Receptor activator of nuclear factor-kappa B ligand-derived microglia healing peptide 1-AcN inhibits osteoarthritis progression in mice

Abstract

Background

Osteoarthritis (OA) is a degenerative disease characterized by subchondral bone sclerosis, chronic inflammation, and cartilage degradation. Abnormal mechanical stress by meniscal deviation activates osteoclasts and induces the release of transforming growth factor-beta (TGF-β), which promotes mesenchymal stem cell (MSC)-mediated type H angiogenesis and osteogenesis, contributing to bone sclerosis and cartilage damage. Subsequently, macrophages recognize cartilage-derived damage-associated molecular patterns (DAMPs) via Toll-like receptor 4 (TLR4), polarizing into the pro-inflammatory M1 phenotype, thereby exacerbating synovitis and cartilage loss. We developed Microglia Healing Peptide 1 with N-terminal acetylation and C-terminal amidation (MHP1-AcN), a modified peptide derived from receptor activator of nuclear factor-kappa B ligand (RANKL), exhibiting both anti-osteoclastic and anti-inflammatory properties. This study aimed to evaluate the therapeutic potential of MHP1-AcN in a murine OA model and elucidate its underlying mechanisms.

Methods

OA was induced in mice via destabilization of the medial meniscus (DMM) surgery. Mice were randomly assigned to three groups (n = 8/group): Sham (sham surgery + saline), Vehicle (DMM + saline), and MHP1-AcN (DMM + MHP1-AcN). MHP1-AcN (600 µg) was administered intraperitoneally five times per week from a day after surgery. Knee joints were harvested at 2, 4, and 8 weeks post-surgery. In vitro, the effects of MHP1-AcN were assessed on osteoclast differentiation, inflammatory cytokine expression, and M1/M2 macrophage polarization using mouse bone marrow-derived macrophages. Additionally, its effects on TGF-β-induced osteogenic differentiation of bone marrow-derived MSCs (BMMSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated.

Results

MHP1-AcN markedly suppressed key pathological features of OA in vivo, including synovial inflammation, osteoclast-driven subchondral bone remodeling, aberrant angiogenesis, and cartilage degeneration. In vitro, MHP1-AcN effectively inhibited TLR4-mediated inflammatory cascades by reducing M1 macrophage polarization and inflammasome activation. Despite being derived from RANKL, MHP1-AcN supressed RANKL-induced osteoclastogenesis through NF-κB pathway suppression. Furthermore, MHP1-AcN attenuated TGF-β-induced osteogenic and angiogenic activities via Smad2 signaling inhibition in BMMSCs and HUVECs.

Conclusion

MHP1-AcN attenuates OA progression by modulating multi-pathways including aberrant bone remodeling, angiogenesis, and macrophage polarization, representing a promising disease-modifying therapeutic candidate for OA.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13075-025-03609-5.

Introduction

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation. With increasing global life expectancy, OA prevalence is rising. Genetic predisposition and metabolic factors such as obesity, as well as mechanical wear and damage caused by repetitive joint loading, the release of inflammatory cytokines, and biochemical changes in the extracellular matrix of cartilage, have been implicated in the progression of OA [1]. Among the various mechanisms of OA, we focused on macrophage-driven inflammation and subchondral bone sclerosis.

Chronic inflammation drives OA progression, with macrophages playing a pivotal role. Macrophages are activated by damage-associated molecular patterns (DAMPs), such as fragments of damaged cartilage. They recognize these signals via Toll-like receptor 4 (TLR4) and polarize to the pro-inflammatory M1 phenotype [2], triggering factors that cause synovitis and cartilage destruction [3, 4].

Subchondral bone changes also influence OA [5, 6]. Abnormal mechanical loading activates osteoclasts, increasing bone turnover and transforming growth factor-beta (TGF-β) release. This promotes mesenchymal stem cell (MSC)-mediated type H angiogenesis and osteogenesis, leading to bone sclerosis and subsequent cartilage degradation [7–10].

