Astaxanthin protects against osteoarthritis via Nrf2: a guardian of cartilage homeostasis

Abstract

Scope: Osteoarthritis (OA) is a progressive disease characterized by cartilage degradation. Astaxanthin (Ast), a natural compound with remarkable antioxidant activity and multiple medical applications due to its activation of Nrf2 signaling, has been studied for application to various degenerative diseases. Currently, however, little is known about its efficacy in treating OA. This study reports the effects of Ast on cartilage homeostasis in OA progression.

Methods: IL-1β, TNF-α, and tert-butyl hydroperoxide (TBHP) were used to impair cartilage homeostasis. Modulating effects of Ast on the Nrf2 signaling pathway, and damage-associated events including extracellular matrix (ECM) degradation, inflammation, oxidative stress, chondrocyte apoptosis, and in vivo cartilage degradation were examined.

Results: Ast attenuated ECM degradation of OA chondrocytes through the Nrf2 signaling, and ameliorated the IL-1β-induced inflammatory response and ECM degradation via blockade of MAPK signaling. Additionally, Ast alleviated TNF-α-induced ECM degradation and chondrocyte apoptosis by inhibiting the NF-κB signaling, suppressed TBHP-induced oxidative stress, and subsequently reduced chondrocyte apoptosis. In vitro results were finally corroborated in vivo by demonstrating that Ast attenuates the severity of cartilage destruction in a mouse model of OA.

Conclusions: Ast could protect against osteoarthritis via the Nrf2 signaling, suggesting Ast might be a potential therapeutic supplement for OA treatment.

Keywords: Nrf2; apoptosis; astaxanthin; cartilage homeostasis; osteoarthritis.

Conflict of interest statement

CONFLICTS OF INTEREST: The authors confirm that there are no conflicts of interest.

Figures

Figure 1

Ast did not affect cell viability and activated Nrf2 in mouse chondrocytes. (A) The cytotoxic effect of Ast (5, 10, 20, 40, and 80 μM) exposure for 24 and 48 h on chondrocytes was determined using a CCK8 assay. (B, C) Chondrocytes were treated with Ast (5, 10, and 20 μM) for 24 h. Expression levels of Cyclin D1, Nrf2, and Keap1 were determined by western blotting and quantified. (D, E) Nuclear translocation of Nrf2 was detected by western blotting and immunofluorescence after treatment of chondrocytes with Ast (10 μM) for 24 h, and the band density of Nrf2 in nucleus was quantified. The nuclear and cytoplasmic fractions used in the western blotting were obtained using a nuclear and cytoplasmic protein extraction kit (P0027, Beyotime, China). The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as *p < 0.05 vs. control; **p<0.01 vs. control; ****p<0.0001 vs. control.

Figure 2

Effects of IL-1β, TNF-α, and TBHP on mouse chondrocytes. (A) The cytotoxic effect of IL-1β (1 and 5 ng/ml), TNF-α (1 and 5 ng/ml), and TBHP (50 and 100 μM) treatment on chondrocytes for 24 h was determined using a CCK8 assay. (B, C) After the indicated treatment for 24 h, the effects of IL-1β (5 ng/ml), TNF-α (5 ng/ml), and TBHP (100 μM) on the expression of MMP13, Collagen II, iNOS, and cleaved-caspase3 were determined by western blotting and quantified. The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as *p < 0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ****p<0.0001 vs. control.

Figure 3

Effects of Ast on OA chondrocytes. Chondrocytes were pretreated with or without Ast (5, 10, and 20 μM) for 2 h and then exposed to IL-1β (5 ng/ml) for 24 h. Then, (A–C) the expression of Collagen II, ADAMTS5, MMP3, and MMP13 was examined by RT-PCR and western blotting and quantified. Chondrocytes were treated with the vehicle or with increasing concentrations of Ast for 2 h followed by (5 ng/ml) TNF-α stimulation. (D, E) The expression of Collagen II, ADAMTS5, MMP3, and MMP13 were determined by western blotting and quantified. After the cells were treated with the vehicle or Ast (5, 10, and 20 μM) for 2 h, and stimulated with 100 μM TBHP, (F, G) the expression of Collagen II, ADAMTS5, and MMP13 was investigated by western blotting and quantified. The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as **p<0.01 vs. control; ***p<0.001 vs. control; ****p<0.0001 vs. control. #p < 0.05 vs. IL-1β or TNF-α or TBHP group; ##p < 0.01 vs. IL-1β or TNF-α or TBHP group; ###p < 0.001 vs. IL-1β or TNF-α or TBHP group; ####p < 0.0001 vs. IL-1β or TNF-α or TBHP group.

