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3-MA attenuates collagen-induced arthritis in vivo via anti-inflammatory effect and autophagy inhibition

BMC Musculoskeletal Disorders volume 26, Article number: 44 (2025) Cite this article

Abstract

Background

Rheumatoid arthritis (RA) is a chronic autoimmune disease which afflicts about nearly 1% of global population. RA results in synovitis and cartilage/bone damage, even disability which aggravates the health burden. Many drugs are used to relieve RA, such as glucocorticoids (GCs), non-steroidal anti-inflammatory drugs (NSAIDs), and disease-modifying anti-rheumatic drugs (DMARDs) in the clinical treatment. However, present clinical drugs have various disadvantages such as poor bioavailability and short biological half-life and drug resistance, or adverse effects. A recent study showed autophagy modulation may be a novel strategy in the treatment of RA. 3-Methylademine (3-MA), is the most widely used autophagy inhibitor, which blocks autophagy at the initiation and maturation stages. The aim of this study is to evaluate the effect of 3-MA in collagen-induced-arthritis (CIA) mice and further elucidate how 3-MA attenuated inflammation, and cartilage/bone damage in arthritis.

Methods

An in-vivo mouse collagen-induced arthritis model was applied to compare differences in ankle destruction among control mice and CIA mice treated with or without 3-MA. Bone and cartilage destruction degree was evaluated by histology and micro-computed tomography (µCT). Further in-vivo assays utilized mouse serum samples to investigate inflammatory levels, oxidative levels, and bone resorption cytokines. At last, an immunofluorescence assay was applied to detect the autophagy level among the three groups.

Results

The in-vivo mouse collagen-induced arthritis model showed that CIA mice revealed apparent hind paw and ankle swelling which was aggravated gradually along with time, while 3-MA treatment attenuated swelling gradually. µCT and histological results showed typical lesions in CIA group while 3-MA treatment alleviated arthritis-related destruction. Serum assay showed that 3-MA significantly reduced inflammatory cytokines levels, suppressed oxidative levels and bone resorption cytokines. Immunofluorescence assay revealed 3-MA significantly inhibited the abnormal autophagy level in CIA mouse ankle.

Conclusions

3-MA protects bone destruction in CIA-induced mice arthritis by anti-inflammatory effect and autophagy inhibition.

Peer Review reports

Background

Rheumatoid arthritis (RA) is a chronic autoimmune disease accompanied by synovitis and cartilage/bone damage [1, 2]. As the most common inflammatory arthritis, RA has about a 0.5 to 1% prevalence among population and it has nearly 3 times higher incidence in women than man [3]. Severe RA is a major cause of disability which aggravates the health burden [3, 4]. RA has several characteristic pathophysiological changes such as synovial hyperplasia, pannus formation, inflammatory cell infiltration and local hypoxia [5,6,7]. Both inflammatory microenvironment and local oxidate stress are beneficial for osteoclastogenesis and result in damage to articular cartilage [8, 9]. At present, glucocorticoids (GCs), non-steroidal anti-inflammatory drugs (NSAIDs), and disease-modifying anti-rheumatic drugs (DMARDs) are widely applied in clinical treatment of RA [10, 11]. However, many RA patients face limitations of current treatments, including poor bioavailability, short half-life, drug resistance, and significant adverse effects [12, 13].

Autophagy is a highly conserved mechanism that maintains homeostasis in all eukaryotic cells. During autophagy process, cells degrade long-lived proteins and cytoplasmic to resist stress. Mounting evidence proved that autophagy regulates the immune system [14]. Autophagy-related genes (ATGs) have been reported involved in several autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) [15,16,17]. Furthermore, osteoclast (OC) differentiation and resorption function have been revealed to be regulated by autophagy [18]. Except for epigenetic regulation of autophagy, many chemical autophagy activators and inhibitors have been applied for autophagy modulation. In recent years, some autophagy activators and inhibitors have been applied to treat autoimmune diseases. Pharmacological autophagy regulation can be seen as a novel therapeutic strategy for attenuating these diseases [19].

3-Methylademine (3-MA), targeting Vps34 in the class III Phosphoinositide 3-kinase (PI3K) complex and blocking autophagy at the initiation and maturation stages, was widely used during the study of many pathophysiological processes [20,21,22]. A study revealed that autophagy activated in the pathogenesis of experimental autoimmune neuritis (EAN) and 3-MA lowered the neurologic severity of EAN [23], which proved the therapeutic potential of 3MA in autoimmune diseases, such as RA. Besides, 3-MA is also used to abolish osteoclast differentiation in vitro because of the autophagy inhibition effect [24]. However, to our knowledge, 3-MA has not been reported in the treatment of RA and its mechanisms of anti-osteoclastogenesis remain unclear. In this study, the effect of 3-MA on RA was revealed in vivo for the first time and further mechanisms of 3-MA against OC differentiation were explored.

