Emodin Attenuates the ECM Degradation and Oxidative Stress of Chondrocytes through the Nrf2/NQO1/HO-1 Pathway to Ameliorate Rat Osteoarthritis

Abstract

Osteoarthritis (OA) substantially reduces the quality of life of the elderly. OA therapy remains a challenge since no treatment options for its causes are so far available. Over recent years, researchers have speculated that emodin may represent a potential treatment strategy for OA. However, it remains unclear whether the mechanism of action of emodin is associated with the inhibition of OA-induced oxidative stress. In the present study, the potential antioxidant mechanism of action of emodin and its protective properties against the development of OA were investigated both in vitro and in vivo. In vitro, emodin inhibited the production of reactive oxygen species (ROS) in chondrocytes induced by hydrogen peroxide (H₂O₂) and reduced the expression of matrix metalloproteinase (MMP)3 and MMP13 in a concentration-dependent manner. It was found that emodin upregulated the Nrf2/NQO1/HO-1 pathway, thereby attenuating the effects of oxidative stress caused by OA. In a rat model of posttraumatic OA induced by anterior cruciate ligament transection (ACLT), emodin reduced the extent of joint swelling. Emodin attenuated oxidative damage in the cartilage by upregulating superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) activity, reducing malondialdehyde (MDA) concentration and inhibiting the expression of the extracellular matrix (ECM) degradation biomarkers cartilage oligomeric matrix protein (COMP), and C-terminal telopeptide of type I collagen (CTX-I) and type II collagen (CTX-II), thereby reducing cartilage damage. In summary, the present study indicates that emodin reduces ECM degradation and oxidative stress in chondrocytes via the Nrf2/NQO1/HO-1 pathway, thereby ameliorating OA in rats.

Figure 1

Establishment of rat posttraumatic OA model and in vivo experiment design. (a) Rats were anesthetized by inhalation of 2% isoflurane in oxygen/nitrous oxide. (b) Depilation and disinfection of the right knee joint of the rat. (c) Make a longitudinal 2-3 cm incision on the inside with a scalpel. (d) After the patella is displaced, the ACL is cut under a surgical microscope. (e) Under sterile conditions, use absorbable slits to suture the wound. (f) The flow chart of the in vivo study design, showing the time of administration of emodin, the treatment period, and the time of euthanasia.

Figure 2

Primary chondrocytes and the effects of emodin on chondrocyte viability with and without H₂O₂. (a) Chemical structure of emodin. (b) Representative images of the morphology of primary rat chondrocytes, respectively, showing the changes in cell morphology and proliferation of primary chondrocytes, first-generation chondrocytes, second-generation chondrocytes, and H₂O₂ stimulation of chondrocytes for 12 h. (c) Representative image of type II collagen immunofluorescence and toluidine blue staining. Type II collagen in the ECM of chondrocytes is green fluorescent. (d) Use CCK-8 kit to detect the cell viability of chondrocytes treated with different concentrations of H₂O₂ (0, 0.1, 0.3, 0.4, 0.5, 1, and 2 mM) and (e) different concentrations of emodin (0, 5, 10, 15, and 20 μM) for 12 h. (f) Adding 5, 10, and 20 μM emodin to chondrocytes treated with 0.4 mM H₂O₂ can increase cell viability. The data were expressed as the means ± SD (n = 3). ^∗P < 0.05 and^∗∗P < 0.01.

Figure 3

Effect of emodin on ROS and the Nrf2/NQO1/HO-1 signaling pathway in chondrocytes. (a, b) Western blot was used to detect the protein expression of Nrf2, HO-1, and NQO1 and to analyze the effect of different concentrations of H₂O₂ on oxidative stress of chondrocytes for 12 h. (c) DCFH-DA fluorescence staining demonstrated that emodin and H₂O₂ inhibited the overproduction of ROS when cocultured. (d) Mean fluorescence intensity of chondrocytes after administration of emodin. (e) Expression levels of cytosolic Nrf2, NQO1, and HO-1 evaluated by Western blots (f) were quantified. (g) Expression levels of nuclear Nrf2 evaluated by Western blots (h) were quantified. Data were expressed as the means ± SD (n = 3). ^∗P < 0.05 and^∗∗P < 0.01.

Figure 4

Effect of emodin on the mRNA and protein expression of MMP3 and MMP13 in chondrocytes induced by H₂O₂. (a) The protein expression levels of MMP3 and MMP13 were evaluated by Western blots and (b) quantified. (c) The mRNA expression levels of MMP3 and (d) MMP13 were evaluated by qPCR. (e) Heatmap of MMP3 and MMP13 expression in chondrocytes induced by H₂O₂. The data were expressed as the means ± SD (n = 3). ^∗P < 0.05 and^∗∗P < 0.01.

Figure 5

Effect of emodin on the swelling of the knee joint in rats. The data are presented as the means ± SD (n = 3). ^∗P < 0.05 and^∗∗P < 0.01 compared with the control group.

Figure 6

Progression of OA in rats induced by ACLT with or without emodin. (a) Macroscopic image and (b) Pelletier score of the rat knee joint 6 weeks after emodin administration. (c, e) The cartilage surface of the femoral condyle and tibial plateau of the control group was smooth without cartilage defects. Rats in the untreated OA group showed severe cartilage damage, the middle layer of cartilage was eroded, and the subchondral bone was exposed. Cartilage loss after emodin treatment is improved, and the degree of ulcers on the cartilage surface is reduced. After 6 weeks of emodin treatment, hematoxylin-eosin (HE) staining, Safranin O staining (scale bar: 200 μm), and (d, f) OARSI score representative knee joint images. In the control group, chondrocytes had no vacuoles and Safranin O stained evenly. In the OA group, chondrocytes were severely damaged, chondrocytes were hypertrophy, and chondrocytes aggregated. After six weeks of treatment, emodin can improve the loss of chondrocytes and inhibit the loss of proteoglycan in ECM. The data are presented as the means ± SD (n = 3). ^∗P < 0.05 and^∗∗P < 0.01.

Figure 7

Effects of emodin on the levels of CAT, GSH, SOD, and MDA in the serum of OA rats. Data were expressed as the means ± SD (n = 3). ^∗P < 0.05 and^∗∗P < 0.01. NS: no significant difference.

Figure 8

Effects of emodin on the levels of CTX-II, COMP, and CTX-I in the serum of OA rats. The data were expressed as the means ± SD (n = 3). ^∗P < 0.05 and^∗∗P < 0.01.

Figure 9

Emodin activates Nrf2/NQO1/HO-1 signaling which inhibits oxidative stress and ECM degradation in OA.