Exosomes derived from hypoxia-preconditioned M2 macrophages alleviate degeneration in knee osteoarthritis through the miR‑124‑3p/STAT3 axis

Abstract

Background

M2 macrophages-derived exosomes (M2φ-Exos) have been demonstrated to effectively alleviate osteoarthritis (OA) in animal models. Hypoxic preconditioning is commonly used to enhance the biological effects of stem cell-derived exosomes, but its impact on M2φ-Exos remains unclear. This study aims to investigate whether hypoxic preconditioning could enhance the biological effects of M2φ-Exos in OA treatment and to explore the underlying molecular mechanisms, with the goal of providing new insights for the development of safe and effective OA therapeutic strategies.

Methods

Exosomes were extracted from the supernatants of M2 macrophages cultured under normoxic or hypoxic conditions using low-temperature differential ultracentrifugation and were designated as Nor-Exos and Hypo-Exos. The exosomes were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. To evaluate the impact of hypoxic preconditioning on the biological effects of M2φ-Exos, the therapeutic effects of Nor-Exos and Hypo-Exos were assessed in an IL-1β-induced chondrocyte inflammation model and a rat knee OA model established by surgical intervention. Exosomal miRNAs with differential expression between Nor-Exos and Hypo-Exos were identified through exosomal miRNA sequencing. The miRNA with the highest upregulation in Hypo-Exos was selected for further functional validation. To investigate the role of this miRNA, miRNA inhibitors were used to knock down its expression in Hypo-Exos, and the subsequent impact of this change on Hypo-Exos activity was evaluated. Bioinformatic tools and dual-luciferase reporter assays were used to predict and verify the downstream target genes of the miRNA. Target gene expression was knocked down using small interfering RNA, and the effect of downregulating target gene expression on the inhibitory effect of low miRNA expression on Hypo-Exos was observed at the cellular level.

Results

Compared to Nor-Exos, Hypo-Exos exhibited more effective therapeutic effects in both inflammatory chondrocytes and OA rats. miR-124-3p was identified as the most upregulated miRNA in Hypo-Exos, and the suppression of miR-124-3p expression significantly inhibited the biological effects of Hypo-Exos. STAT3 was determined to be a downstream target gene of miR-124-3p. Further cellular experiments revealed that downregulation of STAT3 expression in chondrocytes successfully alleviated the inhibitory effect of low miR-124-3p expression on the biological effects of Hypo-Exos.

Conclusions

Hypoxic preconditioning enhances the biological effects of M2φ-Exos in the treatment of OA. The underlying molecular mechanism is associated with increased delivery of miR-124-3p to chondrocytes, which subsequently inhibits the post-transcriptional expression of STAT3. This provides a promising therapeutic strategy for the clinical intervention of OA.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-06808-5.

Introduction

Osteoarthritis (OA) is a prevalent degenerative joint disorder, particularly common among middle-aged and elderly populations [1–4]. With the global trend of population aging and the increasing influence of modern lifestyle factors such as obesity, the incidence of OA has been rising steadily year by year. It has become one of the leading causes of functional impairment and disability worldwide [5]. The development of OA is closely associated with a range of factors, including age, sex, genetics, lifestyle, and environmental influences, and is generally considered to result from a multifactorial interplay [6–9]. The pathophysiology of OA is complex, involving progressive degeneration and damage of multiple joint components. Key pathological features include articular cartilage degradation, synovial inflammation, subchondral bone remodeling, and dysfunction of periarticular structures [10–13]. Clinically, OA is characterized by joint pain, stiffness, swelling, and limited mobility, which significantly impair patients’ daily functioning and work capacity. Consequently, OA imposes a substantial burden on both quality of life and socioeconomic systems globally [14–16].

The clinical management of OA currently encompasses conservative treatment, pharmacological therapy, and surgical intervention [17–19]. Conservative treatment includes health education, exercise therapy, physical therapy, and functional support, primarily aimed at patients with early-stage disease and mild symptoms [20, 21]. Pharmacological treatments commonly used include nonsteroidal anti-inflammatory drugs, opioid analgesics, intra-articular injections (such as corticosteroids, sodium hyaluronate, chitosan, growth factors, and platelet-rich plasma), symptom-modifying slow-acting drugs (such as glucosamine and chondroitin sulfate), and anxiolytics [22, 23]. Although pharmacological treatments can provide symptomatic relief to some extent, they do not fundamentally address the underlying issue. To date, neither the U.S. Food and Drug Administration nor the European Medicines Agency has approved any drugs that can effectively prevent, slow, or reverse the progression of joint structural damage. Furthermore, the long-term use of these drugs may lead to adverse effects in multiple organ systems, including the cardiovascular system, liver, and kidneys. Surgical treatment, which is reserved for advanced-stage OA, includes reparative surgeries and joint replacement procedures. These are typically considered a last resort, as most patients are forced to undergo surgery due to the failure of pharmacological treatments following continued cartilage degeneration. However, surgical interventions are associated with complications, and patient satisfaction often does not fully meet clinical expectations [24, 25]. Therefore, identifying new, effective interventions to delay cartilage degeneration remains a critical challenge in the field of OA treatment.

