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Cartilage
Cartilage
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Cartilage
1947-6035
1947-6043
SAGE Publications Sage CA: Los Angeles, CA

37086004
10.1177/19476035231169940
10.1177_19476035231169940
Basic Research
Studies on Factors Inovolved in OA Progression
Inhibition of Leukotriene A4 Hydrolase Suppressed Cartilage Degradation and Synovial Inflammation in a Mouse Model of Experimental Osteoarthritis
Wu Xiaoxin 12*
Sun Antonia RuJia 23*
Crawford Ross 24
Xiao Yin 35
Wang Yanping 6
Prasadam Indira 2
https://orcid.org/0000-0002-1835-6043
Mao Xinzhan 1
1 Department of Orthopaedic Surgery, The Second Xiangya Hospital of Central South University, Changsha, China
2 Centre for Biomedical Technologies, Faculty of Science and Engineering, Queensland University of Technology, Brisbane, QLD, Australia
3 School of Medicine and Dentistry, Griffith University, Brisbane, QLD, Australia
4 Orthopaedic Department, The Prince Charles Hospital, Brisbane, QLD, Australia
5 Australia-China Centre for Tissue Engineering and Regenerative Medicine, Queensland University of Technology, Brisbane, QLD, Australia
6 Health Management Center, The Xiangya Hospital of Central South University, Changsha, China
Indira Prasadam, NHMRC Emerging Leadership Fellow, Stem Cell and Tissue Engineering Program, Group Leader: Biomimetic Technologies for Cartilage Repair and Arthritis (BT-CRA), SEF Faculty Research Ethics, and Integrity Advisor, Centre for Biomedical Technologies, School of Mechanical, Medical and Process Engineering, Queensland University of Technology, 60 Musk Ave/cnr. Blamey Street, Kelvin Grove, Brisbane, QLD 4059, Australia. Email: i.prasadam@qut.edu.au
Xinzhan Mao, Director of Orthopaedics, Department of Orthopaedic Surgery, The Second Xiangya Hospital of Central South University, 139 Middle RenMin Road, Changsha 410011, China. Email: xinzhan.mao@csu.edu.cn
* Co-first authors.

21 4 2023
6 2024
15 2 184194
18 12 2022
24 3 2023
29 3 2023
© The Author(s) 2023
2023
SAGE Publications Ltd unless otherwise noted. Manuscript content on this site is licensed under Creative Commons Licenses
https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Objective

Chronic inflammation plays an important role in the osteoarthritis (OA) pathology but how this influence OA disease progression is unclear. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator generated from arachidonic acid through the sequential activities of 5-lipoxygenase, 5-lipoxygenase–activating protein, Leukotriene A4 hydrolase (LTA4H) and its downstream product LTB4. The aim of this study is to investigate the involvement and the potential therapeutic target of the LTB4 pathway in OA disease progression.

Design

Both clinical human cartilage samples (n = 7) and mice experimental OA models (n = 6) were used. The levels of LTA4H and leukotriene B4 receptor 1 were first examined using immunostaining in human OA/non-OA cartilage and mice experimental OA models. We also determined whether the LTA4H pathway was associated with cartilage degeneration and synovitis inflammation in OA mice models and human articular chondrocytes.

Results

We found that both LTA4H and LTB4 receptor (BLT1) were highly expressed in human and mice OA cartilage. Inhibition of LTA4H suppressed cartilage degeneration and synovitis in OA mice model. Furthermore, inhibition of LTA4H promoted cartilage regeneration by upregulating chondrogenic genes expression such as aggrecan (ACAN), collagen 2A1 (COL2A1), and SRY-Box transcription factor 9 (SOX9).

Conclusions

Our results indicate that the LTA4H pathway is a crucial regulator of OA pathogenesis and suggest that LTA4H could be a therapeutic target in combat OA.

osteoarthritis
leukotriene A4 hydrolase
inflammation
cartilage degeneration
National Natural Science Foundation of China https://doi.org/10.13039/501100001809 81371997 Prince Charles Hospital Foundation https://doi.org/10.13039/501100018853 RF-01 National Health and Medical Research Council https://doi.org/10.13039/501100000925 APP1176298 Hunan Provincial Science and Technology Department https://doi.org/10.13039/501100002767 2015JC3034 Hunan Provincial Science and Technology Department https://doi.org/10.13039/501100002767 2018JJ2579 typesetterts1
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pmcIntroduction

Osteoarthritis (OA) remains the leading cause of disability, morbidity, and mortality worldwide with no cure. 1 OA is characterized by cartilage degradation, osteophytes, subchondral bone sclerosis and synovitis, causing pain and reduced function of the affected joints. In the past few years, growing evidence has shown that non-resolved persistent synovitis is important in OA radiographic progression. 2 The proper shift from inflammatory to non-inflammatory status is essential to avoid tissue damage and the development of chronic inflammation. 3 Resolution of inflammation is an active process involving a molecular pathway switching from inflammatory eicosanoids and pro-resolving mediators. We demonstrated that promoting inflammation resolution by a pro-resolving lipid mediator, Resolvin D1, attenuated OA-like changes in a mouse model of post-traumatic OA. 4 Although the mechanism of persistent chronic inflammation in OA is incompletely understood, improper/inactivated resolution pathways may play an important role.