Despite promising results in rodent studies, where drugs with either anti-inflammatory effects on macrophages or bone resorption inhibitors exhibited OA-suppressive effects, these findings have not translated to humans [11–13].

Peptide drug development has regained attention. Compared to antibody and protein-based therapeutics, peptide drugs offer lower immunogenicity and production costs. However, orally administered peptides are highly susceptible to gastrointestinal degradation [14]. Technological advancements have led to significant progress in peptide therapeutics. For instance, temperature-sensitive polymers in peptide formulations enable sustained release [15], fatty acid conjugation enhances blood stability and acid resistance, enabling oral peptide drugs [14]. These advances have led to the approval of 80 peptide therapeutics worldwide [16, 17]. Fibroblast growth factor 18 (FGF18) exhibited cartilage-protective effects in mice, and rhFGF18 showed similar effects in rats, leading to human clinical trials [18–21]. Such findings from animal studies have been translated into potential therapeutic applications in humans [22], highlighting peptides as promising drug candidates.

Microglia healing peptide 1 with N-terminal acetylation and C-terminal amidation (MHP1-AcN), a modified physiological peptide derived from receptor activator of nuclear factor-kappa B ligand (RANKL), demonstrates anti-inflammatory effects without inducing osteoclastogenesis [23–25]. Our previous studies have demonstrated the anti-inflammatory effects of MHP1-AcN in murine models of psoriasis and pulmonary fibrosis [26, 27]. We hypothesized that MHP1-AcN might inhibit the development of OA through its anti-inflammatory and anti-osteoclastic mechanisms. The primary objective of this study was to investigate whether MHP1-AcN can inhibit OA development in a mouse model of knee OA. The secondary objective was to explore its detailed mechanism.

Methods

All experiments were approved by the Animal Experimental Committee of The University of Osaka Graduate School of Medicine Faculty of Medicine (No.: 02-057-014, dated: June 13, 2024).

Peptide design and synthesis

We previously identified the anti-inflammatory properties of RANKL [23, 28]. Further investigations revealed that deletion of the CD and partial EF loop from the RANKL molecule produced a compound that retained anti-inflammatory activity without osteoclastogenic effects [24]. This novel compound was designated MHP1. Furthermore, N-terminal acetylation and C-terminal amidation of MHP1 enhanced its anti-inflammatory efficacy, stability, and anti-osteoclastogenic properties (Fig. 1A) [25]. A 3D structural model of RANKL and RANK was constructed using data from the Protein Data Bank Europe (PDBe). This model illustrates the spatial relationship between MHP1 (white dashed line) and the osteoclast activation site (red area) (Fig. 1B).

Fig. 1.

Structure of MHP1-AcN and administration protocol in a mouse model of knee osteoarthritis: its impact on cartilage degeneration. (A) Structural diagrams of soluble receptor activator of nuclear factor-kappa-B ligand (RANKL) and Microglia healing peptides 1-AcN (MHP1-AcN). (B) 3D schematic representations of RANKL, RANK and MHP1. (C) Immunofluorescence staining of FITC-MHP1-AcN in the knee joint. Control (left) and FITC-MHP1-AcN (right). (D) Safranin O staining and Osteoarthritis Research Society International (OARSI) Score. Data are expressed as means with 95% confidence intervals. They are analysed with Kruskal-Wallis test for nonparametric values followed if positive by Dunn’s multiple comparison test. except otherwise specified, P values indicate statistical comparisons against the Vehicle group. *P < 0.05; ***P < 0.001 (n = 8 per group)

MHP1-AcN (Ac-LMVYVVKTSIKIPSSHNLMKGGSTKNWSGNNH2) and FITC-labeled MHP1-AcN were purchased from ILS (Ibaragi, Tsukuba, Japan). The peptides were dissolved in double-distilled H₂O to prepare a 2 mg/mL solution and stored at 4 °C until use.