Figure 4

Nrf2 signaling mediated protective effects of Ast on OA chondrocytes. Chondrocytes were cultured with vehicle or Ast (5, 10 and 20 μM) for 2 h, then stimulated with IL-1β (5 ng/ml), TNF-α (5 ng/ml) or TBHP (100 μM). (A–C) Levels of Nrf2 signaling pathway proteins, including Nrf2, HO-1, and NQO1 were determined by western blotting and quantified. To knock down Nrf2 expression, chondrocytes were transfected with Nrf2 siRNA using Lipofectamine 3000. (D) Transfection efficiency was evaluated by detecting Nrf2 expression using western blotting. After 24h of transfection, chondrocytes were treated with vehicle or Ast (10 μM) for 2 h followed by stimulation with IL-1β (5 ng/ml) for 24 h. (E, F) The expression of Collagen II, ADAMTS5, MMP3, and MMP13 was measured by western blotting and quantified. The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as *p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ****p<0.0001 vs. control. #p < 0.05 vs. IL-1β or TNF-α or TBHP group; ##p < 0.01 vs. IL-1β or TNF-α or TBHP group; ###p < 0.001 vs. IL-1β or TNF-α or TBHP group; ####p < 0.0001 vs. IL-1β or TNF-α or TBHP group. &p < 0.05 vs. (IL-1β or TNF-α or TBHP) + Ast group; &&p < 0.01 vs. (IL-1β or TNF-α or TBHP) + Ast group; &&&p < 0.001 vs. (IL-1β or TNF-α or TBHP) + Ast group.

Figure 5

Effects of Ast on IL-1β-induced inflammatory response. Chondrocytes were treated with Ast (5, 10, and 20 μM) for 2 h followed by stimulation with vehicle or IL-1β (5 ng/mL) for 24 h. (A) Expression of iNOS and COX-2 protein assessed by western blotting and quantified. (B) To detect altered phosphorylation within the MAPK signaling pathway, chondrocytes were serum-starved for 6 h followed by treatment with vehicle or Ast (5,10, and 20 μM) for 2 h. Cells were then stimulated with IL-1β (5 ng/mL) for 30 min. MAPK activation was examined using western blotting and quantified. (C) Phosphorylation of ERK was detected by western blotting after chondrocytes were pre-treated with vehicle or PD0325901 (a MEK inhibitor) for 2 h, followed by stimulation with IL-1β (5 ng/ml). (D) Chondrocytes were treated as indicated for 24 h. The expression of COX2, Collagen II, and MMP3 was determined using western blotting and quantified. The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as *p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ****p<0.0001 vs. control. #p < 0.05 vs. IL-1β group; ##p < 0.01 vs. IL-1β group; ###p < 0.001 vs. IL-1β group; ####p < 0.0001 vs. IL-1β group.