Inflammatory cascades in OA cartilage and synovial joints are associated with various cytokines and lipid mediators, mainly the eicosanoids and pro-inflammatory mediators. 5 Eicosanoid molecules produced mediators have been highlighted and linked to inflammatory and cellular metabolic responses in many studies, of which leukotrienes (LTs) are recognized as having a pivotal role.6 -10 Eicosanoids, including prostaglandins and LTs, are biologically active lipids implicated in various pathological processes, such as inflammation and cancer. 11 Leukotriene A4 hydrolase (LTA4H) is classically recognized as an epoxide hydrolase to generate leukotriene B4 (LTB4) from leukotriene A4 in response to inflammatory stimulus. 12 This enzyme is expressed mainly in neutrophils and other cell types, including endothelial cells and articular chondrocytes.13 -15 Leukotriene A4 is a pro-inflammatory lipid mediator generated from membrane phospholipid by the 5-lipoxygenase (5-LOX) enzymatic action at arachidonic acid (AA) during the initial phases of inflammation. LTB4 regulates inflammatory cytokine expression and recruitment of neutrophils primarily through its G protein-coupled receptor BLT1, which is only activated by LTB4.16,17 The involvement of LTB4 has been studied in various diseases, such as inflammatory arthritis and metabolic disorders.18,19 Given the central role of LTB4 in regulating inflammatory response mechanisms through recruitment and activation of leukocytes, modulation of the LTB4 pathway has been considered a new therapeutic approach in treating several diseases.

Previous studies have shown that an elevated level of LTB4 is found in the synovium, synovial fluid, articular cartilage, and subchondral bone from patients with OA.20,21 It was also revealed that increased expression of LTA4H occurs in human OA synovial fluid. 5 Although the activity of LTA4H and expression of BLT1 was found in human cultured chondrocytes,22,23 the relationship and mechanism of LTA4H and OA have not been well studied. In the current study, we firstly investigated the expression of LTA4H and BLT1 in OA cartilage. Based on these observations, we further studied the involvement of leukotriene in disease pathogenesis using an established OA model. We tested whether LTA4H inhibitor can affect OA development and progression using both in vivo and vitro models.

Materials and Methods

Study Approval

Animal studies were approved by the Institutional Animal Care and Use Committees and Institutional Biosafety Committees of Central South University, China (CSU; 2013-05). Six-week-old male C57BL/6 mice were purchased from the Animal Centre of Central South University (Changsha, Hunan, China). Collection of human OA tissues from discarded knee replacement surgeries was approved by the Queensland University of Technology (QUT) and the St Vincent Private Hospital Ethics Committees (Human ethics number: #1400001024). Informed consent was obtained from all human subjects.

Human Cartilage Specimen Preparation

Human cartilage was obtained from patients with OA (Kellgren-Lawrence grade 4) undergoing total knee replacement. Following our previous publication, the cartilage tissues collected from the relatively smooth region were classified into the grade 1 (G1, non-OA) group, and the cartilage collected from the severely damaged region were classified into the grade 4 cartilage (G4, OA) group. 24 Osteochondral plugs (including cartilage and subchondral bone) were taken from the tibial plateau region. All samples were prepared for histology and immunohistochemical analysis by washing in phosphate-buffered saline (PBS) and immediately fixed in 4% paraformaldehyde. The samples were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH = 7.2–7.4). The specimens were embedded in paraffin, and 5-µm-thick sections were obtained as described in our previous study. 25

Cell Culture and Bestatin Treatment In Vitro

Human chondrocyte cell line C28I2 was seeded into 6-well plates at a density of 1 × 106 cells/well and cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% of penicillin and streptomycin (PS). At 90% confluence, cells were serum-starved for 24 hours and then were incubated in the presence or absence of 10 ng/ml interleukin-1β (IL-1β) or 50 μM bestatin with culture medium for 24 hours. Bestatin is a dipeptide immunomodulator that was discovered in the culture supernatant fraction from Streptomyces olivoreticuli, and it is a well-characterized inhibitor of LTA4H to attenuate LTB4 synthesis in many studies and particularly in animal models.26 -28

Cell Viability Test

Human chondrocyte cell line C28I2 was seeded into 96-well plates at a density of 5000 cells/well with culture medium. After 24 hours, cells were serum-starved for 24 hours and then were incubated in 0, 10, 50, 100, and 200 μM of bestatin separately. Cell viability test and cell count were performed according to our previous publication using CyQUANT proliferation assay (Life Technologies, Thermo Fisher). 29 Briefly, 100 μl of lysis buffer containing the CyQUANT GR dye was added to each well and incubated at 37 °C for 30 minutes. Fluorescence was measured using a fluorescence microplate reader with an excitation of 485 nm and emission of 530 nm.

Preparation of Bestatin

Bestatin was purchased from Cayman Chemical (Michigan, USA), and 2-hydroxypropyl-β-cyclodextrin (HPCD) and polyethylene glycol 400 (PEG-400) were obtained from Sigma Aldrich (Missouri, USA). The oral formulation for bestatin was prepared as previously described. 13 Briefly, 100 µl PEG-400 and 100 mg of bestatin were gently mixed before dissolving in a 20% (w/v) aqueous solution of HPCD. Then, the solution was mixed at room temperature to obtain a homogeneous mixture at 2 mg/ml concentration.