Methods

Chemical compounds and reagents

The complete Freund’s adjuvant and bovine type-II collagen were purchased from Sigma (USA). The Elisa kits for RANKL (F2504-B)、OPG (F2144-B)、IL-1β (F2024-B)、IL-6 (F2163-B)、TNF-α (F2132-B)、MDA (F9264-B)、SOD (F2389-B) were purchased from Shanghai Kexing (Shanghai, China). 3-MA was purchased from MedChemExpress (HY-19312). TRAP (ab235448) and LC3B (ab192890) antibodies were purchased from abcam® (USA).

Laboratory animals and Cell Culture

Healthy 8-week-old male DBA/1J mouse, SPF grade, 18 mouse, purchased from HFK Bio-Technology (Beijing, China), whose source certification was detected by Xishan Biotechnology (Suzhou, China). The mice’s weight ranged from 18 g to 22 g. Mice were kept at a temperature of 21–25 °C and a relative humidity of 50–60%. Mouse in each group could eat and drink freely.

CIA mice model

After a week of adaptive feeding, 18 mice were randomly divided into 3 groups: control group, CIA group, CIA + 3-MA group (30 mg/kg/2d). Preliminary studies used 3-MA ranging from 3 mg/kg/d to 30 mg/kg/d as an autophagy inhibitor [25,26,27,28], so we applied 30 mg/kg/2d dosage design to maintain the high plasma concentration and reduce injection injury and drug toxicity. Bovine type-II collagen solution was emulsified in complete Freund’s adjuvant for subsequent use. Except for the blank group (injected with PBS), the mice in the other groups were all injected into the tail for the first immunization. After 21 days, the second immunization was given to the mice with the same injectant to prepare CFA models as the previous study reported [29]. Meanwhile, 3-MA group started intraperitoneal injection (resolved in sodium carboxymethyl fibrate) every two days until execution. All animals were executed on the 36th day.

Ankle swelling score

CIA mice were scored on a scale of 0–4 for each hind paw using the following criteria: 0 score, normal paw (normal color and no swelling); 1 score, one toe inflamed and swollen (significant change in color and form); 2 score, more than one toe, but not entire paw, inflamed and swollen or mild swelling of entire paw; 3 score, entire paw inflamed and swollen. 4 score, very inflamed and swollen paw or ankylosed paw [29]. If paw ankylose occurrs, the mouse cannot grip the wire at the edge of the cage.

Radiological evaluation

High-resolution microcomputed tomography (µCT) (PerkinElmer, USA) (90 Kv, 180 mA, equidistant resolution 20 μm, exposure time 4.5 min) was used to examine the fixed amputated hind paw. Representative microcomputed tomography images of the mouse’s hind paw and several quantitative analyses were applied to reveal the bone protection effect of 3-MA in CIA mice, such as bone mineral density (BMD), ratio of bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp).

Histological assessment of arthritis

The CIA mice among different groups were sacrificed on day 35 after the first immunization. The hind paws of each mouse were removed and fixed in 10% formalin. After decalcification all samples were embedded in paraffin. Tissues were stained with hematoxylin and eosin. Histopathological grading of joint lesions was performed according to a semiquantitative criteria from 0 to 3, as previously described [23]: inflammatory infiltrate (0, none; 1, mild; 2, moderate; 3, severe infiltrate); synovial lesion (0, no lesion; 1, mild alteration; 2, moderate alteration; 3, severe/complete destruction of the synovia); cartilage destruction (0, none; 1, mild; 2, moderate; 3, severe destruction with loss or complete fragmentation of cartilage); and bone destruction (0, none; 1, mild destruction of subchondral bone; 2, moderate destruction; 3, severe destruction with loss of large areas of bone).

Serum cytokine detection

The serum was collected for ELISA and the biochemical assay. The levels of RANKL, OPG, TRACP5b, TNF-α, IL-1β, and IL-6 in the serum were determined by the ELISA assay. Biochemical kits were used to test the enzyme activities of MDA, SOD, and NO. A multifunctional microplate reader (Molecular Devices, USA) was used to measure the serum indicators. All procedures were performed in accordance with the manufacturer’s instructions.

