. 5 PRF001 protects articular cartilage and subchondral bone in MIA-induced OA rats.Coronal sections of cartilage tissues were stained with H&E. (A) Evenly distributed chondrocytes were observed in the normal control group. (B) Changes in morphology and distribution of chondrocytes in the MIA group and (C, D) concentration-dependent protective effects revealed in PRF001-administered groups (MIA+M, H). The data are expressed as the mean±SEM of 10 animals per group.

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