Induction of OA in Mice and Bestatin Treatment

Mice were group-housed (5 animals/cage) in ventilated cages in a temperature-controlled room on 12-hour light/dark cycles with ad libitum access to standard mouse food and water and routine veterinary assessment. Six-week-old male C57BL/6 mice were purchased following our previous publications.4,25 After that, all the animals were fed with the standard diet from the Animal Center (control diet) for at least 2 weeks as an observation period. Eight-week-old C57BL/6 mice (similar to other studies,30 -32 while 8-week-old of mice were considered as around 18-year-old in human age) 33 were randomly assigned and underwent destabilization of the medial meniscus (DMM) surgery to induce OA as previously described. 25 Briefly, OA was induced by dissecting the medial meniscus ligament to destabilize the medial meniscus in the knee joint of the hind limb on the right side. Mice in the sham group were surgically treated in which the ligament was visualized but not dissected. One week after experimental OA induction, mice were treated orally with bestatin 34 or vehicle (PEG-400 and HPCD). All experimental mice were then received twice-weekly oral bestatin after DMM surgery at a dose of 5 mg/kg for a total of 8 weeks. Bestatin (5 mg/kg) significantly decreased serum levels of LTB4 and BLT1 production in a mouse model and showed no difference to high dose of bestatin (15 mg/kg). 27 Mice were sacrificed 8 weeks after surgery for histological analysis. Bestatin is well absorbed in the gastrointestinal tract and is widely distributed in the body, including the kidney, liver, lung, spleen, and heart. It is metabolized in the liver, and its active metabolites are further metabolized and excreted in the urine.35,36 Overall, bestatin is rapidly absorbed and metabolized and is safe for in vivo experiments.

Histologic Evaluation of Knee Joint in Mice

After euthanasia, the knee joints of hind limbs were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, embedded in paraffin, and sectioned for histological and immunohistochemical analysis. Sections were stained with Safranin-O (Saf-O)/Fast green to evaluate cartilage degradation and synovitis. The severity of OA was assessed in the medial compartment of the knee using a modified Mankin scoring system (0–14) as previously described. 25 The degree of synovitis was scored using a 0 to 6 scoring system that measured the thickness of the synovial lining cell layer on a scale of 0 to 3 (0 = 1–2 cells, 1 = 2–4 cells, 2 = 4–9 cells, and 3 = 10 or more cells) and cellular density in the synovial stroma on a scale of 0 to 3 (0 = normal cellularity, 1 = slightly increased cellularity, 2 = moderately increased cellularity, and 3 = greatly increased cellularity) as previously described. 25 Both the Mankin scoring and synovial inflammation scoring were carried out by 2 independent blinded readers (Indira and Antonia). The sum of both parameters was used for analysis. Synovitis scores obtained from all 4 quadrants (lateral femur, lateral tibia, medial femur, and medial tibia).

Immunohistochemistry

Immunohistochemistry was performed according to our previously published methods.37,38 Briefly, after dewaxing, sections were incubated with proteinase K for 20 minutes for antigen retrieval. The endogenous peroxidase activity was eliminated with 3% H2O2 in PBS for 20 minutes. Sections were then eliminated non-specific protein binding by incubation with 1% Bovine serum albumin for 1 hour at room temperature. Sections were incubated overnight at 4 °C with antibodies against LTA4H (Abcam, Melbourne, VIC, Australia; dilution 1:250) and LTB4 receptor (BLT1, LSBio, Sapphire Bioscience, Redfern, NSW, Australia; dilution 1:50) followed by incubation with anti-rabbit or anti-rat secondary antibody (1:5000 dilution; LI-COR Biosciences, Nebraska, USA) and visualization with 3,3-diaminobenzidine chromogen. To exclude antigen-independent staining, negative controls for which the incubation step with the primary antibody was omitted were examined. The percentage of positive cells was determined by counting the number of immunostained cells and dividing by the total number of cells (100 cells per sample) visualized by a hematoxylin counterstain using ImageJ (National Institute of Health, Bethesda, BA, USA).

Quantitative Real-Time PCR

Total RNA was extracted with TRIzol, and cDNA was synthesized using the SensiFAST cDNA Synthesis Kit according to the manufacturer’s protocol. SYBR Green detection chemistry was used on the QuantStudio Real-Time PCR system (Applied Biosystems, Thermo Scientific, Scoresby, VIC, Australia). Quantitative measurements of all primers used in this study were determined using (2−ΔΔ Ct) method, and GAPDH expression was used as the internal control,27,39 as described previously by our group.37,40 -42

Statistical Analysis and Sample Size Justification

All experiments were performed in triplicate using different samples: 7 donors of human knee cartilage for histologic evaluation; 5 donors for immunohistochemistry; and 6 mice per group were used for animal study. Statistical difference of the results was tested using the unpaired Student’s t-test comparison of 2 variables. One-way analysis of variance followed by post hoc test was used for comparison of more than 2 variables. All analyses were performed using GraphPad Prism 7, and P < 0.05 is significant. All data are presented as mean ± SD. Sample size for each experiment was determined using G power program (https://clincalc.com/stats/samplesize.aspx). Eighty percent power and an alpha level of 0.05 were used for the calculation. Given concerns about the ability to complete the experiments, we increased the sample sizes in the experiments above.

Results

Increased LTA4H and BLT1 in OA Cartilage

To study the leukotriene A4 pathway in the articular cartilage, we first divided the cohort of OA patients into 2 groups based on a histomorphometry analysis for the semi-quantitative estimation of OA extent on Safranin-O-stained cartilage sections. As exemplarily shown in Fig. 1A for intact smooth cartilage surface (grade 1, G1) and severely degenerated and fibrillated OA regions (grade 4, G4), significant cartilage damage associated with elevations of Mankin score (12.14 ± 1.574, n = 7) were observed in G4 OA sites; compared with G1 group (Mankin score = 3.286 ± 0.951; n = 7) ( Fig. 1B ).