In recent years, the role of macrophages in OA therapy and basic research has garnered considerable attention [26–28]. Macrophages are crucial cells in the immune system, originating from monocytes, and are a type of white blood cell. They are widely distributed across various tissues and organs in the body and play a key role as effector cells in the innate immune response, possessing powerful phagocytic capabilities that enable them to eliminate bacteria, viruses, dead cells, and other foreign materials. As research on macrophages has deepened, a comprehensive understanding of their origin, secretion, tissue distribution, plasticity, and polarization characteristics has emerged. The polarization and phenotypic transitions of macrophages have become a hot topic and particular interest is in recent studies [29, 30]. The phenotype of macrophages is closely linked to their surrounding microenvironment, typically comprising two distinct subtypes: classically activated M1 macrophages and alternatively activated M2 macrophages [31, 32]. M1 macrophages play a pivotal role in the early stages of inflammation by secreting various pro-inflammatory cytokines, such as Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Tumor Necrosis Factor-α (TNF-α), and are involved in pathogen phagocytosis and the clearance of apoptotic cells [33]. In contrast, M2 macrophages function in the later stages of inflammation by secreting anti-inflammatory cytokines, such as Interleukin-10 (IL-10) and Transforming Growth Factor-β, thereby controlling inflammation and promoting tissue repair and remodeling [34, 35]. In the context of OA, the disease is often characterized by an increase in the number of M1 macrophages and a reduction in M2 macrophages. This imbalance leads to excessive activation of the inflammatory response, ultimately resulting in cartilage destruction in the joints [36–38].

Exosomes, as important mediators of intercellular signaling, have garnered increasing attention in the scientific community due to their high clinical translational value and potential [39, 40]. First discovered in 1983 in the reticulocytes of sheep, exosomes were named by Johnstone in 1987 [41]. Exosomes are membrane-bound vesicles secreted by living cells, characterized by their disc-like shape and specific functions. They are widely distributed in various body fluids, including blood, urine, saliva, synovial fluid, bile, amniotic fluid, breast milk, semen, and ascitic fluid, and are also found in the supernatants of certain tissues and cell cultures [42]. Exosomes exert their biological functions by carrying a variety of bioactive molecules, such as nucleic acids, proteins, short peptides, biomacromolecules, and lipids [43]. They can be taken up by neighboring receptor cells or travel through the circulatory system to distant cells, thereby regulating both physiological and pathological states of the target cells [44].

Oxygen concentration is crucial for the normal growth of cells. Currently, the oxygen concentration used in in vitro cell culture significantly differs from the oxygen levels found in the physiological microenvironment in vivo [45]. Conventional in vitro cultures typically set the oxygen level at 21%, whereas the oxygen content in the body is usually much lower, particularly in certain pathological conditions where tissues and cells may be in a hypoxic state [46]. Hypoxic preconditioning is a technique in which cells or tissues are exposed to a low-oxygen environment for a short period to enhance their adaptability and survival in abnormal conditions [47, 48]. Hypoxic preconditioning has been extensively studied, particularly in the field of regenerative medicine and stem cell applications. The advantages of hypoxic preconditioning for cells include the following: (i) increased adaptability and survival under hypoxic or stressful conditions; (ii) enhanced vitality and functionality of certain cells, such as increased proliferation and migration, improved differentiation potential, and enhanced immune modulation; and (iii) promotion of greater exosome secretion with improved biological activity of the exosomes.

In current basic research, the mechanisms through which M2 macrophages exert their effects are not solely reliant on cytokine secretion, but also involve exosomes generated via paracrine signaling [49–51]. The immunoregulatory and tissue repair functions of M2 macrophage-derived exosomes (M2φ-Exos) have been demonstrated in various disease models, such as promoting cartilage repair in OA [52], inhibiting periodontal bone loss [53], accelerating fracture healing in diabetes [54], promoting angiogenesis after myocardial infarction [55], enhancing skin wound healing [56], and improving the survival rate of flap transplantation [57]. However, the impact of hypoxic preconditioning on the biological effects of M2φ-Exos and its underlying mechanisms remain unclear. This study focuses on the application of M2φ-Exos in the treatment of OA following hypoxic preconditioning, aiming to explore and elucidate the following aspects: (i) the effects of hypoxic preconditioning on the modulation of chondrocyte biological behavior by M2φ-Exos; (ii) the influence of hypoxic preconditioning on the tissue repair effects of M2φ-Exos in OA; and (iii) the potential molecular mechanisms underlying the biological effects of M2φ-Exos influenced by hypoxic preconditioning.