OA model and experimental protocol

Eight-week-old male C57B/6J mice (CLEA Japan, Fujinomiya, Japan) underwent surgical destabilization of the medial meniscus (DMM) for OA induction. Mice were anesthetized by intraperitoneal injection of 0.3 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol. The anterior portion of the right medial meniscus was resected, while sham surgery involved only joint capsule dissection (Supplementary Fig. S1A). The detailed methodology is described elsewhere [29]. Micro-CT confirmed the lateral displacement of the medial meniscus post-surgery (Supplementary Fig. S1B). Mice were randomly divided into three groups (n = 8 per group): Sham (sham surgery + saline), Vehicle (DMM surgery + saline), and MHP1-AcN (DMM surgery + MHP1-AcN). Based on previous experiments, all treatments were administered intraperitoneally (300 µL, five times per week) (Supplementary Fig. S1C) [27]. Additional anesthesia was administered in accordance with the experimental protocol when animals showed signs of distress. To investigate MHP1-AcN migration, FITC-labeled MHP1-AcN was intraperitoneally administered, confirming its distribution in the bone marrow, synovium and liver (Fig. 1C, Supplementary Fig. S1D). No significant differences in body weight or mechanical sensitivity (von Frey test) were observed among the groups (Supplementary Fig. S1E, F). Mice were sacrificed at 2, 4, and 8 weeks post-surgery. The sample size was determined based on the previous study [30], using the difference in Osteoarthritis Research Society International (OARSI) score at 8 weeks. With a statistical power of 80% and significance level of 0.05, Eight animals per group were sufficient to detect significant differences (effect size > 0.8). This was supported by post-hoc power analysis.

High-resolution micro-CT analysis

Mouse knee joints were evaluated using high-resolution micro-CT (SkyScan 1272; Bruker Corporation, Billerica, MA, USA) with 90 kV voltage and 160 mA current. For the distal femur, the region of interest was set 500 to 1000 μm proximal to the growth plate. For the subchondral bone of the tibia, the region of interest was set from the articular surface to the growth plate. Micro-CT data were analyzed using CTAn software (Bruker Corporation) to assess bone morphometric parameters such as bone volume fraction (BV/TV), trabecular bone pattern factor (Tb.pf), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Evaluation methods followed established protocols [31].

Histological examination

Mouse knee joint sections were prepared for histological evaluation. After fixation, decalcification, and paraffin embedding, sections were stained with Safranin O to assess cartilage degeneration in the medial tibial plateau using the OARSI score (range, 0–6) [32]. The OARSI score was independently evaluated by two blinded assessors. Their consistency was confirmed beforehand. Each sample was assessed using 2–3 slides. Synovitis was graded (0–9) using hematoxylin and eosin (H&E) stained sections as described [33]. Tartrate-resistant acid phosphatase (TRAP) staining was performed according to the manufacturer’s protocol (Cosmo Bio, Tokyo, Japan). The number of TRAP-positive cells per trabecular surface was quantified as previously described [34]. Images were acquired using an APERIO CS2 scanner (Leica, Tokyo, Japan).

Immunohistochemistry

Paraffin-embedded sections were immunohistochemically evaluated as previously described [35]. Antibodies and their concentrations are listed in Supplementary Table S1. The number of positively stained cells was quantified using ImageJ (National Institutes of Health).

Quantitative real-time polymerase chain reaction (PCR)

The detailed methodology for quantitative real-time PCR is described elsewhere [30], and the primer sequences are listed in Supplementary Table S2.

Western blotting

Western blotting was performed as previously described [36] using antibodies listed in Supplementary Table S3. Quantitative analysis was conducted with ImageJ. Protein expression levels were normalized to β-actin and presented graphically.

In vitro osteoclast differentiation assay and TRAP staining

Bone marrow-derived macrophages (BMDMs) were seeded in 6-well plates (1.0 × 10⁶ cells/well), stimulated with 10 ng/mL macrophage colony-stimulating factor (M-CSF) (Cosmo Bio) and 50 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA), and incubated with or without MHP1-AcN for four days. TRAP staining was performed using a TRAP kit (Cosmo Bio) according to the manufacturer’s protocol. TRAP-positive cells with ≥ 3 nuclei were counted as osteoclasts.

