Contributed equally
Pain is the hallmark symptom of osteoarthritis (OA), and current analgesic treatments may be insufficient or have potentially adverse effects. The inhibition of Monoacylglycerol lipase (MAGL) produces anti-inflammatory and anti-nociceptive effects. However, the potential mechanism of MAGL in OA pain remains unclear. In the present study, the synovial tissues were removed from OA patients and mice. Immunohistochemical staining and western blotting were used to detect the expression of MAGL. M1 and M2 polarization markers were detected by flow cytometry and western blotting, and the mitophagy levels were detected by the immunofluorescence staining of mitochondrial autophagosomes with lysosomes and western blotting. The OA mice were intraperitoneally injected with MJN110 to inhibit MAGL once a day for a week. Mechanical and thermal pain thresholds were detected by electronic Von Frey and hot plate methods on days 0, 3, 7, 10, 14, 17, 21, and 28. The accumulation of MAGL in the synovial tissues of OA patients and mice promoted the polarization of macrophages towards an M1 phenotype. Pharmacological inhibition and siRNA knockdown of MAGL promoted polarization of M1 macrophages towards an M2 phenotype. MAGL inhibition increased the mechanical and thermal pain thresholds of OA mice and enhanced the mitophagy levels of M1 macrophages. In conclusion, in the present study, it was shown that MAGL regulated synovial macrophage polarization by inhibiting mitophagy in OA.
Osteoarthritis (OA) is a chronic degenerative joint disease that causes stiffness, restrictions in joint motion, and chronic persistent pain (
Recent studies have shown that synovial inflammation and chondrocyte cell death play prominent roles in OA progression (
Mitophagy, or mitochondrial autophagy, is a selective process that mitigates inflammation and maintains homeostasis by delivering damaged mitochondria to autophagosomes for destruction (
In the present study, the primary aim was to explore the potential role of MAGL in OA pain. MAGL accumulation in synovial tissue and M1 polarization of synovial macrophages was observed in OA patients and monoiodoacetate (MIA) mice model of OA. Next, the effects of pharmacological inhibition and MAGL knockdown on the polarization of macrophages were xassessed. Lastly, it was determined that MAGL regulated the polarization of macrophages by targeting mitophagy. These findings may provide a novel perspective on the mechanism of MAGL for OA pain.
Written informed consent was obtained from each patient for inclusion in the study and the use of their synovial tissues for research. Synovial tissues were collected from patients who underwent total knee replacement due to knee OA (OA group, n=5; one woman, four men; age range, 64–74 years; median age, 69 years) and those who underwent knee arthroscopic surgery due to anterior cruciate ligament injury (Control group, n=5; one woman, four men; age range, 63–71 years; median age, 66 years). This study was performed in accordance with the Ethical Standards of the Declaration of Helsinki and was approved by the Ethics Committee of Suzhou Municipal Hospital (approval no. K-2021-066-K01).
Male C57BL/6 (C57) mice weighing 25–35 g were obtained from the Sino-British SIPPR/BK Lab (Shanghai, China). All mice were provided
The MIA model of OA pain in the mouse was established as previously described (
All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (approval no. Y20210267).
In the logarithmic growth phase, macrophages (RAW264.7, Beyotime Institute of Biotechnology, cat. no. C7505) were selected and seeded into 6-well plates. After cell adhesion, the cell culture medium (DMEM/F12, 10% FBS, 1% Penicillin-Streptomycin) (Gibco; Thermo Fisher Scientific, Inc.) was discarded and washed once with PBS. Then, the M1 polarization induction medium containing 40 ng/ml LPS (MilliporeSigma) or M2 polarization induction medium containing 40 ng/ml IL-4 (MedChemExpress) was added as appropriate (
MAGL-siRNA was designed and synthesized by Gima Gene Company and divided into a Control group (untransfected), a siRNA Negative Control group (NC siRNA, sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′), and three target gene transfection groups (MAGL siRNA 1 sense, 5′-GCUGGACAUGCUGGUAUUUTT-3′ and antisense, 5′-AAAUACCAGCAUGUCCAGCTT-3′; MAGL siRNA 2, sense 5′-CCAUGACCAUGUUGGCCAUTT-3′ and antisense, 5′-AUGGCCAACAUGGUCAUGGTT-3′; and MAGL siRNA 3 sense, 5′-GCCUACCUGCUCAUGGAAUTT-3′ antisense, 5′-AUUCCAUGAGCAGGUAGGCTT-3′). Macrophages in each group were seeded in 6-well plates. The following experiments were performed in accordance with the instructions of the Gima gene product. The siRNA was diluted with buffer solution and gently mixed to prepare the siRNA transfection diluent. Opti-MEM (200 µl) was diluted with Lipofectamine® 3000 (5 µl) (Invitrogen; Thermo Fisher Scientific, Inc.) and mixed and incubated for 5 min at room temperature. The diluted Lipofectamine® with 100 pmol siRNA was gently mixed and incubated for 20 min. The mixed solution of the complex was added to the cell culture plate, and the cells were incubated for 48 h.
