Dihydroartemisinin ameliorates hemarthrosis-induced cartilage degeneration by suppressing chondrocyte senescence via activation of Keap1-Nrf2 signaling pathway

Abstract

Background: Joint bleeding (hemarthrosis) is a major manifestation of joint trauma, especially repeated and spontaneous in hemophilia patients. Hemarthrosis has been identified to induce the excessive reactive oxygen species (ROS) accumulation and permanent damage in articular cartilage. Dihydroartemisinin (DHA), a well-known clinical anti-malaria drug with few sides effects therapy, has been reported to possess anti-oxidative activity. This study was aimed at exploring the effect of DHA on blood-induced cartilage erosion and its underlying mechanisms.

Methods: Two distinct hemarthrosis models were constructed respectively by fresh blood joint injection in WT and joint needle puncture in F8 ^-/- mice, and then treated with DHA (10 or 20 mg/kg/day) for 4 weeks. In vitro chondrocytes treated with frozen-thaw blood and DHA (1, 5 or 10 μM) for 24 h. Histopathological, immunofluorescence and western blotting were investigated to demonstrate the effects of DHA on blood-induced chondrocyte senescence, ROS accumulation and extracellular matrix (ECM) degradation. Additionally, Nrf2 inhibitor (MLB385, 30 mg/kg for once a four days) and Nrf2-siRNA were used to investigate the relationship between DHA and Nrf2/Keap1 signaling in vitro and in vivo, respectively.

Results: DHA remarkably ameliorated the cartilage degeneration in both two hemarthrosis models. Similarly, in vitro experiments confirmed that DHA promoted the synthesis of ECM in blood-stimulated chondrocytes with a dose-dependent manner. DHA also effectively suppressed blood-induced chondrocyte senescence and ROS accumulation. Mechanistically, DHA activated the Nrf2 signaling by accelerating Keap1 ubiquitination and degradation. Furthermore, Nrf2 siRNA and antagonist abolished the anti-senescence and anti-oxidative functions of DHA, resulting the severe cartilage degeneration in bleeding joint of F8 ^-/- mice.

Conclusion: Our findings indicate that DHA effectively reduces chondrocyte senescence and mitigates cartilage destruction following hemarthrosis via activation of Nrf2/Keap1 signaling pathway.

The translational potential of this article: On the one hand, this study highlights the important role of chondrocyte senescence in hemarthrosis-induced cartilage degradation, implying that inhibiting chondrocyte senescence may be a viable therapeutic strategy for blood-induced arthropathy. On the other hand, our findings demonstrate the remarkable chondroprotective effect of DHA in bleeding joint by modulating the Nrf2/Keap1 anti-oxidative signaling pathway, suggesting DHA may serve as a potential candidate drug for the therapy of blood-induced arthropathy.

Keywords: Cartilage degeneration; Chondrocyte senescence; Dihydroartemisinin; Hemarthrosis; Nrf2/Keap1 signaling pathway; Oxidative stress.

Graphical abstract

Fig. 1

DHA delays cartilage degradation in joint hemorrhage mice. (a) Representative images of SO/FG staining and the OARSI score for knee sections of F8^−/− mice treated with or without DHA at 4 weeks post-injury. Red arrow indicates the wear area. Representative images and corresponding quantitative analysis of IHC staining of (b) Col2a1 and (c) Mmp13 in F8^−/− mice treated with or without DHA at 4 weeks post-injury. Red arrow indicates the positive cells; scale bar: 100 μm. (d) Representative images of SO/FG staining and the OARSI score for knee sections of WT mice treated with or without DHA at 4 weeks following blood injection. Red arrow indicates the wear area; scale bar: 100 μm. Representative images and corresponding quantitative analysis of IHC staining of (e) Col2a1 and (f) Mmp13 in WT mice treated with or without DHA at 4 weeks following injury. Red arrow indicates the positive cells; scale bar: 100 μm; each point represents one biological repeats (n = 6) The data were presented as means ± SD and subjected to analysis by one-way ANOVA with Tukey's multiple comparisons test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 2