Materials and methods

Cell cultivation and reagent preparation

RAW264.7 cells, a macrophage line with differentiation potential, were incubated in Dulbecco’s minimum essential medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, New York, USA) and a 1% penicillin-streptomycin solution (Gibco, USA). ATDC5 cells (a chondrogenic cell line) were incubated in DMEM containing nutrient mixture F12 (DMEM/F12; Gibco, USA) supplemented with 10% FBS and a 1% penicillin-streptomycin solution. These cells can be induced to form chondrocytes by adding insulin-transferrin-selenium. Both cell lines were incubated in a humid environment at 37 °C supplemented with 5% CO₂. Recombinant mouse IL-1β, IL-4, and IL-13 were purchased from PeproTech (Rocky Hill, USA), and exosome-depleted FBS was obtained from Umibio Technology Group (Shanghai, China).

Induction of M2 macrophage polarization

The RAW264.7 cells were inoculated in culture plates overnight to facilitate the attachment of these cells to the bottom of the plate. M2 macrophages were subsequently induced by incubating the cells with fresh DMEM containing IL-4 (20 ng/mL) and IL-13 (20 ng/mL) for 24 h [58, 59].

Extraction and identification of M2φ-Exos

After reaching 80% confluence, M2 macrophages were washed with Phosphate Buffered Saline (PBS) and inoculated in DMEM/F12 containing exosome-free FBS under normoxic (21% O₂) or hypoxic (1% O₂) conditions in a triple gas incubator (Thermo Fisher Scientific, USA) for 24 h. The supernatants were collected separately to isolate exosomes via ultracentrifugation (Optima XPN-100, Beckman, USA). Briefly, the supernatant was centrifuged under different rpm (300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min) to remove intact cells and cellular debris. Next, the supernatant was centrifuged at 100,000×g at 4 °C for 70 min, to separate the protein-containing exosomes. The exosomes were subsequently purified by washing with precooled PBS, followed by additional centrifugation at 100,000×g for 70 min. The exosomes were resuspended in PBS and either stored at − 80 °C or used immediately for subsequent experiments.

The morphological characteristics of the obtained Nor-Exos and Hypo-Exos were examined via transmission electron microscopy (TEM; Tecnai G2 Spirit; Hillsboro, USA). Nanoparticle tracking analysis (NTA; Nanosight NS300, Malvern, UK) was used to analyze the distribution of the vesicle diameters of Nor-Exos and Hypo-Exos. Western blotting was performed to identify exosome-specific marker proteins, including CD9, CD63, and TSG101. Information on the antibodies used is shown in Table S1. The protein content of the exosomes was quantified using a bicinchoninic acid (BCA) kit (Beyotime, China), following the manufacturer’s instructions.

Exosome labeling and cellular uptake

The Nor-Exos and Hypo-Exos were labeled using a PKH26 cell linker kit (Umibio Technology Group, China), according to the reported method. Briefly, a red fluorescent dye was added to a solution containing exosomes. The unbound fluorescent dye was removed, and the labeled exosomes were centrifuged at 120,000×g for 90 min. Then, the resuspended exosomes and chondrocytes were cocultured for 6 h. Next, the supernatant was removed, and the cytoskeleton and nucleus were stained with FITC-conjugated phalloidin (Sigma-Aldrich, USA, St. USA) and 4,6-diamidino-2-phenylindole (DAPI; Beyotime, China), respectively. The cells were washed with PBS three times, and their images were captured using a Laser Scaning Confocal Microscope (LSCM; LSM800, ZEISS, Germany).

Establishment and experimental grouping of the KOA model

The animal experimental procedures followed the guidelines of Animal Research: Reporting of In Vivo Experiments and were approved by the Ethics Committee of the Second Affiliated Hospital of Shanxi Medical University (DW2024002). Female Sprague-Dawley rats were acquired from the Animal Center of Shanxi Medical University and raised under sterile conditions. They were fed a commercially available diet and provided with water and libitum. Modified Hulth’s method was used to establish a KOA model in the right knee. Briefly, rats were anesthetized by administering pentobarbital via intraperitoneal injection. Then, a median incision was made to open the skin. Next, a medial incision was made in the parapatellar region to expose the articular cavity, the anterior cruciate ligament was cut, the medial meniscus was removed, and finally, the surgical incision was sutured layer by layer. Penicillin was injected intramuscularly to prevent infection. The rats were randomly distributed into six groups. Four weeks after surgery, group 1 was assigned to the sham group, group 2 was assigned to the vehicle group and administered an intra-articular injection of normal saline once a week for four weeks, and groups 3–6 were administered an intra-articular injection of equivalent volumes of Nor-Exos, Hypo-Exos, miR-NC^KD-Hypo-Exos, and miR^KD-Hypo-Exos, respectively.