Statistical analysis

Analyses were performed using GraphPad Prism 10 software (GraphPad Software, San Diego, CA, USA). In vitro, one-way analysis of variance with the Tukey post hoc test was used to differentiate between each group. Data are presented as mean ± SD. For in vivo analyses, the Kruskal-Wallis test followed by Dunn’s post hoc test was applied. P < 0.05 was considered significant. Data are presented with 95% confidence intervals.

Other experimental methods are described in Supplementary Materials and Methods.

Results

Effects of MHP1-AcN on articular cartilage degeneration

Articular cartilage degeneration was evaluated through OARSI scoring of Safranin O-stained sections (Fig. 1D) (Supplementary Table S4). The Kruskal-Wallis test revealed significant differences among the groups in both the 4-week (H = 20.32, p < 0.0001, mean values: 0.125, 2.875, and 1.000 for the Sham, Vehicle, and MHP1-AcN groups, respectively) and 8-week models (H = 21.00, p < 0.0001, mean values: 0.1875, 3.8750, and 1.3750, respectively), while post hoc Dunn’s tests confirmed significant differences between the Vehicle and the Sham groups and the Vehicle and the MHP1-AcN groups in both models, indicating that MHP1-AcN treatment significantly reduced cartilage degeneration compared to the Vehicle group.

Effects of MHP1-AcN on intra-articular inflammation

As OA progresses, articular chondrocytes express inflammatory cytokines such as interleukin-1β (IL-1β) and matrix metalloproteinase 13 (MMP13), transforming into hypertrophic chondrocytes [37]. In the Vehicle group, IL-1β, MMP13 (from 4 weeks), and type X collagen (COL-X) (from 2 weeks) were increased compared to the Sham group, while MHP1-AcN suppressed these changes (Fig. 2A–C, Supplementary Fig. S2A-C), indicating reduced cartilage inflammation and hypertrophy.

Fig. 2.

Effects of MHP1-AcN on intra-articular inflammation in a mouse model of knee osteoarthritis. (A-C) Immunohistochemical staining and quantification of Interleukin-1β (IL-1β) (A), matrix metalloproteinase 13 (MMP13) (B) and Type X collagen (COL-X) (C) in the articular chondrocytes. (D) Hematoxylin and eosin (H&E) staining and synovitis score of synovial tissue. (E, F) Immunohistochemical staining and quantification of IL-1β (E) and CD86 (F) in synovial tissue. Data are expressed as means with 95% confidence intervals. They are analysed with Kruskal-Wallis test for nonparametric values followed if positive by Dunn’s multiple comparison test. except otherwise specified, P values indicate statistical comparisons against the Vehicle group. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 8 per group)

Synovitis was assessed by H&E scoring (Fig. 2D, Supplementary Fig. S3) and IL-1β, CD86 expression (Fig. 2E, F, Supplementary Fig. S4A, B). Postoperative inflammation at 2 weeks was excluded due to the strong response. By 4 weeks, synovitis scores and IL-1β- and CD86-positive cell proportions increased in the Vehicle group but were significantly reduced by MHP1-AcN. At 8 weeks, synovitis scores further increased in the Vehicle group, while MHP1-AcN maintained suppression.

