Nesfatin-1 suppresses interleukin-1β-induced inflammation, apoptosis, and cartilage matrix destruction in chondrocytes and ameliorates osteoarthritis in rats

Abstract

Osteoarthritis (OA) is a chronic degenerative joint disease, related to the overexpression of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), inflammation, and chondrocyte apoptosis. Nesfatin-1 is an adipokine, which plays an important role in the development of OA, especially in obese people. In the present study, cartilage degradation and apoptosis observed in OA patients was evaluated. Furthermore, the anti-inflammatory and anti-apoptotic effects of nesfatin-1, and its underlying in vitro and in vivo mechanisms were investigated. The results showed that nesfatin-1 increased significantly the expression of collagen type II alpha 1 chain (Col2a1), and reduced the expression of MMPs, ADAMTS5, cyclooxygenase (COX)-2, caspase-3, nitric oxide (NO), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), interleukin (IL)-6, and chondrocyte apoptosis rate, which may be induced by IL-1β in rat chondrocytes. Furthermore, nesfatin-1 treatment prevented cartilage degeneration in the rat OA model. It was found that nesfatin-1 suppressed the IL-1β-induced activation of NF-κB, the mitogen-activated protein kinase (MAPK), and the Bax/Bcl-2 signal pathway in chondrocytes. These results suggest that in vivo nesfatin-1 could play a protective role in the development of OA and can be potentially used for its treatment.

Keywords: apoptosis; inflammation; matrix metalloproteinases; nesfatin-1; osteoarthritis.

Figure 1

Chondrocyte apoptosis, inflammation, and matrix degradation in the OA samples. (A) Representative images of H&E staining; (B) Representative images of immunohistochemistry; (C60) Quantitative optical density analysis of immunohistochemistry. Data represent the mean ± SD (n=5) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. ** p < 0.01 and * p < 0.05 versus normal samples.

Figure 2

Effect of nesfatin-1 and IL-1β on cell viability. (A) Cells treated with increasing concentrations of nesfatin-1 (0, 0.1, 1 to 1000 ng/mL) for 24 h. (B) Pre-treatment with nesfatin-1 for 2 hours, followed by IL-1β treatment for 24 h. Chondrocyte viability was evaluated via CCK-8 analysis. Data represent the mean ± SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test.* p < 0.05 versus IL-1β-treated alone samples.

Figure 3

Apoptosis rate determination using flow cytometry of nesfatin-1- and IL-1β-treated cells. (A) Cells pre-treated with nesfatin-1 (10 ng/mL) for 2 hours, followed by IL-1β treatment for 24 hours, and analyzed by flow cytometry. (B) Flow cytometric analysis. IL-1β-stimulation alone significantly increased the percentage of apoptotic chondrocytes. Annexin V- and PI-positive cells were markedly decreased among nesfatin-1 pre-treated cells. Data represent the mean ± SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. ** p < 0.01 versus the IL-1β group.

Figure 4

Effect of nesfatin-1 on IL-1β-induced activation of apoptosis in rat chondrocytes. (A) Chondrocytes were treated with nesfatin-1 on IL-1β-induced chondrocytes for 6 h and the protein levels of Bcl-2, Bax, cleaved caspase-3, pro-caspase-3, cleaved PARP, and PARP were evaluated via western blot analysis. (B–D) Quantitative analysis of western blot in rat chondrocytes. Data represent the mean ± SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. ** p < 0.01 versus the IL-1β group.

Figure 5

Nesfatin-1 suppressed the IL-1β-induced MMP-3, MMP-9, and MMP-13 expression in rat chondrocytes and the secretion level in the supernatant. Chondrocytes were treated with different concentrations of nesfatin-1 on IL-1β-induced chondrocytes for 24 h. The secretion level of MMPs was evaluated by ELISA (A, B, and C) and the mRNA level of MMPs was evaluated via real-time PCR (D, E, and F). The values are expressed as mean ±SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. **p < 0.01 versus the IL-1β group.

Figure 6

Nesfatin-1 suppressed the IL-1β-induced MMP-3, MMP-9, and MMP-13 expression in rat chondrocytes. Chondrocytes were treated with different concentrations of nesfatin-1 on IL-1β-induced chondrocytes for 24 h. The protein level of MMPs was evaluated via western blot analysis (A, B, C, and D). The values are expressed as mean ±SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. **p < 0.01 versus the IL-1β group.

Figure 7

Nesfatin-1 suppressed the IL-1β-induced ADAMTS5 expression and reversed IL-1β-induced Col2a1 degradation in rat chondrocytes. Chondrocytes were treated with different concentrations of nesfatin-1 on IL-1β-induced chondrocytes for 24 h. The mRNA levels of ADAMTS5 and Col2a1 were evaluated via real-time PCR (D, E) and the protein levels of ADAMTS5 and Col2a1 were evaluated via western blot analysis (A, B, and C). The values are expressed as mean ±SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. **p < 0.01 versus the IL-1β group.

Figure 8

Nesfatin-1 suppressed the IL-1β-induced iNOS and COX-2 expression in rat chondrocytes. Chondrocytes were treated with different concentrations of nesfatin-1 on IL-1β-induced chondrocytes for 24 h. The protein levels of COX-2 and iNOS were evaluated via western blot analysis (A, B, and C). The values are expressed as mean ±SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. *p<0.05, **p < 0.01 versus the IL-1β group.

Figure 9

Nesfatin-1 suppressed the IL-1β-induced IL-6, NO, and PGE2 expression in rat chondrocytes and the supernatant. Chondrocytes were treated with different concentrations of nesfatin-1 on IL-1β-induced chondrocytes for 24 h. The secretion level of IL-6 in the supernatant was evaluated via ELISA (A) and the mRNA level of IL-6 was evaluated via real-time PCR (B). The NO level was evaluated via Griess reaction (C) and the PGE2 level was evaluated via ELISA (D). The values are expressed as mean ±SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. *p<0.05, **p < 0.01 versus the IL-1β group.

Figure 10

Effect of nesfatin-1 on the IL-1β-induced NF-κB and MAPK activation in rat chondrocytes. (A, C). Chondrocytes were treated with different concentrations of nesfatin-1 for 6 h after IL-1β-induction. The protein levels of p38, phosphor-p38, JNK, phosphor-JNK, p65, and phosphor-p65 were evaluated via western blot analysis (B, D, and E). Quantitative analysis of western blot in rat chondrocytes. The values are expressed as mean ±SD (n=3) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test.*p < 0.05, **p < 0.01 versus the IL-1β group.

Figure 11

Specimen evaluation in animal models. (A, D) Representative images of safranin-O-stained rat knee joint sections with different treatments and the related Mankin scores of the three groups. Scale bar=200 μm. Data represent the mean ± SD (n = 30) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. ** p < 0.01 versus the OA group. (B) Representative immunohistochemistry images against MMP-13, COX-2, caspase-3, ADAMTS5, and Col2a1. Scale bar=200 μm; (C) Quantitative optical density analysis of immunohistochemistry for the different groups. Data represent the mean ± SD (n = 30) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test. **p < 0.01 versus the OA group.