Figure 1. Increased leukotriene A4 hydrolase (LTA4H) and leukotriene B4 receptor 1 (BLT1) in osteoarthritis cartilage. (A) Representative Safranin-O (Saf-O) and fast green stained sections of G1 or G4 knee regions. Similar sections were stained with LTA4H and BLT1. Scale bars, 200 μm. (B) Severity of articular cartilage degradation was graded using Mankin scoring system. Graphs represent mean ± SD (n = 7). The percentage of LTA4H (C) and BLT1 (D) positive cells per knee section were counted. Graphs represent mean ± SD (n = 5). (E) Sham and DMM mice knees were stained with LTA4H and BLT1 (n = 6). Positive cells per knee section were counted (F). Scale bars, 20 μm. Graphs represent as mean ± SD. DMM = the surgical destabilization of the medial meniscus. P < 0.05 is considered significant.

We then examined LTA4H and BLT1 expression in OA cartilage of humans and mice DMM OA model. The percentage of LTA4H immunostained chondrocytes was markedly elevated in OA-affected G4 cartilage compared with undamaged G1 regions of patient-matched OA cartilages ( Fig. 1A and C ). BLT1 levels were also significantly increased in severe G4 versus mild G1 cartilage ( Fig. 1A and D ). To further ascertain whether leukotriene plays a role in OA pathogenesis, we examined LTA4H and BLT1 expression in OA cartilage of mice. Similar results were found in experimental OA mice, in which LTA4H and BLT1 were significantly increased in knee cartilage from DMM mice compared to Sham surgery mice ( Fig. 1E and F ). These results indicated that the expression of LTA4H and BLT1 is in accordance with the disease severity.

Inhibition of LTA4H Level Suppressed Articular Cartilage Degradation In Vivo

The articular cartilage of mice with DMM-induced OA exhibited pathological OA changes characterized by Safranin-O loss and detectable cartilage roughness ( Fig. 2A ), with a Modified Mankin score of 11.833 ± 1.169 ( Fig. 2B ). In contrast, the knee joint of sham mice exhibited no visible changes on the articular cartilages with a significant lower Mankin score (0.857 ± 0.690) than that in mice with DMM-induced OA ( Fig. 2A and B ). Bestatin is a well-characterized inhibitor of LTA4H and was shown to attenuate LTB4 synthesis in several previous studies.27,43 Inhibition of LTA4H level in mice with DMM-induced OA suppressed OA progression with a lower Mankin score (8.000 ± 1.095) compared with DMM-induced OA mice with vehicle ( Fig. 2A and B ). These results suggested that increased leukotriene may be one of the reasons causing OA as inhibition of which could suppress OA progress in DMM mice.

Figure 2. Inhibition of LTA4H level suppressed articular cartilage degradation in a mouse model of experimental osteoarthritis. (A) Representative Saf-O and fast green stained sagittal sections of sham and DMM mice knee with/without LTA4H inhibitor. Scale bars in the top panel, 100 μm; bottom panel, 20 μm. (B) Severity of articular cartilage degradation was graded using Mankin scoring system. Graphs represent mean ± SD (n = 6). LTA4H = leukotriene A4 hydrolase; DMM = the surgical destabilization of the medial meniscus. P < 0.05 is considered significant.

Inhibition of LTA4H Level Reduced Synovitis in OA Mice

Mice with DMM-induced OA also revealed an increased influx of synoviocytes and lining layer thickening in synovium compared with the respective control mice ( Fig. 3A ). The synovitis scores reiterated these observations with a significantly higher score in DMM-induced OA mice (Synovitis score: 4.417 ± 0.540 compared with 1.208 ± 0.485) ( Fig. 3B ). The role of leukotriene in synovitis in mice was examined by oral administration of LTA4H inhibitor-bestatin. Bestatin treatment reduced the thickness of the synovial lining cell layer and cellular density in the synovial stroma in mice with DMM (Synovitis score: 2.917 ± 0.585) compared with vehicle-treated mice with DMM (Synovitis score: 4.417 ± 0.540) ( Fig. 3A and B ). However, oral administration of bestatin had no significant effect on synovitis in mice without DMM ( Fig. 3A and B ). These results indicated that bestatin treatment also reduced synovitis.

Figure 3. Inhibition of LTA4H level reduced synovitis in osteoarthritis mice. (A) Representative Saf-O and fast green stained sagittal sections of sham and DMM mice knee with/without LTA4H inhibitor. Scale bars, 100 μm. (B) Severity of articular synovitis inflammation was graded using the Synovitis scoring system. Graphs represent mean ± SD (n = 6). LTA4H = leukotriene A4 hydrolase; DMM = the surgical destabilization of the medial meniscus. P < 0.05 is considered significant.

In Vitro Inhibition of LTA4H Significantly Affect Gene Expression Profile in Chondrocytes

We further tested the effect of LTA4H inhibitor on C28I2 chondrocytes cell line in vitro. To test whether bestatin plays a direct role in chondrocytes, we validated the potential effect of bestatin by gene expression profile analysis. We first tested cell viability under different concentrations of bestatin ( Fig. 4A and B ). Our results indicated that 0 to 200 μM of bestatin treatment showed no toxic effect on chondrocytes for 24 hours. Next, we tested the effects of 50 μM bestatin treatment on chondrocytes phenotype. Bestatin treatment significantly upregulated COL2A1 and inhibited SOX9 expression in chondrocytes in the absence of IL-1β ( Fig. 4D and F ). No significant effects of bestatin on the expression of aggrecan (ACAN), Runt-related transcription factor 2 (RUNX2), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and MMP13 were observed (Fig. 4C, E, G, and H). To establish an in vitro OA model, the cells were stimulated with IL-1β (10 ng/ml). The expression of RUNX2 gene caused by IL-1β in chondrocytes was significantly inhibited by bestatin ( Fig. 4E ). The gene expression of ACAN, COL2A1, and SOX9 was robustly upregulated by bestatin, but there were no significant changes in the cartilage degradation marker, ADAMTS5 ( Fig. 4G ). In response to bestatin treatment, MMP13 showed a decreased gene expression; however, this effect was not significant ( Fig. 4H ).