Effects of MHP1-AcN on subchondral bone

Abnormal mechanical stress induces osteoclast activation in the subchondral bone, leading to the dissolution of the surrounding bone matrix [38]. The release of TGF-β subsequently stimulates MSCs through Smad2 phosphorylation, promoting angiogenesis and osteophyte formation [8]. Micro-CT evaluation of subchondral bone revealed that BV/TV increased significantly in the Vehicle group at 4 and 8 weeks, indicating subchondral bone sclerosis, which was suppressed by MHP1-AcN. Similar trends were observed in Tb.pf (Fig. 3A). MHP1-AcN induced only slight but statistically significant changes in Tb.Th and Tb.sp at 4 and 8 weeks, respectively, and had no effect on Tb.N at any time point. (Supplementary Fig. S5A). Notably, MHP1-AcN did not affect BV/TV in distal femur (Supplementary Fig. S5B), suggesting specific inhibition of subchondral bone increase. Osteoclasts in the subchondral bone were evaluated using TRAP staining. The osteoclast count peaked at 2 weeks, declining thereafter. MHP1-AcN reduced osteoclasts throughout the study (Fig. 3B, Supplementary Fig. S6A). CD31 expression, a marker of vascular endothelial cells and an indicator of subchondral angiogenesis, was reduced by MHP1-AcN at 2 and 4 weeks, but not at 8 weeks. However, no significant difference was observed between the MHP1-AcN and sham groups at 8 weeks (Fig. 3C, Supplementary Fig. S6B), indicating reduced angiogenesis. P-Smad2/3, induced by TGF-β from bone matrix following osteoclast activation, was also inhibited by MHP1-AcN (Fig. 3D, Supplementary Fig. S7A). Osteocalcin, indicating subchondral sclerosis, increased at 2 weeks post-DMM but was reduced by MHP1-AcN, with no inter-group differences at 4 and 8 weeks (Fig. 3E, Supplementary Fig. S7B).

Fig. 3.

Effects of MHP1-AcN on subchondral bone in a mouse model of knee osteoarthritis. (A) Micro-CT analysis of subchondral bone, showing bone volume fraction (BV/TV) and trabecular bone pattern factor (Tb.pf) at 2, 4, and 8 weeks post-surgery. (B-E) Immunohistochemical staining and quantification in subchondral bone at 2, 4, and 8 weeks post-surgery of (B) Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, (C) CD31-positive endothelial cells, (D) p-Smad2/3-positive cells, and (E) osteocalcin-positive osteoblasts. Data are expressed as means with 95% confidence intervals. They are analysed with Kruskal-Wallis test for nonparametric values followed if positive by Dunn’s multiple comparison test. except otherwise specified, P values indicate statistical comparisons against the Vehicle group. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 8 per group)

Effects of intra-articular administration of MHP1-AcN

Intra-articular administration of MHP1-AcN failed to prevent cartilage degeneration, subchondral sclerosis, or osteoclast activation (Supplementary Fig. S8A-C). These findings suggest that the inhibitory effect of MHP1-AcN on OA progression is achievable only through systemic administration, not local intra-articular delivery.

Effects of MHP1-AcN on lipopolysaccharide (LPS)-induced inflammation of macrophages in vitro

DAMPs generated within the joint are recognized by TLR4 on synovial macrophages, leading to polarization of macrophages from M0 to M1 phenotype. Since LPS is also recognized by TLR4 in a manner similar to DAMPs, the effect of MHP1-AcN was evaluated using LPS as a stimulant. Quantitative real-time PCR revealed that MHP1-AcN suppressed the increase in M1 macrophage markers, such as inducible nitric oxide synthase (iNOS) and CD86. It also suppressed the upregulation of NLR family pyrin domain containing 3 (NLRP3) and Caspase-1, thereby inhibiting the activation of the inflammasome pathway. This resulted in a trend toward the restoring M2 macrophage markers, such as CD206 and arginase, while suppressing the production of inflammatory cytokines, including IL-1β, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and cyclooxygenase-2 (COX-2). Moreover, MHP1-AcN alone did not alter the expression of M1/M2 markers or inflammatory cytokines. (Fig. 4A). Western blotting confirmed a dose-dependent suppression of iNOS, while NLRP3 and IL-1β expression levels were dose-dependently diminished (Fig. 4B). Flow cytometry demonstrated that MHP1-AcN shifted LPS-induced M1 macrophages toward the M2 phenotype (Fig. 4C, Supplementary Fig. S9A-C).

Fig. 4.