Immunohistochemistry

Indirect immunohistochemistry techniques (with peroxidase in paraffin) were used on all samples.

Anti-mouse ACP5 antibody ab235448 from abcam®. The chromogen utilized in this process was diaminobenzidine (DAB), which imparts a brown color, along with the peroxidase substrate (H2O2). Following immunohistochemical staining, the samples were counterstained with Gill III’s haematoxylin–eosin, subsequently dehydrated, and rinsed with xylene. DPX (acrylic resin) was used as a coverslip mounting medium.

Immunofluorescence

Immunofluorescence was utilized to validate diagnostic biomarkers in this study. Mouse joint sections were deparaffinized and treated with 10 mM citrate buffer (Solarbio, China). The sections were then blocked with 5% BSA (Solarbio, China) for 1 h before incubation with primary antibody solution (diluted according to the antibody datasheet) overnight at 4° C. The next day, the incubated sections were retrieved and subjected to a 1-hour incubation with a fluorescence-labeled secondary antibody solution provided by Bioworld (Nanjing, China), which corresponded to the specific first antibodies used in the experiment. The sections were washed three times with 1×TBST solution for 10 min after each incubation. DAPI staining was applied for nuclear counterstaining, and an anti-fluorescence quencher was added to seal the cover glass. The samples were observed and imaged using a PANNORAMIC SCAN II scanner (3DHISTECH, Hungary).

Statistical analysis

The data were expressed as the means ± standard deviation (SD). Statistical analyses were performed by one-way ANOVA to compare variance between groups in SPSS 20.0. Values of p < 0.05 were considered statistically significant. All the graphs were accomplished by GraphPad Prism 6.0.

Statement

This research has been approved by the authors’ affiliated institutions.

Results

3-MA reduced the clinical scores of collagen-induced rheumatoid arthritis in mouse

In this study, an animal model of CIA was used to evaluate the therapeutic effects of 3-MA in RA treatment. The scheme for collagen-induced arthritis is shown in Fig. 1A. Measurement started on day 14, then we arranged the second immunization and 3-MA treatment on day 21 and continued until day 35. Obvious swelling of the ankle could be found at day 14 after the first immunization (Fig. 1A). As it shown in Fig. 1B and C, clinical scores significantly reduced after 2 weeks 3-MA treatment, while the CIA model group’s clinical scores continued to rise in the last 2 weeks.

Fig. 1
figure 1

The scheme for collagen-induced rheumatoid arthritis in mouse and clinical scores (*P<0.05, ***P<0.001)

Full size image

3-MA attenuated ankle erosion and bone loss in CIA mouse

As revealed in Fig. 2A, obvious ankle joint erosion and bone loss occurred in the CIA model group (with PBS intraperitoneal injection), while the 3-MA group significantly protect the joint from erosion. Related bone parameters showed the same effect, 3-MA could reverse the damages to the shinbone and cartilage, such as a rise the value of BV/TV, BMD, Tb. N while reducing the Tb. Sp and Tb. Th (Fig. 2B).

Fig. 2
figure 2

Ankle erosion and bone loss results. (A) µCT reconstruction image. (B) Quantitative analysis of bone mineral density (BMD), ratio of bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp)

(*P<0.05, **P<0.01, ***P<0.001)

Full size image

Histological evaluation of the CIA mouse

As it showed, after HE staining, the CIA mouse revealed typical arthritis histopathological features, including synovial lining hyperplasia, severe inflammatory infiltration, pannus formation, disappearance of the tidemark of cartilage and even complete fragmentation of cartilage (indicated by red arrows). 3-MA treatment reduced all these pathological features (Fig. 3A). The numbers of TRAP-positive cells in synovia from CIA mice were significantly higher than in 3-MA treatment mice (Fig. 3B). While 3-MA attenuated the OC formation in number and size (indicated by blue arrows) (Fig. 3B). Besides, detailed semi-quantitative analyses of several histopathological features were applied. Similar to the clinical evaluation, morphological signs of arthritis in 3-MA-treated mice were drastically reduced compared with CIA mice (Table 1).