ELISA

The concentrations of IL-1β, IL-6, and TNF-α in the joint fluid were quantified using ELISA kits (cat#H002-1-2, H007-1-2, H052-1-2, Nanjing Jiancheng Bioengineering Institute, China) following the methodology outlined by the manufacturer. The optical density of the extracted lysate supernatant was measured and recorded using a microplate reader (ELx800, BioTek, USA).

Plate test

A hot plate (Ugo Basile S.R.L.) was used to measure paw withdrawal latency (PWL) in the rats. After the rats adapted to the surrounding environment and the hot plate device, they were placed on a metal surface maintained at 55 ± 0.1 °C. The surface of the device was covered with transparent glass to ensure that the behavior of the rats could be observed. The latency of jumping or licking the hind paws was recorded, and 30 s was used as the cutoff time to prevent injury to the paws.

Gait analysis

A CatWalk XT 10.6 system (Noldus Information Technology, Netherlands) was used to assess abnormal gait. Each rat was allowed to walk freely along the walking path. The system camera captured the paw prints and generated gait parameters. Each paw print was manually examined and labeled as right hind (RH), left hind (LH), right front (RF), or left front (LF). The footprint area, maximum contact area, swing duration and mean intensity were analyzed, and the RH/LH ratio for each index was calculated to eliminate the effects of individual differences. The same detection settings were used for all rats.

X-ray and micro-CT analysis

After the rats were euthanized, their knee joints were collected and placed in 4% paraformaldehyde for 48 h to facilitate fixation. The samples were scanned using an X-ray imager at a voltage of 32 kV and an exposure time of 6 ms. The samples were also analyzed by high-resolution micro-computed tomography (Micro-CT-80, Switzerland) at a voltage of 70 kV, current of 100 µA, and a scan thickness of 15.6 μm. Knee joint reconstruction was performed using three-dimensional model visualization software. The subchondral bone of the tibia was identified as the region of interest (ROI) and highlighted in yellow for further quantitative analysis. The standardized parameters of the bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were analyzed.

Histopathological and immunohistochemical analysis

After the samples were fixed, they were decalcified in 10% ethylenediaminetetraacetic acid (pH 7.4) for four weeks. Next, the appropriately sized medial compartment of the knee joint was embedded in paraffin. The tissue sections were cut in the sagittal position at a thickness of 5 μm for subsequent staining with hematoxylin and eosin (H&E) and safranin O-fast green (S&F) stain. H&E staining was performed to quantify the synovial tissue score and the ratio of hyaline cartilage (HC) to calcified cartilage (CC). The synovial tissue score was based on the thickness of the synovial lining cells (range 0–3) and the density of the synovial matrix (range 0–3), with a maximum score of 6 (Table S1). HC and CC were defined as the distance between the articular surface and tidemark and the distance between the subchondral bone plate and tidemark, respectively, and the ratio of HC to CC can reflect the relative thickness of the HC. S&F staining was performed to quantify the Osteoarthritis Research Society International (OARSI) score (Table S2). Immunohistochemical staining was conducted using antibodies against collagen type II (COL2) and matrix metalloproteinase-13 (MMP13), and the specific antibodies used are listed in Table S3. The ImageJ software was used to quantitatively analyze the percentage of positively stained cells in the articular cartilage.

Construction and experimental grouping of the OA-like cell model

To simulate the inflammatory response in vitro, chondrocytes were stimulated with IL-1β (10 ng/mL) for 24 h. Exosomes were added simultaneously at a concentration of 1 × 10⁹ particles/mL to evaluate their effects on the biological behavior of chondrocytes. The specific groups used were as follows: Control, IL-1β, Nor-Exos, Hypo-Exos, miR-NC^KD-Hypo-Exos, miR^KD-Hypo-Exos, miR^KD-Hypo-Exos + siNC, and miR^KD-Hypo-Exos + siSTAT3.