Effects of MHP1-AcN on lipopolysaccharide (LPS)-induced inflammation of macrophages in vitro. (A) Effects of MHP1-AcN on mRNA expression of inflammatory markers in LPS-stimulated Bone marrow-derived macrophages (BMDMs). Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 3). (B) Effects of MHP1-AcN on protein levels of inflammatory mediators in LPS-stimulated BMDMs. (C) Effects of MHP1-AcN on M1/M2 polarization markers in LPS-stimulated BMDMs as assessed by flow cytometry

Effects of MHP1-AcN on RANKL-induced osteoclast differentiation in vitro

Quantitative real-time PCR revealed that MHP1-AcN suppressed RANKL-induced expression of osteoclast markers, including TNF receptor-associated factor 6 (TRAF6), cellular oncogene fos (c-Fos), nuclear factor of activated T-cells c1 (NFATc1), Cathepsin-K, TRAP, and MMP9. It also slightly reduced the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and platelet-derived growth factor-BB (PDGF-BB), which recruits CD31-positive cells for bone formation (Fig. 5A) [39]. MHP1-AcN dose-dependently inhibited osteoclast formation (Fig. 5B, Supplementary Fig. S10A). Western blotting revealed reduction of NF-κB pathway components (p-p65, p-Erk1/2) and osteoclast transcript factors (c-Fos and NFATc1) (Fig. 5C, Supplementary Fig. S10B).

Fig. 5.

Effects of MHP1-AcN on RANKL-induced osteoclast differentiation in vitro. (A) Effects of MHP1-AcN on mRNA expression of osteoclast differentiation markers in BMDMs. Data are expressed as mean ± SD (n = 3). (B) Effects of RANKL and MHP1-AcN on TRAP-positive multinucleated cell formation in BMDMs. Representative images of TRAP staining are shown. (Scale bar: 500 μm) Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 4). (C) Effects of MHP1-AcN on RANKL-induced mitogen-activated protein kinase (MAPK) signaling pathways and osteoclast marker proteins in BMDMs. Western blotting analysis of key signaling molecules and osteoclast-specific proteins

Effects of MHP1-AcN on angiogenesis and bone formation in vitro

MHP1-AcN reduced TGF-β-induced Smad2 phosphorylation in HUVECs (Fig. 6A) and inhibited HUVEC migration, indicating reduced angiogenesis (Fig. 6B).　In addition, MHP1-AcN slightly reduced Rac1, Cdc42, and S1P receptor, which are key regulators of cytoskeletal dynamics and angiogenic responses in endothelial cells (Supplementary Fig. S11A). In BMMSCs, MHP1-AcN reduced TGF-β-induced Smad2 phosphorylation (Fig. 6C) and inhibited bone formation, shown by alkaline phosphatase (ALP) and alizarin red staining (Fig. 6D, E, Supplementary Fig. S11B). These effects occurred without cytotoxicity in any cell type (Supplementary Fig. S11C). Thus, MHP1-AcN inhibits subchondral angiogenesis and MSC osteogenesis by reducing Smad2 phosphorylation, thereby potentially attenuating subchondral sclerosis.

Fig. 6.

Effects of MHP1-AcN on angiogenesis and bone formation in vitro. (A) Effects of MHP1-AcN on transforming growth factor-β (TGF-β)-induced Smad2 phosphorylation in HUVECs. (B) Effects of MHP1-AcN on HUVECs migration. The graph represents the quantification of the wound closure percentage after 24 h. (Scale bar: 500 μm) (C) Effects of MHP1-AcN on TGF-β-induced Smad2 phosphorylation in bone marrow mesenchymal stem cells (BMMSCs). (D) Effects of MHP1-AcN on osteogenic differentiation of BMMSCs was evaluated by alkaline phosphatase (ALP) staining and qPCR analysis of ALP mRNA expression. (E) Effects of MHP1-AcN on osteogenic differentiation of BMMSCs is shown by Alizarin Red staining. The staining was quantified for evaluation. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 3–4 for each experiment)

Effects of MHP1-AcN on mouse primary chondrocytes in vitro

In vitro studies using mouse primary chondrocytes showed MHP1-AcN was non-toxic and suppressed LPS-induced IL-6 and MMP13 mRNA expression. However, MHP1-AcN did not affect MMP3, SOX9, type II collagen (COL-II), or Aggrecan expression, nor TNF-α or IL-1β responses (Supplementary Fig. S12A–D). These findings suggest that MHP1-AcN may have limited effects on chondrocytes, further supported by the failure of intra-articular MHP1-AcN administration to prevent OA progression.