Figure 4. In vitro inhibition of LTA4H significantly affect gene expression profile in chondrocytes. Cell viability (A), cell counts (B), and qualitative polymerase chain reaction analysis of ACAN (C), COL2A1 (D), RUNX2 (E), SOX9 (F), ADAMTS5 (G), and MMP13 (H) messenger RNA in C28I2 chondrocytes cell line with/without LTA4H inhibitor under normal and inflammation condition (interleukin-1β treatment). Data represented as mean ± SD (n = 6). NS = not significant; LTA4H = leukotriene A4 hydrolase; ACAN = aggrecan; COL2A1 = collagen 2A1; RUNX2 = Runt-related transcription factor 2; SOX9 = SRY-Box transcription factor 9; ADAMTS5 = a disintegrin and metalloproteinase with thrombospondin motifs 5. P < 0.05 is considered significant.

Discussion

Growing evidence shows that the resolution of inflammation represents a critical therapeutic target in OA treatment. 44 The involvement of lipid mediators in inflammatory events has been brought into focus with several studies pointing to its role in a multitude of cellular responses, including cell growth and death and inflammation via receptor-mediated pathways. 45 A constitutive low-grade inflammatory response accompanies the OA, and the leukotriene signalling is now considered a potent chemoattractant agent related to acute inflammatory responses and maintenance of chronic inflammation.46,47 In this study, we examined the role of the leukotriene pathway in the development of OA. We found that the level of LTA4H and LTB4 receptor BLT1 was increased in articular cartilage from OA patients and mice with post-traumatic OA. Moreover, inhibition of LTA4H decreased cartilage tissue damage and synovial inflammation. These results indicate that upregulation of LTB4 could be an important event in OA pathogenesis, and its upregulation may be involved in the mechanisms that trigger both cartilage degeneration and synovial inflammation.

First, the production of LTA4H and LTB4 in OA has been previously shown. Paredes et al. have shown that human OA subchondral osteoblasts constitutively produce LTB4, whereas He et al. and Lascelles et al. have demonstrated a high amount of LTB4 in the synovial tissue from human and animal models of OA, respectively.20,48,49 Overexpression of LTA4H in human OA synovial fluid was also observed, and treatments with chemical inhibitors of 5-lipoxygenase- activating protein/5-lipoxygenase (FLAP/5-LOX) were shown to inhibit the severity of OA.5,50,51 Our experiment found that human OA cartilage/chondrocytes expressed increased LTA4H and LTB4 receptor BLT1. These findings are in corroboration with a study performed by Martel-Pelletier et al. that revealed the presence of FLAP and 5-LOX (as determinants of LTB4 synthetic capacity) in chondrocytes. 51 Besides, Hansen et al. found a decreased level of BLT1 in OA chondrocytes. This might be because the samples they compared were cultured cells, which showed a significant variation between cartilage and cultured chondrocytes in their study. 22 In the mouse model of post-traumatic OA, we observed significant changes in cartilage damage and synovitis and correlated with an increase in the expression of LTA4H and BLT1. Similarly, Attur et al. showed that the level of lipoxygenase-derived products such as LTB4 was found to be significantly increased in OA cartilage. 52 Although LTB4 expresses mainly in inflammatory and immune cells, including macrophages, granulocytes, mast cells, and T and B lymphocytes, our study and other studies all demonstrate that the inflammatory leukotrienes are also produced by human OA chondrocytes. 52 LTB4 is stimulated by diverse signalling molecules, including pro-inflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and IL-1β, and itself. 12 The LTB4 synthesis in OA chondrocytes is increased by growth factors such as transforming growth factor β1 (TGFβ1) and 1,25(OH)2D3 in the inflammatory response. 51 Thus, LTA4H and LTB4 in OA tissues could be considered an important event in OA pathogenesis and pathophysiology.