Cell proliferation assay

Cell proliferation activity was measured using a Cell Counting Kit-8 (CCK-8; Keygen Biotech Co., Ltd, China). Briefly, chondrocytes were inoculated in a 96-well plate at a density of 4,000 cells per well overnight and then subjected to various treatments for one, two, and three days. Then, 100 µL of fresh medium containing 10 µL of CCK-8 solution was added to the plate, which was then placed in an incubator for 2 h. The optical density of each well was subsequently measured at 450 nm using a microplate reader.

Cell proliferation activity was also evaluated using a 5-ethynyl-2ʹ-deoxyuridine (EDU) kit (RiboBio, China). Briefly, chondrocytes were inoculated in 24-well plates at a density of 3.0 × 10⁴ cells per well overnight and subjected to various treatments for 24 h. Next, 100 µL of 50 µM EDU solution was added, and the mixture was cultured for 2 h. Then, 100 µL of the Apollo^® reaction mixture was added to each well and cultured in the dark for another 30 min. The cells were subsequently counterstained with Hoechst 33,258 for 30 min, and proliferation images were captured using a fluorescence microscope (Leica DMIL LED, Germany). The ImageJ software was used for counting cells to compute the percentage of EDU-positive cells.

Cell migration assay

A scratch wound healing test was conducted to evaluate the ability of the cells to migrate. Chondrocytes were inoculated in six-well plates at a density of 1 × 10⁶ cells per well and allowed to reach 100% confluence overnight. A sterile 200 µL pipette tip was used to create a scratch on the surface of the cell layer. The floating cells were rinsed with PBS and subjected to various treatments. The cells were incubated in a serum-free medium for 24 h. Images were captured using an optical microscope at 0 h and 24 h post-wounding. The healing ability of the wound gap was analyzed using the ImageJ software.

Transwell migration assays were conducted to further analyze the cell migration ability. First, chondrocytes were resuspended in a culture medium containing 1% FBS and inoculated at a density of 3 × 10⁴ cells per well in the upper chamber of a 24-well perforated dish (Corning, USA; pore size: 8 μm). Subsequently, 750 µL/well of culture medium supplemented with 10% FBS was added to the lower chamber. After subjecting the cells to various treatments for 24 h, a cotton swab was used to remove cells that had not migrated through the filter membrane. The cells that had migrated through the filter membrane were stained with 0.5% crystal violet for 30 min. The migratory ability was assessed by visualizing the stained cells through an optical microscope (Nikon, Japan), and the migrated cells were counted using the ImageJ software.

Cell apoptosis assay

A TdT-mediated dUTP nick-end labeling (TUNEL) apoptosis detection kit (Roche, Switzerland) was used to determine the cell apoptosis rate. The chondrocytes were inoculated at a density of 1 × 10⁵ cells per well in a 12-well plate and incubated overnight. After the cells were subjected to various treatments for 24 h, a TUNEL staining reaction was conducted in the dark for 30 min. The nuclei were stained with DAPI for 5 min and photographed under a fluorescence microscope. The number of apoptotic cells and the total number of cells in a randomized field of vision were quantified using the ImageJ software, and the proportion of TUNEL-positive cells was calculated.

An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (BD Biosciences, USA) was used to determine the percentage of apoptotic cells. Chondrocytes were inoculated at a density of 1 × 10⁶ cells per well in a six-well plate and incubated overnight. Then, the cells were subjected to various treatments for 24 h, after which they were centrifuged at 400 ×g for 5 min. Next, the cells were resuspended in 200 µL of PBS and reacted with 10 µL of Annexin V-FITC and 10 µL of PI in the dark at 4 °C for 30 min. The cells were resuspended again in 300 µL of PBS and analyzed by flow cytometry. The data were quantified and presented using the NovoExpress software.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was obtained from cells and exosomes using TRIzol reagent (Invitrogen, USA), and the quality and concentration of the RNA were measured using a NanoDrop ND-1000 spectrophotometer. The optical density 260/280 ratio was within the range of 1.8–2.0, which indicated the suitability of the RNA for further analysis. Next, cDNA was synthesized using a reverse transcription kit (Takara Bio, Shiga, Japan) for mRNA and a tailing reaction kit (Sangon Biotech, China) for miRNA. U6 was used as the internal control for miR-328-3p, while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control for the other genes. The SYBR Green reagent was used, and all samples were subjected to RT-qPCR analysis using the ABI7500 system (Applied Biosystems, Inc., USA). The relative level of expression was determined by the 2^−ΔΔCT method. The primer sequences are provided in supplementary material (Table S4).