The cells were incubated in blocking buffer (0.5% BSA in 1× PBS) for 30 min at room temperature and stained with FITC-conjugated anti-inducible nitric oxide synthase (iNOS; 1:20, BD Biosciences, cat. no. 610330) and PE-conjugated anti-arginase 1 (Arg1; 1:20, R&D Systems, cat. no. IC5868P) at 4°C in the dark for 30 min. The cells were analyzed using a BD FACSAria™ II flow cytometer (BD Biosciences) and the BD FACSDiva™ software version 8.0 (BD Biosciences).
Immunohistochemistry was performed as previously described (
H&E staining was performed and evaluated as previously described (
Western blotting was performed as previously described (
The synovial tissues were permeabilized in 0.1% Triton X-100 for 20 min and then blocked with 5% bovine serum albumin for 1 h at room temperature. Then the synovial tissues were incubated with anti-MAGL (1:200, Abcam, cat. no. ab246902) and anti-iNOS (1:200; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-17139) antibodies overnight at 4°C, followed by incubation with the secondary antibody (1:1,000, Abcam, cat. nos. ab150084 and ab150113) for 2 h at room temperature in the dark. An Olympus Fluoview FV3000 (Olympus Corporation) confocal microscope was used to capture images.
The synovial tissues were cut into 1 mm3 segments, preserved in 3% glutaraldehyde for 2 h at 4°C, treated with 1% osmic acid for 2 h at 4°C, and dried with various acetone concentrations before encasing in resin. The samples were sliced into extremely thin sections (90 nm) using an ultramicrotome. A Hitachi H-7560 (Hitachi) transmission electron microscope was used to capture images.
The mechanical pain threshold of mice was measured using an electronic von Frey (Ugo Basile S.R.L, cat. no. 38450) (
The thermal pain threshold of mice was measured using a hot plate (Ugo Basile S.R.L., cat. no. 35250) as described previously (
All experiments were performed at least three times. Data are presented as the mean ± SD. Statistical analysis was performed using GraphPad Prism version 8.0.1 (GraphPad Software, Inc.). Normality and homogeneity were evaluated using Shapiro-Wilk and Levene tests, respectively. Differences between groups were compared using an unpaired Student's t-test (two groups), a Mann-Whitney U test (two groups), a Kruskal-Wallis followed by Dunn's test, or a one-way or two-way ANOVA followed by a Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
To investigate the potential role of MAGL in OA, we first collected synovial tissues from patients who underwent total knee replacement due to knee OA (OA group, n=5) and from patients who underwent arthroscopic knee surgery due to anterior cruciate ligament injury (Control group, n=5) to perform H&E staining. As shown in
To further determine the polarization phenotype of synovial macrophages from knee OA patients, the expression levels of the M1 polarization markers, iNOS and CD80, and the M2 polarization markers, Arg1 and CD206, were determined by immunohistochemical staining. As shown in
To further investigate the role of MAGL in OA, MJN110, a potent pharmacological inhibitor of MAGL, was intraperitoneally injected in MIA-induced OA mice once a day for a week. First, the expression levels of MAGL in the synovial tissue were detected. As shown in
To further identify the role of MAGL inhibition in OA pain treatment, H&E staining, and mechanical and thermal threshold tests were performed. Based on the staining results (
Next, the function of MAGL
Subsequently, western blotting was used to examine the expression of MAGL in the macrophages in the three groups. As shown in
Mice macrophages (LPS polarization inducing) were transfected with MAGL siRNA or NC siRNA (
As shown in the electron microscopy images in
Knee OA is a common degenerative disease characterized by joint pain and cartilage destruction, which is more common in the elderly. The incidence of OA has increased with the advent of an aging society but is also becoming increasingly diagnosed in the younger population as well (
MAGL regulates the endocannabinoid system by hydrolyzing 2-AG and plays a vital role in inflammatory responses, analgesia, and neuroprotection (
Synovial inflammation plays a vital role in the pathological process of OA and is an essential factor in OA pain. The infiltration of inflammatory cells in synovial tissues further aggregates a variety of inflammatory and immune cells, especially macrophages, which are widely involved in the inflammatory cascade (
Mitophagy degrades damaged mitochondria in cells and regulates cell metabolism through autophagy, which is critical for cell function and mitochondrial network function, and it plays a vital role in the maintenance of homeostasis and protects nerve cells by removing damaged mitochondria (
The present study has several limitations. First, the study did not include knockout mice. The gene knockout mice may help further elucidate the role of MAGL in regulating mitophagy and polarization of synovial macrophages. Second, male C57 mice were used to establish the MIA-OA model in these experiments. The role of MAGL in OA pain in aged female mice should thus be also assessed. Third, the detailed molecular mechanism of MAGL inhibiting mitophagy requires elucidation.