DHA inhibits blood-induced ECM degradation in chondrocytes. Quantification of mRNA levels for (a)Adamts5,(b)Mmp3, (c)Mmp13, (d)Col2a1, (e)Aggrecan, and (f)Sox9 in mouse chondrocytes treated with DHA (1, 5 and 10 μM); each independent biological repeat (n = 3–4) with 3 technical repeats. (g) Western blot and corresponding quantification analysis of proteins for (h) Adamts5, (i) Mmp3, (j) Mmp13, (k) Col2a1, (l) Aggrecan, and (m) Sox9 in mouse chondrocytes treated with DHA (1, 5 and 10 μM); each independent biological repeat (n = 4) with 3 technical repeats. (n) Representative immunofluorescence images of Col2a1 and Mmp13 expressions in mouse chondrocytes treated with DHA (1,5 and 10 μM). Corresponding quantification of (o) Col2a1 and (p) Mmp13 fluorescence intensity; scale bar: 100 μm, each independent biological repeat (n = 4) with 3 technical repeats. The above data were presented as means ± SD and subjected to analysis by one-way ANOVA with Tukey's multiple comparisons test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 3

DHA reduced blood-induced oxidative stress in chondrocytes in vitro and in vivo. (a) Representative images and (b) corresponding quantitative analysis of ROS staining in mouse chondrocytes treated with DHA (1, 5 and 10 μM); scale bar: 100 μm; n = 3. Quantitative analysis of (c) SOD and (d) GSH/GSSH ratio in mouse chondrocytes treated with DHA (1, 5 and 10 μM); each independent biological repeat (n = 3) with 3 technical repeats. (e) Western blot and corresponding quantification analysis of proteins for (f) Nox1, (g) Nox2, and (h) Gpx4 in mouse chondrocytes treated with DHA (1,5 and 10 μM); each independent biological repeat (n = 4) with 3 technical repeats. (i) Representative images and (j) corresponding quantitative analysis of IHC staining of Nox1 and Nox2 in F8^−/− mice treated with or without DHA at 4 weeks post-injury. Red arrow indicates the positive cells; scale bar: 100 μm. (k) Representative images and (l) corresponding quantitative analysis of IHC staining of Gpx4 in F8^−/− mice treated with or without DHA at 4 weeks post-injury. Red arrow indicates the positive cells; scale bar: 100 μm. (m) Representative images and (n) corresponding quantitative analysis of ROS staining in F8^−/− mice at 4 weeks post-injury. Scale bar: 100 μm. For the data i-n, each point represents one biological repeats (n = 6). The data were presented as means ± SD and subjected to analysis by one-way ANOVA with Tukey's multiple comparisons test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 4

DHA reduced blood-induced senescence in chondrocytes in vitro and in vivo. (a) Heatmap of mRNA-seq analysis showing the expressions of senescence-related genes in chondrocytes induced by blood. (b) Western blot and corresponding quantification analysis of protein for (c) P21 in mouse chondrocytes treated with S1623 (10 μM); each independent biological repeat (n = 4) with 3 technical repeats. (d) Western blot and corresponding quantification analysis of proteins for (e) P16, (f) P21, and (g) P53 in mouse chondrocytes treated with DHA (1, 5 and 10 μM); each independent biological repeat (n = 3–4) with 3 technical repeats. Quantification of mRNA levels for (h)P16,(i)P21, and (j)P53 in mouse chondrocytes treated with DHA (1,5 and 10 μM); each independent biological repeat (n = 3) with 3 technical repeats. (k) Representative images and (l) corresponding quantitative analysis of SA-β-gal staining in mouse chondrocytes treated with DHA (1,5 and 10 μM); scale bar: 100 μm; each independent biological repeat (n = 3–4) with 3 technical repeats. (m) Representative images and (n) corresponding quantitative analysis of IHC staining of P16 and P21 in F8^−/− mice treated with or without DHA at 4 weeks post-injury; red arrow indicates the positive cells; scale bar: 100 μm. For the data m-n, each point represents one biological repeats (n = 6). The data were presented as means ± SD and subjected to analysis by one-way ANOVA with Tukey's multiple comparisons test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 5

