Quercetin-3-O-β-D-glucuronide attenuates osteoarthritis by inhibiting cartilage extracellular matrix degradation and inflammation

Abstract

Objective: Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage damage. In order to find a safer and more effective drug to treat OA, we investigated the role of quercetin-3-O-β-D-glucuronide (Q3GA) in OA.

Methods: We used qRT-PCR and western blots to detect the effects of Q3GA on extracellular matrix (ECM) and inflammation related genes and proteins in interleukin-1β (IL-1β) induced chondrocytes. We determined the effect of Q3GA on the NF-κB pathway using western blots and immunofluorescence. Moreover, the effect of Q3GA on the Nrf2 pathway was evaluated through molecular docking, western blots, and immunofluorescence experiments and further validated by transfection with Nrf2 siRNA. Subsequently, we established a rat model of OA and injected Q3GA into the joint cavity for treatment. After 5 weeks of Q3GA administration, samples were obtained for micro-computed tomography scanning and histopathological staining to determine the effects of Q3GA on OA rats.

Results: We found that Q3GA reduced the degradation of ECM and the expression of inflammatory related proteins and genes in primary chondrocytes of rats induced by IL-1β, as well as the expression of nitric oxide (NO) and reactive oxygen species (ROS). It inhibited the activation of the NF-κB pathway by increasing the expression of Nrf2 in the nucleus. In addition, Q3GA inhibited cartilage degradation in OA rats and promoted cartilage repair.

Conclusion: Q3GA attenuates OA by inhibiting ECM degradation and inflammation via the Nrf2/NF-κB axis.

The translational potential of this article: The results of our study demonstrate the promising potential of Q3GA as a candidate drug for the treatment of OA and reveal its key mechanisms.

Keywords: Extracellular matrix; Inflammation; Nrf2; Osteoarthritis; Quercetin-3-O-β-D-glucuronide.

Graphical abstract

Figure 1

Effects of quercetin-3-O-β-D-glucuronide (Q3GA) on the viability of chondrocytes. (A) Chemical structure of Q3GA. (B, C) MTT kit was used to determine the cytotoxic effects of different concentrations of Q3GA (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160 μM) on chondrocytes for 24 and 48 h, n = 5.

Figure 2

Q3GA on the expression of genes and proteins in the extracellular matrix (ECM) and inflammation in chondrocytes. (A) QRT–PCR was used to detect the mRNA expression of Col2a1, Acan, Adamts5, Mmp13, and Mmp3, n = 3. (B) Western blots were used to detect the protein expression of Col2a1, ACAN, ADAMTS5, MMP13, and MMP3, n = 3. (C) QRT–PCR was used to detect the mRNA expression of Inos and Cox2, n = 3. (D) Western blots were used to detect the protein expression of iNOS and COX2, n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with the IL-1β induced group.

Figure 3

The effect of Q3GA on the NF-κB pathway in IL-1β-induced chondrocytes. (A) The level of NO in the supernatant of chondrocytes, n = 3. (B) The level of ROS in chondrocytes (scale bar: 50 μm). (C) The protein expression levels of IκBα, p-p65, and p65 in whole cells, as well as the expression level of p65 in the nucleus, n = 3. (D) The nuclear translocation of p65 was detected using immunofluorescence staining (scale bar: 50 μm). *P < 0.05, and ***P < 0.001, compared with the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with the IL-1β induced group.

Figure 4

The effect of Q3GA on the Nrf2 pathway in IL-1β-induced chondrocytes. (A) The 3D structure of Q3GA. (B) The 3D structure of the Nrf2 protein. (C) Molecular docking results of Q3GA with potential candidate proteins. (D) A combined 3D structure of Q3GA and Nrf2. (E) Western blots revealed the protein expression of Nrf2 in the nucleus and HO-1 in whole cells, n = 3. (F) Immunofluorescence assay was used to detect the expression of Nrf2 (scale bar: 50 μm). #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with the IL-1β-induced group.

Figure 5

The effect of knockdown of Nrf2 on IL-1β-induced chondrocytes. (A) The mRNA expression of Nrf2 and Ho-1 in chondrocytes treated with or without Nrf2 siRNA was detected using qRT–PCR, n = 3. (B) The protein expression of Nrf2 and HO-1 in chondrocytes treated with or without Nrf2 siRNA was detected using western blots, n = 3. (C) The protein expression of p-p65 and p65 in chondrocytes was detected using western blots, n = 3. (D) The protein expression of ACAN, ADAMTS5, MMP13, MMP3, and iNOS was detected using western blots, n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 6

Q3GA ameliorates OA in the MNX rat model. (A) The 3D micro-CT images of the sagittal view of the medial compartment of the knee joint six weeks after surgery, scale = 1 mm. (B) Quantitative analysis of BV/TV, Tb.N, Tb.Sp, n = 6. (C) The OARSI score for assessing the degree of cartilage damage, n = 6. (D) Cartilage staining of S&F, H&E, and Masson (scale bar: 250 μm). ***P < 0.001, compared with the control group; #P < 0.05, ##P < 0.01 and ###P < 0.001, compared with the OA group.

Figure 7

Q3GA inhibits ECM degradation in OA rats. (A) Immunohistochemical staining assay for ACAN, ADAMTS5, MMP13, and Nrf2 in the cartilage. (B) Quantitative analysis of ACAN, ADAMTS5, MMP13, and Nrf2 using immunohistochemistry staining, (scale bar: 100 μm). ***P < 0.001, compared with the control group; #P < 0.05, ##P < 0.01 and ###P < 0.001, compared with the OA group.