Western blotting analysis

The cell lysates from the radioimmunoprecipitation assay containing protease inhibitors and phosphorylated protease inhibitors were added to the cell culture dishes to extract all proteins from the cells. The protein concentration was measured using a BCA kit. The selection of an appropriate separation gel depends on the weight of the molecules being analyzed. The samples were separated by gel electrophoresis with a 10% SDS-PAGE protein sampling buffer. The proteins were subsequently transferred to a polyvinylidene fluoride membrane (Milipore, USA). The antigen mixture was blocked with 5% skim milk powder for 1 h at room temperature, after which it was incubated with the specific primary antibodies at 4 °C overnight (Table S3). Then, the membrane was washed three times in Tris-buffered saline with Tween 20 (TBST). The membrane was subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After washing three times in TBST, protein bands were visualized using enhanced chemiluminescence reagents and a bioimaging system (Bio-Rad, USA). Protein expression was quantified semi-automatically using the ImageJ software.

Immunofluorescence staining

The cells were processed with a 4% paraformaldehyde solution with 0.1% Triton X-100, and then, 5% goat serum (Beyotime, China) was added to block nonspecific binding. The cells were incubated with primary antibodies (Table S3) at 4 °C for 12–16 h. Then, Alexa Fluor 488-conjugated secondary antibodies were added dropwise, followed by nuclear staining with DAPI for 15 min in the dark. Cells from three randomly selected fields of view were photographed under a CLSM, and the expression was measured using the ImageJ software.

Exosomal miRNA sequencing and analysis

Exosomal miRNA sequencing and subsequent bioinformatics profiling were performed by LC-Bio Technology Co., Ltd (Hangzhou, China). The miRNAs were extracted from the exosomes using TRIzol reagent. The sequencing library was constructed using the TruSeq Small RNA Sample Preparation Kit (Illumina, USA). The sequencing was conducted on the Illumina HiSeq 2500 platform, using the 50-cycle SE configuration. Single-end sequencing reads were cleaned using a quality filter, an adapter cutter, and a length filter. The fold change cutoff for differentially expressed miRNAs was set at ≥ 1.5 for upregulated and downregulated genes. The potential miRNA target genes were predicted using miRanda (http://www.microrna.org/) and TargetScan (https://www.targetscan.org/).

MiRNA, siRNA, and cell transfection

The miR-124-3p inhibitor and its negative control (inhibitor-NC) were chemically synthesized by RiboBio Co., Ltd. The small interfering RNA targeting mouse STAT3 (siSTAT3) and its scrambled control (siNC) were purchased from GenPharma (Shanghai, China). The cells were transfected using the Lipofectamine 3000 reagent (Invitrogen, USA). The transfection efficiency was confirmed through RT-qPCR assays.

Dual-luciferase reporter gene assay

GenePharma constructed the 3’UTR sequence of the wild-type (WT) and mutant (MUT) STAT3 fragments containing the miR-124-3p-binding site into the pmirGLO-reporter vector according to the base sequence. These strains were denoted as pmirGLO-STAT3-WT and pmirGLO-STAT3-MUT, respectively. Next, HEK293T cells transfected with the miR-124-3p inhibitor or inhibitor-NC were inoculated in 96-well plates and then co-transfected with pmirGLO-STAT3-WT or pmirGLO-STAT3-MUT. The luciferase activity of the lysates was evaluated following the procedures outlined in the luciferase assay kit. The ratio of firefly relative luciferase units to Renilla relative luciferase units was used to determine the binding strength.

Statistical analysis

All data are presented as mean ± standard deviation, and statistical analysis and visualization were performed using GraphPad Prism software. After testing for normality, comparisons between two groups were conducted using a two-tailed Student’s t-test, while comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by post-hoc pairwise comparisons with the least significant difference method. A p-value of less than 0.05 was considered statistically significant.

Results

Induction and characterization of M2 macrophages

IL-4 and IL-13 were added to induce the polarization of RAW264.7 cells toward M2 macrophages. The polarization efficiency was determined by comprehensively analyzing the expression of related characteristic markers by conducting RT-qPCR and Western blotting. The results of the RT-qPCR analysis revealed that induction by IL-4 and IL-13 efficiently upregulated the expression of M2 macrophage markers (Cd206 and Arg-1) compared to that in the control group, whereas the expression of the mRNA of the total macrophage marker (Cd68) remained unaltered (Fig. 1A). The western blotting revealed a pattern similar to that found by the RT-qPCR (Fig. 1B and C). These findings indicated that RAW264.7 cells were polarized to M2 macrophages, which provided a foundation for collecting supernatants and preparing exosomes.

Fig. 1.