DHA promotes Keap1 ubiquitination and degradation to suppress Nrf2 signaling in blood-induced chondrocytes in vitro. (a) Western blot and corresponding quantification analysis of proteins for (b) Nrf2, (c) Keap1 and (d) HO-1 in mouse chondrocytes treated with DHA (1, 5 and 10 μM); each independent biological repeat (n = 4) with 3 technical repeats. (e) Representative immunofluorescence images and (f) corresponding quantitative analysis of nuclear Nrf2 protein in mouse chondrocytes treated with DHA (1, 5 and 10 μM); scale bar: 100 μm; each independent biological repeat (n = 4) with 3 technical repeats. (g) Western blot analysis for Nrf2 protein in cytosolic and nuclear fractions, and (h) corresponding quantification analysis of in mouse chondrocytes treated with DHA (10 μM); each independent biological repeat (n = 4) with 3 technical repeats. (i–j) Molecular docking revealed the binding free energy of DHA to Keap1 protein. (k) Western blot and corresponding quantification analysis of proteins for (l) Keap1 in mouse chondrocytes treated with DHA (10 μM) and CHX (50 μM); each independent biological repeat (n = 4) with 3 technical repeats. (m) The Ubiquitination level of Keap1 was determined in the indicated group in chondrocytes; each independent biological repeat (n = 4) with 3 technical repeats. The data were presented as means ± SD and subjected to analysis by one-way ANOVA with Tukey's multiple comparisons test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 6

Nrf2 mediated the effect of DHA on ROS and senescence in blood-induced chondrocytes. (a) Representative images and (b) corresponding quantitative analysis of ROS staining in blood-induced chondrocytes treated with or without DHA, siRNA-NC, siRNA-Nrf2-345, or siRNA-Nrf2-1565; scale bar: 100 μm; each independent biological repeat (n = 4) with 3 technical repeats. Quantitative analysis of (c) SOD and (d) GSH/GSSH ratio in blood-induced chondrocytes treated with or without DHA, siRNA-NC, siRNA-Nrf2-345 or siRNA-Nrf2-1565; each independent biological repeat (n = 4) with 3 technical repeats. (e) Western blot and corresponding quantification analysis of proteins for (f) Nrf2, (g) Gpx4, (h) Nox1, (i) Nox2, (j) P16, and (k) P21 in blood-induced chondrocytes treated with or without DHA, siRNA-NC, siRNA-Nrf2-345, or siRNA-Nrf2-1565; each independent biological repeat (n = 4) with 3 technical repeats. (l) Representative images and (m) corresponding quantitative analysis of SA-β-gal staining in blood-induced chondrocytes treated with or without DHA, siRNA-NC, siRNA-Nrf2-345, or siRNA-Nrf2-1565; scale bar: 100 μm; each independent biological repeat (n = 4) with 3 technical repeats. The data were presented as means ± SD and subjected to analysis by one-way ANOVA with Tukey's multiple comparisons test, ∗∗∗P < 0.001.

Fig. 7

DHA activates Nrf2/Keap1 signaling in cartilage from F8^−/− mice following joint bleeding. Representative images and corresponding quantitative analysis of IHC staining of (a) Nrf2, (b) Keap1 and (c) HO-1 in F8^−/− mice treated with or without DHA at 4 weeks post-injury. Red arrow indicates the positive cells; scale bar: 100 μm. For the data a-c, each point represents one biological repeats (n = 6). (e) Representative images of SO/FG staining for knee sections of F8^−/− mice treated with or without DHA or ML385 at 4 weeks post-injury and the OARSI score. Red arrow indicates the wear area; scale bar: 100 μm. (f) Representative images and corresponding quantitative analysis of ROS staining in F8^−/− mice treated with or without DHA or ML385 at 4 weeks post-injury. Scale bar: 100 μm. Representative images and corresponding quantitative analysis of IHC staining of (g) P16 and (h) HO-1 in F8^−/− mice treated with or without DHA or ML385 at 4 weeks post-injury. Red arrow indicates the positive cells; scale bar: 100 μm. (i) Representative 3D reconstruction and corresponding quantification analysis of BV/TV (%) of the subchondral bone at F8^−/− mice treated with or without DHA or ML385 at 4 weeks post-injury. Scale bar: 100 μm. For the data e-i, each point represents one biological repeats (n = 3). The data were presented as means ± SD and subjected to analysis by one-way ANOVA with Tukey's multiple comparisons test; ns: no significance; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.