The inflammatory process in diseases is due to an imbalance between pro-inflammatory and anti-inflammatory molecules and a deficiency of bioactive lipid mediators in the resolution phase of inflammation. 17 It has been reported that LTB4 contributes to persistent inflammation in arthritis by delivering IL-1 into the joint through neutrophils. 53 More recent studies have shown that BLT1−/− mice were protected entirely from collagen-induced arthritis development and systemic insulin resistance in diet-induced obese mice.18,19 Our previous study has shown that intra-articular treatment of pro-resolving lipid mediator resolvin D1 diminishes the progression of OA in mice. 4 Leukotriene has also been shown to increase the production of pro-inflammatory cytokines, IL-1β, and TNF-α in dose-dependent human OA synovial membrane.48,54 Modulating the secretion of lipid mediators may therefore attenuate the severity of the disease. In this study, we considered the LTA4H as the candidate to investigate the effect of leukotriene activity on OA progression since it is the essential enzyme that catalyzes the distal step in LTB4 generation. Blocking LTA4H by bestatin in vivo has shown protective and preventive effects in various diseases such as pulmonary hypertension and oesophagal adenocarcinomas.13,26 Another advantage of bestatin is the low toxicity. 26 Therefore, we used the LTA4H inhibitor bestatin and tested if inhibiting LTB4 production has beneficial effects in mice with post-traumatic OA. In our study, bestatin significantly inhibited the presence of LTA4H and BLT1 production in articular cartilage of mice with DMM-induced OA. Our results indicate that LTA4H inhibition decreases cartilage degradation and synovitis in a mouse model of post-traumatic OA. This decrease in synovial inflammation could be because LTB4 is a critical mediator in the cascade of complement, lipids, cytokines, and chemokines. In BLT1 knock-out mice, LTB4 has shown crucial roles in T helper 2 (Th2)-type cytokine interleukin-13 (IL-13) production from lung T cells and recruitment of antigen-specific effector CD8+ T cells. 55 Therefore, blockade of the LTB4-BLT1 pathway might be a promising therapeutic strategy in OA patients. However, clinical trials to date have shown that compared with traditional disease-modifying antirheumatic drugs, oral LTB4 receptor and 5-LOX inhibitors have little clinical benefit 56 ; however, whether or not this is the case with OA patients needs further investigation. The limitations of this study were that our results couldn’t reveal that the mechanism-of-action of bestatin is via attenuated synovitis or reduced synovial-driven cartilage degeneration. Further experimental design was needed to investigate the role of bestatin, synovitis, and cartilage degeneration. Another limitation was that DAPI staining for nuclei detection was no used in our study. The quantification of the immunohistochemistry (IHC) staining in this study was calculated by counting the number of positively stained cells per 100 cells per slide visualized by hematoxylin counterstain to avoid difference of cellularity between samples. This is also one of the well-established methods for IHC quantification in several publications.57 -60 We have been trying to quantify by using staining intensity or IDO value. However, because chondrocytes only accounts for around 5% of the cartilage area, it would bring us much background signal for quantification.

In conclusion, our study applied oral LTB4 receptor inhibitor bestatin for feeding mice after OA surgery. Reduced cartilage damage and synovium inflammation were observed in mice joints after 8 weeks, indicating that oral bestatin uptake in early OA might be a potential drug for OA therapy targeting Leukotriene A4 hydrolase.

Author Contributions: Conceptualization: I.P., X.M., R.C., Y.X., and Y.W.; methodology and formal analysis: X.W., I.P., and A.S.; drafting of the article: A.S., X.W., and I.P.; review and editing: all authors; supervision: I.P.; acquisition of funding: I.P., X.M. and Y.W. All authors have read and agreed to the published version of the manuscript.

Acknowledgments and Funding: The authors thank the team of the central animal facility of the Central South University for expert animal care. The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This research was funded by Hunan Provincial Science and Technology Department, China (2018JJ2579, 2015JC3034) and National Natural Science Foundation of China (81371997), and The Prince Charles Hospital Research Foundation fellowship (RF-01) and NHMRC Investigator Fellowship (APP1176298) for Indira Prasadam.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval: Animal studies were approved by the Institutional Animal Care and Use Committees and Institutional Biosafety Committees of Central South University, China (CSU; 2013-05). Collection of OA tissues from discarded knee replacement surgeries were approved by the Queensland University of Technology and the St Vincent Private Hospital Ethics Committees (Human ethics number: #1400001024).

ORCID iD: Xinzhan Mao https://orcid.org/0000-0002-1835-6043
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References