Identification of M2 macrophages and characterization of exosomes. (A) RT-qPCR to evaluate the mRNA expression levels of Cd68, Cd206, and Arg1 in RAW264.7 treated with IL-4 and IL-13. (B-C) Western blotting to detect the proteins expression levels of CD68, CD206, and ARG1 in RAW264.7 treated with IL-4 and IL-13. (D) Schematic diagram of exosomes extraction by ultracentrifugation. (E) Representative transmission electron microscopy images of two exosomes. Scale bar: 100 nm. (F) NTA to determine the size distribution of two exosomes. (G-H) Western blot analysis to detect the exosomal proteins expression of CD9, CD63, and TSG101 in two exosomes. (I) BCA assay to measure the protein concentration in two exosomes. (J) Cellular uptake of PKH26-labeled exosomes into chondrocytes. Chondrocytes were incubated with PKH26-lablled exosomes (red), chondrocytes were co-stained with phalloidin (green) and nuclei were stained with Hoechst 33,342 (blue). Scale bar: 25 μm. (K) Quantitative analysis of fluorescence intensity of uptake. Data are presented as the mean ± standard deviation (SD) (n = 3). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

Hypoxia enhances the release of exosomes from M2 macrophages

As exosomes serve as messengers that carry genetic information, they are integral to intercellular communication and exchange. The contents and functions of exosomes depend on the parental cells from which they are derived. To determine the effect of oxygen concentration on the secretion of exosomes from M2 macrophages. The supernatants were collected from a solution containing M2 macrophages incubated under normoxic and hypoxic conditions, and the exosomes were extracted following the described methods (Fig. 1D). The exosomes were then analyzed using TEM, NTA, and western blotting. TEM revealed that both Nor-Exos and Hypo-Exos displayed a typical tea saucer shape (Fig. 1E). NTA revealed that the average sizes of Nor-Exos and Hypo-Exos were 142.2 nm and 161.7 nm, respectively (Fig. 1F). Western blotting revealed robust expression of the exosome surface markers CD9, CD63, and TSG101; the level of expression increased significantly after exposure to hypoxia (Fig. 1G and H). Additionally, the protein concentration in the Hypo-Exos group was considerably greater than that in the Nor-Exos group (Fig. 1I). These findings indicated that hypoxic preconditioning significantly increased exosome release from M2 macrophages.

Hypoxia promotes chondrocytes uptake of labeled exosomes

We assessed the differences in the ability of chondrocytes to take up Nor-Exos and Hypo-Exos. The exosomes were labeled with PKH26 and subsequently cocultured with chondrocytes. The red fluorescence in the cytoplasm indicated that the exosomes were internalized by chondrocytes, and the fluorescence intensity was substantially greater in Hypo-Exos than in Nor-Exos (Fig. 1J). Additionally, real-time fluorescence microscopy also revealed significant differences (Fig. 1K). These findings indicated that hypoxic preconditioning facilitates the internalization of exosomes by chondrocytes.

Hypo-Exos inhibit inflammatory infiltration and alleviate cartilage degeneration in vivo

A rat KOA model was established to evaluate the therapeutic effects of Nor-Exos and Hypo-Exos in vivo. The specific treatment modalities, assessment techniques, and designated time points are shown in Fig. 2A.

Fig. 2.

Inflammation, behavioral and imaging evaluation of Hypo-Exos on KOA rats in vivo. (A) Schematic diagram of in vivo evaluation. (B-D) ELISA to quantify inflammatory factors (IL-6, IL-1β, and TNF-α) in joint fluid. (E) Hot plate to measure paw withdrawal latency. (F) Representative images of Catwalk. (G-J) Quantitative analysis of gait indexes. (K) Representative images of X-ray and 3D reconstruction based on micro-CT. Scale bar: 1 mm. (L-O) Quantitative analysis of the region of interest in the subchondral bone of tibia. Data are presented as the mean ± SD (n = 5). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

The level of inflammation is closely associated with the function and condition of chondrocytes. To assess the degree of inflammation in the knee joint, synovial fluid was harvested and analyzed to quantify the concentrations of the inflammatory factors IL-6, IL-1β, and TNF-α. The ELISA showed a considerable increase in the expression of inflammatory factors in the vehicle group. The reduction was more pronounced in the Hypo-Exos group than in the Nor-Exos group (Fig. 2B-D). These findings suggested that Hypo-Exos may more effectively inhibit inflammatory infiltration.

Patients suffering from KOA often present with greater pain sensitivity and gait abnormalities. Hence, we performed the hot plate test and CatWalk test to determine whether these effects occurred in the rats. The hot plate test showed that the vehicle group displayed a significant increase in pain sensitivity, as determined by a reduction in PWL. This index subsequently recovered in the Nor-Exos and Hypo-Exos groups, with the Hypo-Exos group demonstrating better outcomes (Fig. 2E). The CatWalk test showed that the KOA model could induce abnormal gait patterns (Fig. 2F), as determined by a significant reduction in the RH/LH ratio in the paw print area, maximum contact area, and swing phase. Compared to the Nor-Exos group, the Hypo-Exos group presented a greater increase in the RH/LH ratio (Fig. 2G-I). No noticeable differences were observed in the mean intensities across all groups (Fig. 2J). These findings indicated that Hypo-Exos significantly reduced abnormal pain and gait.