In conclusion, the present study demonstrated that MAGL accumulated in the synovial tissues of patients and mice with OA, and inhibition of MAGL promoted the polarization of synovial macrophages from an M1 towards an M2 phenotype and alleviated pain in OA mice. Based on these results, it is proposed that MAGL regulates synovial macrophage polarization by inhibiting mitophagy in OA.
Not applicable.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
CW and DG conceived, designed the study and revised the manuscript. CG and MC performed the experiments, analyzed the data and drafted the manuscript. XL analyzed the data. CG, MC and CW confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
This study was performed in accordance with the Ethical Standards of the Declaration of Helsinki and was approved by the Ethics Committee of Suzhou Municipal Hospital (approval no. K-2021-066-K01). All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (approval no. Y20210267).
All patients provided informed consent for publication of their data.
The authors declare no competing interests.
osteoarthritis
monoacylglycerol lipase
2-arachidonoylglycerol
monoiodoacetate
inducible nitric oxide synthase
arginase 1
hematoxylin and eosin
PTEN-induced kinase 1
MAGL accumulates in OA patients and polarizes macrophages towards an M1 phenotype. (A) Representative images of the synovial tissues stained with H&E (upper panel, ×200, scale bar 50 µm; lower panel, ×400, scale bar 20 µm). (B) Inflammation scores of the synovial tissues. (C and D) Immunohistochemical staining for MAGL in synovial tissues (upper panel, ×200, scale bar 50 µm; lower panel, ×400, scale bar 20 µm). (E) Western blots of MAGL expression in synovial tissues. (F) Western blotting analysis of MAGL in synovial tissues of each group. (G-J) Immunohistochemical staining of iNOS, CD80, Arg1, and CD206 in synovial tissues (left ×200, scale bar 50 µm; right ×400, scale bar 20 µm). (K and L) Immunofluorescence staining of MAGL (red) with iNOS (green) in the synovial tissues (scale bar 100 µm). n=5 per group. *P<0.05, **P<0.01 vs. control group. ns, not significant. MAGL, monoacylglycerol lipase; OA, osteoarthritis.
Inhibition of MAGL promotes the polarization of macrophages towards an M2 phenotype and alleviates pain behaviors in OA mice. (A-F) Immunohistochemical staining of MAGL, iNOS, and Arg1 in synovial tissues (upper panel, ×200, scale bar 50 µm; lower panel, ×400, scale bar 20 µm). (G) Representative images of the synovial tissues stained with H&E upper panel, ×200, scale bar 50 µm; lower panel, ×400, scale bar 20 µm). (H) Inflammation scores of the synovial tissues. (I) The mechanical pain threshold of mice was measured by electronic von Frey. (J) The thermal pain threshold of mice was measured using a hot plate (n=5 per group). *P<0.05, **P<0.01 vs. MIA-OA group; #P<0.05 vs. control group. ns, not significant; MAGL, monoacylglycerol lipase; OA, osteoarthritis; H&E, hematoxylin and eosin; MIA, monoiodoacetate.
MAGL results in accumulation of M1 polarized macrophages
MAGL knockdown promotes M1 to M2 polarization
MAGL knockdown enhances the mitophagy of M1 macrophages