Vindoline Attenuates Osteoarthritis Progression Through Suppressing the NF-κB and ERK Pathways in Both Chondrocytes and Subchondral Osteoclasts

Abstract

Disruption of extracellular matrix (ECM) homeostasis and subchondral bone remodeling play significant roles in osteoarthritis (OA) pathogenesis. Vindoline (Vin), an indole alkaloid extracted from the medicinal plant Catharanthus roseus, possesses anti-inflammatory properties. According to previous studies, inflammation is closely associated with osteoclast differentiation and the disorders of the homeostasis between ECM. Although Vin has demonstrated effective anti-inflammatory properties, its effects on the progression of OA remain unclear. We hypothesized that Vin may suppress the progress of OA by suppressing osteoclastogenesis and stabilizing ECM of articular cartilage. Therefore, we investigated the effects and molecular mechanisms of Vin as a treatment for OA in vitro and in vivo. In the present study, we found that Vin significantly suppressed RANKL-induced osteoclast formation and obviously stabilized the disorders of the ECM homeostasis stimulated by IL-1β in a dose-dependent manner. The mRNA expressions of osteoclast-specific genes were inhibited by Vin treatment. Vin also suppressed IL-1β-induced mRNA expressions of catabolism and protected the mRNA expressions of anabolism. Moreover, Vin notably inhibited the activation of RANKL-induced and IL-1β-induced NF-κB and ERK pathways. In vivo, Vin played a protective role by inhibiting osteoclast formation and stabilizing cartilage ECM in destabilization of the medial meniscus (DMM)-induced OA mice. Collectively, our observations provide a molecular-level basis for Vin's potential in the treatment of OA.

Keywords: ERK pathway; NF-κB pathway; extracellular matrix; osteoarthritis; osteoclastogenesis; vindoline.

FIGURE 1

The chemical structure of Vin and results of the cell viability assays (A) The chemical structure of Vin. (B–D) Cell viability of bone marrow macrophages (BMMs), RAW264.7 and ATDC5 cells after treated with different concentrations of Vin for 48 h (E) ATDC5 cells were co-treated with IL-1β (10 ng/ml) and various concentrations of Vin for 48 h, and the cell viability was detected by CCK-8 assay. (*p < 0.05; **p < 0.01 vs the control group; #p < 0.05; ##p < 0.01 vs. the IL-1β-treated group).

FIGURE 2

Vin inhibits the formation of osteoclasts induced by RANKL in BMMs, and stabilizes the disorders of the ECM homeostasis stimulated by IL-1β. (A) Tartrate-resistant acid phosphatase (TRAP) staining results for BMMs cultured with M-CSF (30 ng/ml), RANKL (50 ng/ml), and different concentrations of Vin (0, 5, 10, and 20 μM) for 5–7 days. Scale bar = 200 μm. (B,C) TRAP-positive multinuclear cell numbers and area. (D) Toluidine blue staining results for ATDC5 cultured with IL-1β (10 ng/ml) and various concentrations of Vin (0, 5, 10, and 20 μM) for 7–9 days by high density culture method. Scale bar = 200 μm. (E) The relative intensity of blue staining. (*p < 0.05; **p < 0.01 vs. the control group; #p < 0.05; ##p < 0.01 vs. the IL-1β-treated group).

FIGURE 3

Vin suppresses osteoclast-related and IL-1β-induced gene expression. (A) The osteoclast-related gene expressions (c-fos, NFATc1, TRAP, DC-STAMP, calcitonin receptor, and V-ATPase d2) were analyzed using quantitative PCR. (B) The IL-1β-induced gene expressions (COL2a1, SOX9, aggrecan, MMP13, ADAMTS4 and ADAMTS5) were assessed by quantitative PCR. (*p < 0.05; **p < 0.01 vs the control group; #p < 0.05; ##p < 0.01 vs the M-CSF and RANKL groups or the IL-1β-treated group).

FIGURE 4

Vin suppresses the signaling pathways of NF-κB and ERK induced by RANKL, and inhibits c-fos and NFATc1 expressions induced by RANKL. (A) RAW264.7 cells were pretreated with or without Vin (20 μM) for 2 h followed by 0 or 50 ng/ml RANKL for 10 min. (B) The protein levels of p-ERK/ERK, p-p65/p65, IκBα/GAPDH were quantified by ImageJ software. (C) Nuclear translocation of p65 in RAW264.7 was determined using immunofluorescence. Scale bar = 10 μm. (D) RAW264.7 cells were pretreated with or without Vin (20 μM) for 2 h followed by 0 or 50 ng/ml RANKL for 48 h. (E) ImageJ was used to quantify the protein levels of NFATc1/GAPDH and c-fos/GAPDH. (*p < 0.05; **p < 0.01).

FIGURE 5

Vin suppresses NF-κB and ERK pathways stimulated by IL-1β, and inhibits the degradation of COL2a1 and aggrecan, and the expression of MMP13. (A) ATDC5 cells were pretreated with or without Vin (20 μM) for 2 h followed by 0 or 10 ng/ml IL-1β for 30 min. (B) The protein levels of p-ERK/ERK, p-p65/p65, IκBα/GAPDH were quantified by ImageJ software. (C) Nuclear translocation of p65 in ATDC5 was determined using immunofluorescence. Scale bar = 10 μm. (D) ATDC5 cells were pretreated with or without Vin (20 μM) for 2 h followed by 0 or 10 ng/ml IL-1β for 48 h. (E) ImageJ quantification of COL2a1/GAPDH, aggrecan/GAPDH and MMP13/GAPDH protein levels. (*p < 0.05; **p < 0.01).

FIGURE 6

Vin protects against cartilage degeneration and osteoclast activity in a DMM-induced OA mouse model in vivo. (A) The effect of Vin on DMM-induced cartilage degeneration was observed by Safranin O-Fast Green staining. (B) Osteoclasts in subchondral bone were stained by TRAP (C–F) Immunofluorescence (p-p65, p-ERK and aggrecan) and immunohistochemical (MMP13) staining of chondrocytes in articular cartilage. (G–L) Quantitative analysis of OARSI scores and positively stained cells in articular cartilage. All scale bar = 200 μm. (*p < 0.05; **p < 0.01).

FIGURE 7

The schema elucidates Vin as a promising therapeutic to treat OA via suppressing the NF-κB and ERK pathways in both chondrocytes and subchondral osteoclasts.