Dioscin alleviates the progression of osteoarthritis: an in vitro and in vivo study

Abstract

Osteoarthritis (OA) is a common joint disease and is the main cause of physical disability in the elderly. Currently, there is no adequate therapeutic strategy to reverse the progression of OA. Many natural plant extracts have received attention in the treatment of OA due to their potential anti-inflammatory properties, and reduced incidence of adverse events. Dioscin (Dio), a natural steroid saponin, has been demonstrated to inhibit the release of inflammatory cytokines in mouse and rat models of various diseases, and has a protective effect in chronic inflammatory diseases. However, whether Dio alleviates OA progression remains to be explored. In this research, our purposes were to investigate the therapeutic potential of Dio in OA. The results demonstrated that Dio exerted anti-inflammatory effects by repressing NO, PGE₂, iNOS and COX-2. Moreover, the application of Dio could repress IL-1β-induced overexpression of matrix metalloproteinases (MMPs, including MMP1, MMP3, and MMP13) and ADAMTS-5, and improve the synthesis of collagen II and aggrecan, which contribute to the maintenance of chondrocyte matrix homeostasis. The underlying mechanism involved the inhibition of the MAPK and NF-κB signaling pathways by Dio. Furthermore, the treatment of Dio significantly improved the pain behaviors of rat OA models. The in vivo study revealed that Dio could ameliorate cartilage erosion and degradation. These results collectively indicate that Dio can be used as a promising and effective agent for the therapy of OA.

Keywords: Cartilage matrix; Chondrocyte; Dioscin; Inflammatory cytokines; Osteoarthritis.

Fig. 1

Effects of Dio on cell viability. A Chemical structure of Dio. B Rat chondrocytes were treated with different concentrations of Dio in the absence or (C) presence of IL-1β (10 ng/mL) for 24 h. The cell viability was evaluated using a CCK-8 kit..^*P < 0.05 vs. control group; n = 3

Fig. 2

Dio suppresses IL-1β-induced inflammatory cytokines expression in chondrocytes. Chondrocytes were exposed to IL-1β (10 ng/mL) with or without Dio (200, 400, and 800 ng/mL) for 24 h. A Griess reaction was used to determine the NO concentration. B PGE2 level was detected by ELISA. C Gene expressions of COX-2 and iNOS were detected by qRT-PCR. D Representative western blots and (E) quantitative analysis of COX-2 and iNOS in each group. ^#P < 0.05 vs. control group; ^*P < 0.05 vs. IL-1β group; ^**P < 0.01 vs. IL-1β group; n = 3

Fig. 3

Dio inhibits IL-1β-induced upregulation of MMPs and ADAMTS-5. Chondrocytes were exposed to IL-1β (10 ng/mL) with or without Dio (200, 400, and 800 ng/mL) for 24 h. A Gene expressions of MMP1, MMP3, MMP13, and ADAMTS-5 were detected by qRT-PCR. B Representative western blots and (C) quantitative analysis of MMP1, MMP3, MMP13, and ADAMTS-5 in each group. ^#P < 0.05 vs. control group; ^*P < 0.05 vs. IL-1β group; ^**P < 0.01 vs. IL-1β group; n = 3

Fig. 4

Dio ameliorates IL-1β-induced cartilage matrix degradation. Chondrocytes were exposed to IL-1β (10 ng/mL) with or without Dio (200, 400, and 800 ng/mL) for 24 h. A Representative western blots and (B) quantitative analysis of collagen II and aggrecan in each group. C Collagen II and (D) aggrecan were observed by immunofluorescence after the cells treated with IL-1β (10 ng/mL) in absence or presence of Dio (800 ng/mL). ^#P < 0.05 vs. control group; ^*P < 0.05 vs. IL-1β group;.^**P < 0.01 vs. IL-1β group; n = 3

Fig. 5

Dio blocks IL-1β-induced MAPK signaling pathway activation. Chondrocytes were exposed to IL-1β (10 ng/mL) with or without Dio (200, 400, and 800 ng/mL) for 30 min. A Representative western blots and (B) quantitative analysis of P38, P-P38, ERK, P-ERK, JNK, and P-JNK in each group. ^#P < 0.05 vs. control group; ^*P < 0.05 vs. IL-1β group; ^**P < 0.01 vs. IL-1β group; n = 3

Fig. 6

Dio inhibits IL-1β-mediated activation of NF-κB signaling pathway. Chondrocytes were exposed to IL-1β (10 ng/mL) with or without Dio (200, 400, and 800 ng/mL) for 30 min. A Representative western blots and (B and C) quantitative analysis of P65, P-P65, IKK-β, P- IKKα/β, IκBα and P- IκBα in each group. D The nuclear translocation of P65 was observed by immunofluorescence after the cells treated with IL-1β (10 ng/mL) in absence or presence of Dio (800 ng/mL). ^#P < 0.05 vs. control group; ^*P < 0.05 vs. IL-1β group; ^**P < 0.01 vs. IL-1β group; n = 3

Fig. 7

Effects of Dio on pain-related behaviors in rat OA models. A Evolution of paw withdrawal threshold (PWT) over time in all groups (n = 8 rats/group) from Week 0 (before surgery) to Week 8. B Evolution of Weight-bearing asymmetry over time in all groups (n = 8 rats/group) from Week 0 (before surgery) to Week 8. ^*P < 0.05 vs. OA group; ^**P < 0.01 vs. OA group; ^***P < 0.001 vs. OA group

Fig. 8

Dio alleviated osteoarthritis progression in rat OA models. A The H&E and Safranin-O-Fast green staining demonstrated that the OA group showed obvious damage to the cartilage compared with the sham group. However, the administration of Dio significantly ameliorated the cartilage damage compared with the OA group. B The immunohistochemical staining of collagen II, aggrecan, MMP13 and iNOS also confirmed the positive effects of Dio on cartilage. C The OARSI scores of each group. D Quantitative of collagen II, aggrecan, MMP13, and iNOS in the cartilage samples from each group. ^#P < 0.05 vs. sham group; ^**P < 0.01 vs. OA group

Fig. 9

Schematic diagram of the protective effects of Dio on OA. IL-1β is an inflammatory cytokine which is excessive released in the joints of OA patients. The cytokine can induce the expression of pro-inflammatory factors (iNOS, COX2, MMPs and ADAMTS5) and downregulate the synthesis of Collagen II and Aggrecan in chondrocytes. Moreover, IL-1β functions by activating the MAPK and NF-κB signaling pathways. Dio can block IL-1β-induced MAPK and NF-κB signaling pathway activation, resulting in the inhibition of IL-1β-induced overexpression of MMPs and ADAMTS-5 and the improvement of the production of Collagen II and Aggrecan, which contribute to the maintenance of chondrocyte matrix homeostasis