Assumed mechanisms of MHP1-AcN in knee OA development

The graphical abstract (Fig. 7) shows MHP1-AcN inhibiting OA progression by disrupting the cycle of meniscus dislocation-induced mechanical stress and DAMPs release.

Fig. 7.

Graphical abstract: Effects of MHP1-AcN in knee OA progression. MHP1-AcN inhibits osteoarthritis progression by disrupting the cycle of meniscus dislocation-induced stress and inflammation, suppressing macrophage polarization, inflammatory mediators, angiogenesis, and osteoclast activity, while modulating TGF-β signaling in MSCs, thereby addressing both joint inflammation and abnormal subchondral bone remodeling

Discussion

MHP1-AcN inhibits OA progression through anti-inflammatory effects and suppression of subchondral bone sclerosis. Previous research has demonstrated that MHP1-AcN competes with CD14, which mediates downstream signaling of TLR4 [40]. Our study elucidated the detailed mechanism by which MHP1-AcN inhibits TLR4-mediated M1 macrophage polarization, inflammasome activation, and the release of pro-inflammatory cytokines (Supplementary Fig. S13A). Suppressing macrophage M1 polarization has been shown to inhibit OA progression in rodent models [11]. This occurs through shifting macrophages from pro-inflammatory M1 to anti-inflammatory M2 phenotype, reducing synovitis and cartilage degradation. Herein, MHP1-AcN was found to inhibit M1 polarization, thereby attenuating synovitis. Numerous studies have shown that OA progression can be suppressed through inhibition of NLRP3, a critical component of the inflammasome pathway [41]. Our results indicate MHP1-AcN’s anti-inflammatory effects occur through reduction of NLRP3 expression and inhibition of inflammasome pathway. However, the in vitro experiments using chondrocytes yielded only limited anti-inflammatory effects, and the failure of intra-articular injection to suppress cartilage degeneration suggests that the anti-inflammatory effects of MHP1-AcN are primarily mediated through systemic effects on macrophages, rather than local effects on chondrocytes.

Extensive research has implicated subchondral bone remodeling in the pathogenesis of OA [42]. Early OA involves increased osteoclast-driven bone resorption, followed by subchondral sclerosis [43]. Bone resorption inhibitors are thus promising OA therapeutics [12, 13]. Although MHP1-AcN is a peptide derived from RANKL, it is suggested to inhibit the downstream NF-κB pathway by binding to RANK and acting as an antagonist (Supplementary Fig. S13B) [40]. Importantly, MHP1-AcN suppressed excessive subchondral bone formation without affecting systemic bone mass in the distal femur, suggesting its specificity in targeting abnormal mechanical stress-induced osteoclast activation and sclerosis in subchondral bone. Furthermore, given that MHP1-AcN prevented bone loss in the ovariectomy model　mouse [44], it is plausible that this compound contributes to the normalization of bone metabolism.

TGF-β released during bone resorption induces type H angiogenesis, promoting cartilage destruction and osteophyte formation [10, 45]. Osteoclast suppression inhibits angiogenesis and OA onset [46], with PDGF-BB and Smad2/3 pathways mediating these processes [47, 48]. MHP1-AcN may have reduced TGF-β release through osteoclast inhibition, and reduced MSC-mediated angiogenesis and bone formation.

This study has limitations. Further investigation is required to determine the relative contributions of RANK-mediated and CD14-mediated pathways to MHP1-AcN’s anti-inflammatory effects. Our data suggest that MHP1-AcN binds to RANK and exerts anti-inflammatory effects (Supplementary Fig. S14A). Although systemic administration of MHP1-AcN alleviated joint inflammation, serum inflammatory cytokine levels in this OA model were minimal, making their detection by ELISA challenging (Supplementary Fig. S14B, C).