1 Woolf AD Pfleger B. Burden of major musculoskeletal conditions. Bull World Health Organ. 2003;81 (9 ):646-56.
2 Kortekaas MC Kwok WY Reijnierse M Kloppenburg M. Inflammatory ultrasound features show independent associations with progression of structural damage after over 2 years of follow-up in patients with hand osteoarthritis. Ann Rheum Dis. 2015;74 (9 ):1720-4.
3 Headland SE Norling LV. The resolution of inflammation: principles and challenges. Semin Immunol. 2015;27 (3 ):149-60.
4 Sun AR Wu X Liu B Chen Y Armitage CW Kollipara A , et al . Pro-resolving lipid mediator ameliorates obesity induced osteoarthritis by regulating synovial macrophage polarisation. Sci Rep. 2019;9 :426.30674985
5 Jónasdóttir HS Brouwers H Kwekkeboom JC van der Linden HMJ Huizinga T Kloppenburg M , et al . Targeted lipidomics reveals activation of resolution pathways in knee osteoarthritis in humans. Osteoarthritis Cartilage. 2017;25 (7 ):1150-60.
6 Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 2001;294 :1871-5.
7 Abdellatif KR Chowdhury MA Dong Y Chen QH Knaus EE. Diazen-1-ium-1,2-diolated and nitrooxyethyl nitric oxide donor ester prodrugs of anti-inflammatory (E)-2-(aryl)-3-(4-methanesulfonylphenyl)acrylic acids: synthesis, cyclooxygenase inhibition, and nitric oxide release studies. Bioorg Med Chem. 2008;16 :3302-8.
8 Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science. 1983;220 :568-75.
9 Shin K Hwang JJ Kwon BI Kheradmand F Corry DB Lee SH. Leukotriene enhanced allergic lung inflammation through induction of chemokine production. Clin Exp Med. 2015;15 (3 ):233-44.
10 Filgueiras LR Brandt SL Wang S Wang Z Morris DL Evans-Molina C , et al . Leukotriene B4-mediated sterile inflammation promotes susceptibility to sepsis in a mouse model of type 1 diabetes. Sci Signal. 2015;8 :ra10.
11 Greene ER Huang S Serhan CN Panigrahy D. Regulation of inflammation in cancer by eicosanoids. Prostaglandins Other Lipid Mediat. 2011;96 (1-4 ):27-36.21864702
12 Henderson WR Jr. The role of leukotrienes in inflammation. Ann Intern Med. 1994;121 :684-97.
13 Tian W Jiang X Tamosiuniene R Sung YK Qian J Dhillon G , et al . Blocking macrophage leukotriene b4 prevents endothelial injury and reverses pulmonary hypertension. Sci Transl Med. 2013;5 :200ra117.
14 Low CM Akthar S Patel DF Löser S Wong CT Jackson PL , et al . The development of novel LTA(4)H modulators to selectively target LTB(4) generation. Sci Rep. 2017;7 :44449.28303931
15 Lin HN O’Connor JP. Immunohistochemical localization of key arachidonic acid metabolism enzymes during fracture healing in mice. PLoS One. 2014;9 (2 ):e88423.
16 Tager AM Luster AD. BLT1 and BLT2: the leukotriene B(4) receptors. Prostaglandins Leukot Essent Fatty Acids. 2003;69 :123-34.
17 Fullerton JN Gilroy DW. Resolution of inflammation: a new therapeutic frontier. Nat Rev Drug Discov. 2016;15 (8 ):551-67.
18 Shao WH Del Prete A Bock CB Haribabu B. Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice. J Immunol. 2006;176 :6254-61.
19 Spite M Hellmann J Tang Y Mathis SP Kosuri M Bhatnagar A , et al . Deficiency of the leukotriene B4 receptor, BLT-1, protects against systemic insulin resistance in diet-induced obesity. J Immunol. 2011;187 :1942-9.
20 Paredes Y Massicotte F Pelletier JP Martel-Pelletier J Laufer S Lajeunesse D. Study of the role of leukotriene B()4 in abnormal function of human subchondral osteoarthritis osteoblasts: effects of cyclooxygenase and/or 5-lipoxygenase inhibition. Arthritis Rheum. 2002;46 :1804-12.
21 Hashimoto A Endo H Hayashi I Murakami Y Kitasato H Kono S , et al . Differential expression of leukotriene B4 receptor subtypes (BLT1 and BLT2) in human synovial tissues and synovial fluid leukocytes of patients with rheumatoid arthritis. J Rheumatol. 2003;30 (8 ):1712-8.
22 Hansen AK Indrevik JT Figenschau Y Martinez-Zubiaurre I Sveinbjörnsson B. Human articular chondrocytes express functional leukotriene B4 receptors. J Anat. 2015;226 (3 ):268-77.
23 Amat M Díaz C Vila L. Leukotriene A4 hydrolase and leukotriene C4 synthase activities in human chondrocytes: transcellular biosynthesis of Leukotrienes during granulocyte-chondrocyte interaction. Arthritis Rheum. 1998;41 (9 ):1645-51.
24 Fan X Wu X Trevisan Franca De Lima L Stehbens S Punyadeera C Webb R , et al . The deterioration of calcified cartilage integrity reflects the severity of osteoarthritis—a structural, molecular, and biochemical analysis. FASEB J. 2022;36 (2 ):e22142.
25 Sun AR Wu X Crawford R Li H Mei L Luo Y , et al . Effects of diet induced weight reduction on cartilage pathology and inflammatory mediators in the joint tissues. Front Med. 2021;8 :628843.
26 Chen X Li N Wang S Wu N Hong J Jiao X , et al . Leukotriene A4 hydrolase in rat and human esophageal adenocarcinomas and inhibitory effects of bestatin. J Natl Cancer Inst. 2003;95 :1053-61.
27 Zhao S Yao K Li D Liu K Jin G Yan M , et al . Inhibition of LTA4H by bestatin in human and mouse colorectal cancer. EBioMedicine. 2019;44 :361-74.
28 Zhao S Yao K Liu K Huang L Jiang Y Li J , et al . Bestatin cream impairs solar simulated light‒driven skin inflammation and skin carcinogenesis in mice. J Invest Dermatol. 2021;141 (11 ):2699-709.
29 Wu X Crawford R Xiao Y Mao X Prasadam I. Osteoarthritic subchondral bone release exosomes that promote cartilage degeneration. Cells. 2021;10 :251.33525381
30 Samvelyan HJ Madi K Törnqvist AE Javaheri B Staines KA. Characterisation of growth plate dynamics in murine models of osteoarthritis. Front Endocrinol. 2021;12 :734988.
31 Loeser RF Olex AL McNulty MA Carlson CS Callahan M Ferguson C , et al . Disease progression and phasic changes in gene expression in a mouse model of osteoarthritis. PLoS One. 2013;8 (1 ):e54633.
