Cuprorivaite microspheres inhibit cuproptosis and oxidative stress in osteoarthritis via Wnt/β-catenin pathway

Abstract

This study aims to evaluate the therapeutic potential of cuprorivaite microspheres for osteoarthritis (OA), in particular, potential molecular mechanisms were investigated. The microspheres were developed from Ca(NO₃)₂•4H₂O, Cu(NO₃)₂•3H₂O, and silica gel, and further therapeutic effects were tested in vitro on mouse primary chondrocytes treated with interleukin-1β (IL-1β) to mimic OA, and in vivo on OA mice induced via anterior cruciate ligament transection (ACLT) surgery. The microspheres were shown to mitigate IL-1β-induced apoptotic, inflammatory, oxidative stress and cuproptosis markers while enhancing cell viability and extracellular matrix (ECM) components in chondrocytes. Moreover, the microspheres ameliorated histopathological damage, reduced inflammatory, oxidative stress and cuproptosis markers, and enhanced ECM biomarker levels in OA mice, implicating their role in suppressing cuproptosis and oxidative stress. The aforementioned effects of the cuprorivaite microspheres were demonstrated by using SKL2001, an agonist of the Wnt/β-catenin pathway. The results suggest cuprorivaite microspheres as a promising intervention for OA and cartilage regeneration, highlighting their therapeutic effects on cellular and molecular levels.

Keywords: Cuproptosis; Cuprorivaite microspheres; Osteoarthritis; Oxidative stress; Wnt/β-catenin pathway.

Graphical abstract

Fig. 1

Characterization of Cuprorivaite (Egyptian blue, CaCuSi₄O₁₀) microspheres. (A) Flowchart of CaCuSi₄O₁₀ microspheres synthesis. (B) The schematic crystal structure of CaCuSi₄O₁₀ using the spatial model. (C) Representative SEM images for CaCuSi₄O₁₀. (D) EDS mapping. (E) XRD examination and a representative gross image of CaCuSi₄O₁₀. (F) Particle size distributions of the microspheres. (G) Cu²⁺ release detected by inductively-coupled plasma emission spectrometer.

Fig. 2

Toxicity examination of in vitro and in vivo. (A–C) Blood routine tests include hemoglobin (HGB), aspartate aminotransferase (AST), alanine transaminase (ALT), white blood cell (WBC), and platelet (PLT). (D) Hematoxylin and eosin staining for heart, liver, spleen, lung and kidney. (E) Cell counting kit-8 test for cell viability. Compared with the control group, ns indicates not significant, ∗∗∗P < 0.001.

Fig. 3

Anti-inflammatory role of Cuprorivaite (CaCuSi₄O₁₀) microspheres in IL-1β-stimulated chondrocytes. (A) Cell counting kit-8 test for cell proliferation. (B) Fluorescein diacetate for cell viability detection. (C) Cell apoptosis by flow cytometry. (D) ELISA detection for TNF-α, IL-6 and MMP13 in cell supernatant. (E) Western blot detection for TNF-α, IL-6 and MMP13 in cell lysates. (F) RT-qPCR quantification for in TNF-α, IL-6 and MMP13. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 4

Anti-cuproptosis and anti-oxidative effects of Cuprorivaite (CaCuSi₄O₁₀) microspheres in IL-1β-stimulated chondrocytes. (A) RT-qPCR quantification for extracellular matrix components including collagen II and SOX9. (B) Intracellular copper content. (C) RT-qPCR quantification for cuproptosis biomarkers including ATP7B and FDX1. (D) Western blot detection for ATP7B and FDX1. (E) ROS generation detected by flow cytometry. (F) Oxidative stress detection including MDA, SOD and GSH. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 5

Role of Cuprorivaite (CaCuSi₄O₁₀) microspheres via Wnt/β-catenin pathway in IL-1β-stimulated chondrocytes. (A–C) RT-qPCR quantification for Wnt1 (A), GSK3β (B) and β-catenin (C). (D) Western blot detection for Wnt1, GSK3β and β-catenin in cell lysates. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 6

Cuprorivaite (CaCuSi₄O₁₀) microspheres improved IL-1β-induced injury in chondrocytes via inhibiting Wnt/β-catenin pathway. (A) Cell counting kit-8 test for cell viability. (B) Fluorescein diacetate for cell viability detection. (C) Cell apoptosis by flow cytometry. (D) ELISA detection for TNF-α, IL-6 and MMP13 in cell supernatant. (E) Western blot detection for TNF-α, IL-6 and MMP13 in cell lysates. (F) RT-qPCR quantification for extracellular matrix components including collagen II and SOX9. (G) Intracellular copper content. (H) RT-qPCR quantification for cuproptosis biomarkers including ATP7B and FDX1. (I) Western blot detection for ATP7B and FDX1. (J) Oxidative stress detection including MDA, SOD and GSH. (K) RT-qPCR quantification for Wnt1, GSK3β and β-catenin. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 7

Validation of mechanism of Cuprorivaite (CaCuSi₄O₁₀) microspheres in OA. (A) Hematoxylin and eosin staining. (B) Safranin-O staining/fast green staining. (C) OARSI score. (D) ELISA detection for TNF-α, IL-6 and MMP13 in the serum. (E) Western blot detection for TNF-α, IL-6 and MMP13 in cartilage tissue. (F) RT-qPCR quantification for extracellular matrix components including collagen II and SOX9 in cartilage tissue. (G) Copper content in cartilage tissue. (H) RT-qPCR quantification for cuproptosis biomarkers including ATP7B and FDX1 in cartilage tissue. (I) Western blot detection for ATP7B and FDX1 in cartilage tissue. (J) Representative images of FDX1 expression in cartilage tissue detected by immunohistochemistry. (K) Oxidative stress detection including MDA, SOD and GSH in cartilage tissue. (L) RT-qPCR quantification for Wnt1, GSK3β and β-catenin in cartilage tissue. ∗∗∗P < 0.001.

Fig. 8

The potential mechanism by which Cuprorivaite microspheres improved cartilage injury in OA. By Figdraw.