Figure 6

Effects of Ast on TNF-α-induced apoptosis and ECM degradation. (A, B) Protein levels of BAX, Bcl-2, and cleaved caspase-3 were determined by western blotting, and apoptotic chondrocytes were stained with Annexin V-FITC/PI and examined by flow cytometry after treated as above for 24 h. Apoptosis rate was calculated and the data were expressed in (C). FL1 represents Annexin V-FITC and FL2 represents PI. To detect the activation of NF-κB signaling, chondrocytes were serum-starved for 6 h followed by treatment with the vehicle or Ast (5,10, and 20 μM) for 2 h. Cells were then stimulated with TNF-α (5 ng/ml) for 15 min. (D) Activation of the NF-κB signaling pathway was measured by western blotting and quantified. (E) Phosphorylation of p65 was detected by western blotting after the chondrocytes were pre-treated with the vehicle or QNZ (an inhibitor of the NF-κB pathway) for 2 h, followed by stimulation with TNF-α (5 ng/ml). (F, G) Chondrocytes were treated as indicated for 24 h. The expression of Collagen II, MMP3 and cleaved-caspase3 was determined using western blotting and quantified. The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as *p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control. #p < 0.05 vs. TNF-α group; ##p < 0.01 vs. TNF-α group; ###p < 0.001 vs. TNF-α group.

Figure 7

Effects of Ast on TBHP-induced oxidative stress, and chondrocyte apoptosis. Chondrocytes were treated with Ast (10 μM) for 2 h, then stimulated with TBHP (100 μM) for 24 h. The samples with the same concentration of chondrocytes were used to detect intracellular ROS. Intracellular ROS was stained by DCFH-DA followed by fluorescence spectrophotometry and flow cytometric analysis. (A) Green fluorescence indicates that the intracellular ROS production. (B) Representative data of flow cytometric measurement of ROS production. (C) Dot plots graphs show the mean fluorescence intensity (MFI) of ROS in chondrocytes. (D, E) Mitochondrial membrane potential (MMP) was detected by fluorescence and flow cytometric analysis after chondrocytes were incubated with JC-1. Red fluorescence represents JC-1 aggregates in healthy mitochondria, while green fluorescence is emitted by JC-1 monomers, representing MMP dissipation. Merged images exhibit co-localization of JC-1 aggregates and monomers. FL1 represents JC-1 green and FL2 represents JC-1 red. (F) Dot plots graphs represent flow cytometric analysis of MMP levels, represented as the ratio of red/green MFI. (H) To measure chondrocyte apoptosis, cells were stained with Annexin V-FITC/PI and apoptotic cells were counted by flow cytometry. FL1 represents Annexin V-FITC and FL2 represents PI. Apoptosis rates were evaluated, and the results are expressed as dot plots graphs (I). (G) Chondrocytes were treated with vehicle or Ast (5, 10, and 20 μM) for 2 h, then stimulated with TBHP (100 μM) for 24 h. Protein levels of BAX, Bcl-2, and cleaved-caspase3 were examined by western blotting and quantified. (J) Expression of cleaved-caspase 3 protein in chondrocytes under treatment with scrambled siRNA (NC), TBHP (100 μM), N-acetylcysteine (NAC)+TBHP, Nrf2-siRNA+TBHP, respectively. The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as *p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control. #p < 0.05 vs. TBHP group; ##p < 0.01 vs. TBHP group.

Figure 8

Effects of Ast on cartilage degradation in vivo. Mice received intra-articular injections of Ast (20 mg/kg) or vehicle for 8 weeks after DMM surgery. (A) Histological analysis of cartilage degradation was evaluated by safranin O staining. Representative safranin O staining of cartilage (scale bar: 200 μm and 50 μm). (B) Osteoarthritis Research Society International (OARSI) scores of three groups. (C) Nrf2 expression in cartilage was detected by immunofluorescence staining (scale bar: 50 μm). (D) Dot plots graphs represent rates of nuclear Nrf2 in total chondrocytes from each cartilage section. The data are presented as dot plots from three independent experiments. Significant differences among different groups are indicated as **P < 0.01; ***P<0.001; ****P < 0.0001.

Figure 9

Schematic diagram of Ast-mediated protective effects on cartilage homeostasis. Ast attenuated ECM degradation of OA chondrocytes through the Nrf2 signaling, and ameliorated the IL-1β-induced inflammatory response and ECM degradation via blockade of MAPK signaling. In addition, Ast alleviated TNF-α-induced ECM degradation and chondrocyte apoptosis by inhibiting the NF-kB signaling, suppressed TBHP-induced oxidative stress, and subsequently reduced chondrocyte apoptosis.