Polydatin retards the progression of osteoarthritis by maintaining bone metabolicbalance and inhibiting macrophage polarization

Abstract

Background: Polydatin (PD), also known as tiger cane glycoside, is a natural compound extracted from the Japanese knotweed plant, which is often referred to as white resveratrol. It exhibits anti-inflammatory, antioxidant, and anti-apoptotic effects in the treatment of various diseases. However, the potential molecular mechanisms of PD in osteoarthritis have not been clearly elucidated.

Methods: Anterior cruciate ligament transection (ACLT) surgery was performed to establish an osteoarthritis animal model. Female mice at the age of 12 weeks were intraperitoneally injected with different concentrations of PD (20 and 40 mg/kg). In vitro models were established by isolating mouse articular chondrocytes, which were subsequently treated with lipopolysaccharide or IL-1β for 24 h for subsequent experiments. In addition, different concentrations of PD were administered for 12 h. Morphological changes were observed by toluidine blue staining, joint bone metabolism changes were observed by tartrate-resistant acid phosphatase staining, immunohistochemistry was used to observe the expression levels of inflammatory factors and extracellular matrix. MicroCT analysis was conducted to assess changes in the microstructure of subchondral bone trabeculae, and Western blot was performed to measure the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway and markers of M1 polarization in macrophages.

Results: PD significantly delays the progression of osteoarthritis induced by ACLT, effectively inhibits IL-1β-induced joint inflammation, bone metabolic remodeling and extracellular matrix degradation. In addition, paeoniflorin markedly suppresses the transmission of the NF-κB signaling pathway and reverses M1 polarization in macrophages induced by IL-1β.

Conclusion: Taken together, PD might be a potential therapeutic agent for the prevention and treatment of osteoarthritis.

Keywords: NF-κB; Polydatin; cartilage; extracellular matrix; osteoarthritis.

FIGURE 1

PD attenuated cartilage destruction and inflammatory development in ACLT mice. After ACLT surgery for 3 days, mice were intraperitoneally injected with different concentrations of PD (20 and 40 mg/kg) and maintained for twelve weeks. (A) Representative images of Toluidine Blue in knee joints. Immunohistochemistry assay for (B) IL-6 and (C) TNF-α in the knee joints among all groups. (D, E) Immunohistological analysis showed that the expression of IL-6 and TNF-α in the ACLT group was greater than that in the PD groups. (F) The quantification of Osteoarthritis Research Society International (OARSI) score in different groups. Note: *P < 0.05, **P < 0.01.

FIGURE 2

PD can regulate bone metabolism and improve the microstructure of knee subchondral bone trabeculae, thus delaying the progression of osteoarthritis. (A) Representative micro-CT image of tibial plateau subchondral bone among all groups. Tartrate acid phosphatase (TRAP) staining of tibial plateau subchondral bone. The osteoclasts were obtained by counting the number of TRAP-positive cells (N. Trap+). (B–F) The results of proximal tibia BMD, bone volume (BV)/total volume (TV), trabecular number (Tb.N; mm⁻¹), trabecular thickness (Tb.Th; μm), and trabecular separation (Tb.Sp; μm) in different groups. (G) Quantitative analysis of TRAP-positive cells for tibial plateau subchondral bone. Note: *P < 0.05, **P < 0.01; scale bars = 2 mm as indicated.

FIGURE 3

PD can maintain the knee space and delay the progression of osteoarthritis. (A) Schematic diagram of lateral and medial compartment height measurement of the knee joint. (B, C) The results of lateral and medial compartment height measurement in all groups. Note: *P < 0.05, **P < 0.01.

FIGURE 4

Polydatin inhibits IL-1β-induced chondrocyte necroptosis and matrix degradation in vitro. (A–E) Western blot and quantitative analysis of necroptosis-related markers (Cleaved caspase-3, PIPK1, PIPK3) and inflammatory factors (TNF-α) in chondrocytes cells treated with different doses of PD. (F–H) RT-qPCR analysis of Aggrecan, ADAMTS-4 and ColII in chondrocytes treated with different doses of PD. Note: *P < 0.05, **P < 0.01.

FIGURE 5

PD can significantly inhibit the accumulation of IL-1β-induced ROS by suppressing the NF-κB signaling pathway. (A–E) The expression levels of NF-κB signaling pathway-related proteins (p-IκBα, IκBα, p-p65, and p65) were detected by Western blot. (F–G) ROS levels were detected by flow cytometry in all groups. Note: *P < 0.05, **P < 0.01.

FIGURE 6

Molecular docking between PD and NF-κB (Typical docking mode and binding site images. (A):The protein residues are represented by the ribbon model. (B):The space filling model intuitively reflects the spatial position of PD in the inhibitory binding pocket of NF-κB. (C):Docking modes and binding sites.

FIGURE 7

Polydatin inhibits IL-1β-induced macrophage M1 polarization. (A–E) The expression levels of repolarization-related markers (p-STAT1, STAT1, p-STAT3, and STAT3 proteins) were detected by Western blot. (F–H) M1 macrophage marker proteins (IL-6, TNF-α, and iNOS) were measured by RT-qPCR. Note: *P < 0.05, **P < 0.01.

FIGURE 8

The schematic illustration for PD ameliorates inflammation response via NF-κB/ROS pathway in ACLT-induced mice osteoarthritis model.