Fig. 3
figure 3

Histological evaluation of the CIA mouse. (A) HE staining results. Severe inflammatory infiltration (red stars), pannus formation (red arrows). (B) Immunohistochemistry results. TRAP-positive cells in synovia (blue arrows)

Full size image
Table 1 Histological assessment of arthritis
Full size table

3-MA attenuated oxidative stress level, proinflammatory cytokines level and bone resorption cytokines in CIA mice serum

The serum levels of proinflammatory cytokines such as IL-6, IL-1β, and TNF-α significantly increased in CIA group compared with the control group (P < 0.01). While 3-MA treatment decreased the serum levels of these inflammatory cytokines in the CIA mouse (P < 0.01)(Figure 4A). Compared with the control group, the CIA group significantly increased serum oxidative stress markers (MDA and NO) and bone resorption markers (RANKL and TRACP) While 3-MA treatment relieved these oxidative stress and bone resorption markers (Fig. 4B and C). While the anti-oxidant and bone protection markers in serum such as SOD and OPG showed exact opposite trends among the three groups (Fig. 4B and C).

Fig. 4
figure 4

Serum cytokine results. (A) Inflammatory cytokines level among groups. (B) oxidative stress cytokines level among groups. (C) Bone resorption markers level among groups

(*P<0.05, **P<0.01, ***P<0.001)

Full size image

Autophagy marker LC3B was suppressed by 3-MA treatment

Immunofluorescence results of the ankle joints of the three groups of mice showed that LC3B were highly expressed in the CIA group mice compared to the control group, while 3-MA treatment significantly suppressed the LC3B level in the hind paws in CIA mice (Fig. 5).

Fig. 5
figure 5

Immunofluorescence results of the ankle joints

Full size image

Discussion

CIA mouse model has been extensively applied to elucidate pathogenic mechanisms relevant to human RA due to reproducibility and similar pathological features [30]. It has been established that RA involves a complex interaction between synovial inflammation, cartilage erosion and bone resorption [31]. Based on clinical symptoms, pathological findings, and radiological evaluations, we revealed the common autophagy inhibitor, 3-MA has significant suppression of collagen-induced arthritis. We speculated about several underlying molecular mechanisms based on both the present in vivo study and published data as follows.

Despite the etiology and pathogenesis of RA not been fully elucidated, several pathological factors have been confirmed to play a vital role in the RA process, such as immunological inflammatory reaction and oxidative stress. One crucial cell in the immunological inflammatory reaction is the RA fibroblast-like synoviocytes (RA-FLS). RA-FLS abnormal proliferation induced synovial hyperplasia and pannus formation which caused microenvironment hypoxia and cartilage damage during chronic synovial inflammation [32]. Previous studies revealed that RA-FLS usually exhibit excessive activation of autophagy and resistance to apoptosis compared with normal FLS [33, 34]. Further studies proved that hydroxychloroquine and Chloroquine, two late-stage autophagy inhibitors, have been demonstrated to have anti-inflammatory effects and be effective in RA, particularly when used in combination with other immunosuppressive drugs [35, 36]. In our study, HE staining revealed 3-MA treatment significantly inhibited RA-FLS excessive proliferation and invasion.

Another vital cell participant in immunological inflammatory reaction is the macrophage because the severity of the RA and damage is significantly related to the number and activation of macrophages [37]. Autophagy regulates the polarization of macrophages and the expression of inflammatory cytokines [38, 39]. It was found that the fluorescence intensity of autophagy in monocytes and neutrophils of RA patients was significantly higher than that of normal subjects, and the intensity was positively correlated with RA inflammation. When the patient’s condition was relieved, the autophagy level also decreased [40]. In our study, histological evaluation revealed the number of inflammatory cells decreased after 3-MA treatment. Serum detection showed decreased inflammatory cytokines, such as TNF-α, IL-6, IL-1β in 3-MA group compared to the CIA group. Current research proved the microenvironment inflammation in RA lesion areas have a positive correlation with oxidative stress which is dominated by M1 macrophage [41, 42]. RA patients have excessive free radicals in the body with increasing the level of the oxidation marker malondialdehyde (MDA), and the antioxidant enzyme superoxide dismutase (SOD) system disruption. Thereafter, excessive oxidative stress aggravates bone destruction since ROS regulates OC differentiation and maturity [43, 44]. In this study, we detected the serum levels of oxidant and anti-oxidant. CIA group significantly up-regulated the expression of oxidants, such as MDA and NO, and reduced the expression of anti-oxidant, such as SOD. While 3-MA treatment inhibited the oxidant expression and recovered the anti-oxidant level. We speculated that 3-MA inhibited the autophagy level of the CIA mouse which was confirmed by immunofluorescence assay, resulting in the inhibition of macrophages polarization and the downregulated the level of inflammatory cytokines thus alleviating oxidative stress level. However, the specific mechanism of the 3-MA inhibition effect on macrophage and RA-FLS remains for further exploration.