Larger animal models are needed before clinical trials. We have already conducted a pharmacokinetic study using non-human primates. In this study, MHP1-AcN was administered at 7.5 mg/kg over 10 min, followed by a continuous infusion of 2.4–4.8 mg/kg over 24 h. No major safety concerns were observed, except for transient flushing in some primates within 1 to 8 min after intravenous administration, which resolved within 24 h. Further investigation is required to evaluate the safety of long-term administration in large animal models.

This study used a surgery-induced meniscal dislocation OA model, mimicking meniscal injury or degenerative dislocation often involved in early stage of knee OA [49]. Compared to the anterior cruciate ligament transection model, the DMM model induces milder OA, making it a more physiologically relevant model [50]. Additionally, compared to chemically induced models such as the monosodium iodoacetate model, the DMM model better replicates OA pathology in vivo [51].

Furthermore, this study employed a prophylactic administration protocol. Therefore, the efficacy of MHP1-AcN after OA onset remains unexamined. However, recent trends in the clinical management of musculoskeletal diseases are shifting from conventional symptomatic treatment to preventive medicine. Therapeutic approaches such as MHP1-AcN, which have the potential to improve early-stage OA pathology, may address the unmet medical needs in OA treatment.

Conclusions

MHP1-AcN, a novel RANKL-derived peptide, attenuates DMM-induced OA progression in mice through inhibition of M1 macrophage polarization, osteoclast differentiation, and aberrant subchondral bone formation. These findings suggest that MHP1-AcN has the potential to be a promising disease-modifying agent for OA.

Electronic supplementary material

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Abbreviations

ALP

alkaline phosphatase

BV/TV

bone volume/total volume

c-Fos

cellular oncogene fos

COL

collagen

COX-2

cyclooxygenase-2

DAMPs

damage-associated molecular patterns

DC-STAMP

dendritic cell-specific transmembrane protein

DMM

destabilization of the medial meniscus

ELISA

Enzyme-Linked Immunosorbent Assay

FGF

Fibroblast growth factor

H&E

hematoxylin and eosin

HUVECs

human umbilical vein endothelial cells

IL

interleukin

iNOS

inducible nitric oxide synthase

M-CSF

macrophage colony-stimulating factor

MHP1-AcN

Microglia Healing Peptide 1 with N-terminal acetylation and C-terminal amidation

MMP

matrix metalloproteinase

MSC

mesenchymal stem cell

NFATc1

nuclear factor of activated T-cells c1

NLRP3

NOD-like receptor family pyrin domain containing 3

OA

Osteoarthritis

OARSI

Osteoarthritis Research Society International

PDGF-BB

platelet-derived growth factor-BB

RANKL

receptor activator of nuclear factor-kappa B ligand

Tb.N

trabecular number

Tb.pf

trabecular bone pattern factor

Tb.Sp

trabecular separation

Tb.Th

trabecular thickness

TGF-β

transforming growth factor-beta

TLR4

Toll-like receptor 4

TNF-α

tumor necrosis factor-alpha

TRAF6

TNF receptor-associated factor 6

TRAP

Tartrate-resistant acid phosphatase

Author contributions

YF and KE take responsibility for the integrity of the work as a whole, from inception to the completed manuscript. Project administration; YF, MS, and KE. Conceptualization; YF, MS, YE, TN, MH and KE. Data curation; YF, TKurihara, NJ, NO, and KE. Formal analysis: YF and KE. Investigation; YF, MS, YE, TN, NO, and KE. Methodology; YF, MS, AG, NJ, NO, TM, TKurihara, NO, AS, and KE. Visualization; YF, YE, TN, and KE. Writing - original draft; YF and KE. Writing - review & editing; MS and KE. Supervision; TKanamoto, KN, SO and KE. Resources; MS, TN, KN, SO and KE.

Funding

This research was funded by the Health and Labor Sciences Research Grant of Japan (grant number; 23K15703) and Asahi Kasei Pharma. The funders had no role in the study design, decision to publish, or manuscript preparation.

Data availability

The datasets used and/or analysed in the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All experiments were approved by the Animal Experimental Committee of The University of Osaka Graduate School of Medicine Faculty of Medicine (No. 02-057-014, dated: June 13, 2024).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.