32 Li H You Y Jiang B Li H Li X Wu W , et al . Wang-Bi tablet ameliorates DMM-induced knee osteoarthritis through suppressing the activation of p38-MAPK and NF-κB signaling pathways in mice. Evid Based Complement Alternat Med. 2021;2021 :3930826.34426743
33 Wang S Lai X Deng Y Song Y. Correlation between mouse age and human age in anti-tumor research: significance and method establishment. Life Sci. 2020;242 :117242.31891723
34 Ichinose Y Genka K Koike T Kato H Watanabe Y Mori T , et al . Randomized double-blind placebo-controlled trial of bestatin in patients with resected stage I squamous-cell lung carcinoma. J Natl Cancer Inst. 2003;95 :605-10.
35 Scornik OA Botbol V. Bestatin as an experimental tool in mammals. Curr Drug Metab. 2001;2 (1 ):67-85.11465152
36 Abe F Alvord G Koyama M Matsuda A Talmadge JE. Pharmacokinetics of bestatin and oral activity for treatment of experimental metastases. Cancer Immunol Immunother. 1989;28 (1 ):29-33.2909281
37 Prasadam I van Gennip S Friis T Shi W Crawford R Xiao Y. ERK-1/2 and p38 in the regulation of hypertrophic changes of normal articular cartilage chondrocytes induced by osteoarthritic subchondral osteoblasts. Arthritis Rheum. 2010;62 (5 ):1349-60.
38 Sun AR Panchal SK Friis T Sekar S Crawford R Brown L , et al . Obesity-associated metabolic syndrome spontaneously induces infiltration of pro-inflammatory macrophage in synovium and promotes osteoarthritis. PLoS One. 2017;12 (8 ):e0183693.
39 Orth-He EL Huang HC Rao SD Wang Q Chen Q O’Mara CM , et al . Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation. Cell Rep. 2023;42 :111965.36649711
40 Prasadam I Mao X Wang Y Shi W Crawford R Xiao Y. Inhibition of p38 pathway leads to OA-like changes in a rat animal model. Rheumatology. 2012;51 (5 ):813-23.
41 Prasadam I Crawford R Xiao Y. Aggravation of ADAMTS and matrix metalloproteinase production and role of ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes—possible pathogenic role in osteoarthritis. J Rheumatol. 2012;39 (3 ):621-34.
42 Prasadam I Zhou Y Shi W Crawford R Xiao Y. Role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis. Rheumatology (Oxford). 2014;53 (12 ):2280-7.
43 Méndez LR Arrebola Y Valdés-Tresanco ME Díaz-Guevara L Bergado G Sánchez B , et al . Bestatin and bacitracin inhibit porcine kidney cortex dipeptidyl peptidase IV activity and reduce human melanoma MeWo cell viability. Int J Biol Macromol. 2020;164 :2944-52.
44 Goldring MB Otero M. Inflammation in osteoarthritis. Curr Opin Rheumatol. 2011;23 :471-8.
45 Serhan CN Chiang N Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol. 2008;8 (5 ):349-61.
46 Ying W Wollam J Ofrecio JM Bandyopadhyay G El Ouarrat D Lee YS , et al . Adipose tissue B2 cells promote insulin resistance through leukotriene LTB4/LTB4R1 signaling. J Clin Invest. 2017;127 :1019-30.
47 Peters-Golden M Henderson WR Jr. Leukotrienes. N Engl J Med. 2007;357 :1841-54.
48 He W Pelletier JP Martel-Pelletier J Laufer S Di Battista JA. Synthesis of interleukin 1beta, tumor necrosis factor-alpha, and interstitial collagenase (MMP-1) is eicosanoid dependent in human osteoarthritis synovial membrane explants: interactions with antiinflammatory cytokines. J Rheumatol. 2002;29 (3 ):546-53.
49 Lascelles BD King S Roe S Marcellin-Little DJ Jones S. Expression and activity of COX-1 and 2 and 5-LOX in joint tissues from dogs with naturally occurring coxofemoral joint osteoarthritis. J Orthop Res. 2009;27 (9 ):1204-8.
50 Jovanovic DV Fernandes JC Martel-Pelletier J Jolicoeur FC Reboul P Laufer S , et al . In vivo dual inhibition of cyclooxygenase and lipoxygenase by ML-3000 reduces the progression of experimental osteoarthritis: suppression of collagenase 1 and interleukin-1beta synthesis. Arthritis Rheum. 2001;44 (10 ):2320-30.
51 Martel-Pelletier J Mineau F Fahmi H Laufer S Reboul P Boileau C , et al . Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B(4) in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids. Arthritis Rheum. 2004;50 (12 ):3925-33.
52 Attur M Dave M Abramson SB Amin A. Activation of diverse eicosanoid pathways in osteoarthritic cartilage: a lipidomic and genomic analysis. Bull NYU Hosp Jt Dis. 2012;70 (2 ):99-108.22891999
53 Chou RC Kim ND Sadik CD Seung E Lan Y Byrne MH , et al . Lipid-cytokine-chemokine cascade drives neutrophil recruitment in a murine model of inflammatory arthritis. Immunity. 2010;33 :266-78.
54 Kremer JM. Fish oil for OA? Don’t give up yet. Ann Rheum Dis. 2016;75 (7 ):e42.
55 Miyahara N Miyahara S Takeda K Gelfand EW. Role of the LTB4/BLT1 pathway in allergen-induced airway hyperresponsiveness and inflammation. Allergol Int. 2006;55 (2 ):91-7.
56 Díaz-González F Alten RH Bensen WG Brown JP Sibley JT Dougados M , et al . Clinical trial of a leucotriene B4 receptor antagonist, BIIL 284, in patients with rheumatoid arthritis. Ann Rheum Dis. 2007;66 (5 ):628-32.
57 Jing Y Nguyen MM Wang D Pascal LE Guo W Xu Y , et al . DHX15 promotes prostate cancer progression by stimulating Siah2-mediated ubiquitination of androgen receptor. Oncogene. 2018;37 :638-50.
58 Weis S Heindl M Carvalho T Jentho E Lorenz J Sommerer I , et al . Azithromycin does not improve disease severity in acute experimental pancreatitis. PLoS One. 2019;14 :e0216614.
59 Lin Y He X Zhou D Li L Sun J Jiang X. Co-delivery of doxorubicin and itraconazole by Pluronic® P123 coated liposomes to enhance the anticancer effect in breast cancers. RSC Adv. 2018;8 :23768-79.
60 Chen L He Z Qin L Li Q Shi X Zhao S , et al . Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity. PLoS One. 2011;6 (9 ):e24407.