Cartilage and bone damage are mainly caused by OC in RA. Both immunological inflammatory reaction and oxidative stress will enhance OC formation. For example, TNF-α has been proven to not only promote the differentiation of macrophages or bone marrow stromal stem cells into OCs but also improve the bone resorption activity OCs [45]. Microenvironment hypoxia and ROS accumulation can directly activate of OC formation and activity [46, 47]. Besides, previous studies proved OC differentiation and function need autophagy activation and could be regulated by autophagy [18]. Our in vivo results revealed 3-MA inhibited OC formation in CIA mice measured by microCT, and HE staining. The OC suppression effect by 3-MA in CIA mice could be attributed to two mechanisms, one is the direct autophagy suppression, which could be confirmed by immunofluorescence results, and the other is the anti-inflammatory and anti-oxidative effect mentioned above which led to the changes in serum RANKL/OPG and TRACP. It should be mentioned that more in-vitro experiments are required to explore the further mechanism of 3-MA in the treatment of RA, for example, which kind of cells played a crucial part during the process and which signal paths were regulated.

Conclusions

In summary, treatment with 3-MA significantly decreased the severity of arthritis and improved clinical, histological and radiological findings in established CIA mice. Besides, autophagy inhibitor 3-MA reduced the proliferation and invasion of RA-FLS, and alleviated inflammatory cell infiltration and ROS levels thus protected cartilage/bone from erosion from excessive osteoclastogenesis. These findings show that in vivo administration of 3-MA suppresses arthritis in established CIA mice, suggesting that autophagy inhibitor treatment may offer a new promising candidate for RA.

Data availability

All data that support the findings of this study have been reviewed and deposited by Tianjin Institute of Environmental and Operational Medicine. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

3-MA:

3-Methylademine

RA:

Rheumatoid arthritis

RA-FLS:

RA fibroblast-like synoviocytes

CIA:

Collagen-induced-arthritis

μCT:

Microcomputed tomography

GCs:

Glucocorticoids

NSAIDs:

Non-steroidal anti-inflammatory drugs

SLE:

Systemic lupus erythematosus

DMARDs:

Disease-modifying anti-rheumatic drugs

IBD:

Inflammatory bowel disease

OC:

Osteoclast

PI3K:

Class III Phosphoinositide 3-kinase

EAN:

Autoimmune neuritis

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Acknowledgements

Chong Feng and Zi-rou Wang contributed equally to this study. This work was supported by grants from the National Natural Science Foundation of China (NSFC) (grant nos. 31971106, BWS211013, 21WS09002, and JK20211A010213).

Funding

This work was supported by grants from the National Natural Science Foundation of China (NSFC) (grant nos. 31971106, BWS211013, 21WS09002, and JK20211A010213).

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Author notes

  1. Chong Feng and Zi-rou Wang are the co-first author.

Authors and Affiliations

  1. Tianjin Institute of Environmental and Operational Medicine, Tianjin, 300050, China

    Chong Feng, Zi-rou Wang, Chen-yu Li & Xin-xing Wang

  2. School and Hospital of Stomatology, Tianjin Medical University, Tianjin, 300070, China

    Chong Feng & Xiang-yu Zhang

  3. Department of Orthodontics, Tianjin Stomatological Hospital, School of Medicine, Nankai University, Tianjin, 300041, China

    Chong Feng

  4. Tianjin Key Laboratory of Oral and Maxillofacial Function Reconstruction, Tianjin, 300041, China

    Chong Feng

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  5. Xin-xing Wang
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Contributions

1. Chong Feng and Xin-xing Wang conceived the subject. 2. Chong Feng and Zi-rou Wang finished most experiments. 3. Chen-yu Li helped Chong Feng and Zi-rou Wang in the experiments and data analysis. 4. Chong Feng and Zi-rou Wang finished the draft. 5. Xiang-yu Zhang and Xin-xing Wang revised manuscript. 6. All authors reviewed the manuscript.

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Correspondence to Xiang-yu Zhang or Xin-xing Wang.

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Ethical approval for this study was obtained from *INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE OF YI SHENGYUAN GENE TECHNOLOGY (TIANJIN) (APPROVAL YSY-DWLL-2022132)*.

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Feng, C., Wang, Zr., Li, Cy. et al. 3-MA attenuates collagen-induced arthritis in vivo via anti-inflammatory effect and autophagy inhibition. BMC Musculoskelet Disord 26, 44 (2025). https://doi.org/10.1186/s12891-025-08274-y

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Keywords

BMC Musculoskeletal Disorders

ISSN: 1471-2474

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