Schisandrin A Inhibits the IL-1β-Induced Inflammation and Cartilage Degradation via Suppression of MAPK and NF-κB Signal Pathways in Rat Chondrocytes

Abstract

Osteoarthritis (OA) is a common joint disease in the elderly population. Its development has been reported to be associated with cartilage degradation and inflammatory responses. Schisandrin A, a bioactive lignin in Schisandra sphenanthera, has shown its anti-inflammatory potential in various inflammation diseases. However, the effects of Schisandrin A on OA remain to explore. In this study, rat chondrocytes were treated with IL-1β (10 ng/ml) with or without different concentrations of Schisandrin A for 24 h. Cell viability was evaluated by CCK-8 assay. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) was measured by the Griess reaction and ELISA. The MAPK/NF-κB-related signaling molecules expression and the protein production of inducible nitric oxide synthase (iNOS), cyclooxygenase (Cox)-2, MMPs (MMP1, MMP3, MMP13), ADAMTS5, Collagen II, aggrecan, and Sox9 were detected by Western blot. Protein expression of Collagen II, aggrecan, and p65 nuclear translocation was evaluated by immunofluorescence. In vivo, intra-articular injection of 50 μM Schisandrin A or equal volume of vehicle was performed on rat OA models. Severity of cartilage damage was evaluated by HE and Safranin-O-Fast green staining. Our results revealed that Schisandrin A could suppress the IL-1β-induced production of NO and PGE2 in rat chondrocytes. Consistent with these findings, the upregulation of iNOS and Cox2 could also been decreased by Schisandrin A. Additionally, Schisandrin A could inhibit IL-1β-induced cartilage matrix catabolic enzymes including MMPs and ADAMTS5. Moreover, the IL-1β-induced downregulation of Collagen II, aggrecan, and Sox9 could be ameliorated by Schisandrin A. Mechanistically, Schisandrin A functioned by suppressing MAPK and NF-κB signal pathways. In vivo, Schisandrin A prevented cartilage damage in rat OA model. In conclusion, this study elucidates that Schisandrin A inhibits the IL-1β-induced inflammation and cartilage degradation via suppression of MAPK and NF-κB signal pathways, indicating its potential role in OA therapy.

Keywords: ADAMTS5; Cox-2; MAPK; MMPs; NF-κB; Schisandrin A; iNOS; osteoarthritis.

FIGURE 1

Effects of Schisandrin A on cell viability. (A) Rat chondrocytes were exposed to Schisandrin A (25, 50 μM) alone or (B) with IL-1β (10 ng/ml) for 24 h and cell viability was determined by CCK-8 assay. ^∗∗P < 0.01 vs. control group.

FIGURE 2

Effects of Schisandrin A on IL-1β-induced production of NO, PGE2, iNOS, and COX2 in rat chondrocytes. Cells were treated with Schisandrin A (25, 50 μM) in the presence or absence of IL-1β (10 ng/ml) for 24 h. Cell culture supernatants were collected. (A) Griess reaction was used to measure the NO concentration (n = 3). (B) PGE2 level was accessed by ELISA (n = 3). (C) Expression of iNOS and COX2 were detected by Western blot. (D) Relative protein expression was qualified by ImageJ software, GAPDH was served as the loading control (n = 3). ^#P < 0.05 vs. control group; ^∗P < 0.05 vs. IL-1β group; ^∗∗P < 0.01 vs. IL-1β group.

FIGURE 3

Effects of Schisandrin A on IL-1β-induced MMPs and ADAMTS5 protein expression. Chondrocytes were exposed to Schisandrin A (25, 50 μM) with or without IL-1β (10 ng/ml) for 24 h. (A) Western blot was employed to determine the expression of MMP1, MMP3, MMP13, and ADAMTS5. (B) Relative protein expression was qualified by Image-J software, GAPDH was used as the internal control (n = 3). ^#P < 0.05 vs. control group; ^∗P < 0.05 vs. IL-1β group; ^∗∗P < 0.01 vs. IL-1β group.

FIGURE 4

Effects of Schisandrin A on IL-1β-induced cartilage degradation. (A) Cells were treated with Schisandrin A (25, 50 μM) in the presence or absence of IL-1β (10 ng/ml) for 24 h. Protein levels of Collagen II, Aggrecan and Sox9 were determined by Western Blot. (B) Relative protein expression was qualified by Image-J software, GAPDH was used as the loading control (n = 3). (C) Aggrecan and (D) Collagen II were observed by Immunofluorescence after cells were treated with IL-1β (10 ng/ml) with or without Schisandrin A (50 μM) for 24 h. ^#P < 0.05 vs. control group; ^∗P < 0.05 vs. IL-1β group; ^∗∗P < 0.01 vs. IL-1β group.

FIGURE 5

Effects of Schisandrin A on MAPK signaling pathway. Rat chondrocytes were exposed to Schisandrin A (25, 50 μM) with or without IL-1β (10 ng/ml) for 30 min. (A) Phosphorylations of ERK, p38, JNK were determined by Western blot. (B) Relative protein expression was qualified by ImageJ software, ERK, p38, and JNK were used as the internal control, respectively (n = 3). ^#P < 0.05 vs. control group; ^∗∗P < 0.01 vs. IL-1β group.

FIGURE 6

Effects of Schisandrin A on NF-κB signaling pathway. Cells were exposed to Schisandrin A (25, 50 μM) with or without IL-1β (10 ng/ml) for 30 min. (A) Protein levels of p-IκBα, IκBα, p-p65, p65 were detected by Western blot. (B) Relative protein expression was qualified by ImageJ software, GAPDH and p65 were used as the loading control, respectively (n = 3). (C) p65 translocation was observed by Immunofluorescence. (D) Quantitative analysis of p65 nuclear location/total ratio of three groups. ^#P < 0.05 vs. control group; ^∗P < 0.05 vs. IL-1β group; ^∗∗P < 0.01 vs. IL-1β group.

FIGURE 7

Effects of Schisandrin A on rat Osteoarthritis. (A) Marcroscopic images of rat knee joint samples of three groups. (B) Microscopic photos of HE stained rat knee joint sections of three groups. (C) Microscopic photos of Safranin-O-Fast green stained rat knee joint sections of three groups. (D) The OARSI scores of each group. ^#P < 0.05 vs. sham group; ^∗∗P < 0.01 vs. vehicle group.

FIGURE 8

Schematic diagram of the effect of Schisandrin A on IL-1β-Induced Inflammation and Cartilage Degradation. IL-1β induces proinflammatory factors including iNOS, COX2, MMPs, and ADAMTS5 which subsequently trigger the release of NO, PGE2 and degradation of Collageren II, Aggrecan. Further, IL-1β functions by activating MAPK and NF-κB signaling pathways characterized by upregulation of p-ERK, p-p38, p-JNK, p-IκBα, p-p65 and degradation of IκBα, and translocation of p65. However, Schisandrin A can reverse this process.