The degree of cartilage degeneration was evaluated using X-ray and micro-CT imaging techniques. As shown in Fig. 2K, rats in the Nor-Exos and Hypo-Exos groups presented a lower prevalence of osteophyte hyperplasia and cystic degeneration than those in the vehicle group. Furthermore, CT-based quantitative analysis of the ROI revealed a substantial decrease in BV/TV, Tb.N, and Tb.Th and a significant increase in Tb.Sp in the vehicle group. In contrast, these parameters were significantly better in the Nor-Exos and Hypo-Exos groups, with the Hypo-Exos group showing greater improvement (Fig. 2L-O). These findings indicated that Hypo-Exos effectively reduce cartilage degeneration.

The sections were stained with H&E and the S&F stain to determine the histological changes. The histological evaluation of the synovium showed a significant degree of synovial inflammation in the vehicle group; the degree of inflammation decreased significantly after exosomes were injected (Fig. 3A and E). The histological evaluation of cartilage revealed severe degeneration in the vehicle group, as determined by a decrease in thickness, an increase in the fibrotic surface area, and an irregular distribution of chondrocytes. In contrast, the Nor-Exos and Hypo-Exos groups showed lower cartilage degradation, greater cartilage integration, and more uniform surfaces (Fig. 3B and C). The mean HC/CC and OARSI score of the Hypo-Exos were considerably greater than those of the vehicle and Nor-Exos groups (Fig. 3F and G). The expression of ECM proteins was analyzed by immunohistochemical staining. Compared to the vehicle and Nor-Exos groups, the Hypo-Exos group showed a higher expression of COL2 and lower expression of MMP13 (Fig. 3D and H). These findings indicated that Hypo-Exos demonstrate a more pronounced cartilage protection.

Fig. 3.

Histological evaluation of Hypo-Exos on KOA rats in vivo. (A) H&E staining of synovium to score synovial activation. Scale bar: 50 μm (20×), 20 μm (40×). (B) H&E staining of articular cartilage to observe the morphology and structure. Scale bar: 100 μm (10×), 20 μm (40×). (C) S&F staining of articular cartilage observe extracellular matrix and collagen fibers. Scale bar: 100 μm (10×), 20 μm (40×). (D) Immunohistochemical analysis for COL2 and MMP13 in articular cartilage. Scale bar: 20 μm. (E) Quantitative analysis of the synovial score in each group. (F) Quantitative analysis of the HC/CC ration in each group. (G) Quantitative analysis of the OARIS score in each group. (H) Quantitative analysis of COL2 and MMP13 in each group. Data are presented as the mean ± SD (n = 5). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

Hypo-Exos can attenuate IL-1β-induced chondrocyte injury in vitro

To determine why Hypo-Exos play positive functional roles in vivo. Chondrocytes were cultured with a complete culture medium containing IL-1β to establish an OA-like cell model in vitro. The biological effects of Hypo-Exos on chondrocytes were investigated using various assessment techniques.

For cells proliferation activity, EDU-positive cells was strongly reduced following treatment with IL-1β. In contrast, the Nor-Exos and Hypo-Exos exhibited a considerable increase. Moreover, Hypo-Exos exhibited more pronounced proliferation-promoting effects than Nor-Exos (Fig. 4A and B). CCK-8 provided further evidence supporting the role of Hypo-Exos in promoting chondrocyte proliferation (Fig. 4C). For cells migratory capacity, the transwell assays showed that Hypo-Exos had greater efficacy, with more migrating cells in the Hypo-Exos group than in the other groups (Fig. 4D and E). Moreover, the scratch assay revealed that distance traveled by migrating cells was greater in the Hypo-Exos group than in the IL-1β and Nor-Exos groups (Fig. 4F and G). Concerning cell apoptosis, both Nor-Exos and Hypo-Exos significantly reduced the proportion of TUNEL-positive cells. Hypo-Exos had a stronger inhibitory effect on the apoptosis than Nor-Exos (Fig. 5A and B). Additionally, Annexin V-FITC/PI staining revealed a significant reduction in the percentage of apoptotic cells in the Hypo-Exos group than in the IL-1β and Nor-Exos groups (Fig. 5C and D).

Fig. 4.

Hypo-Exos promotes the proliferation and migration of chondrocytes in vitro. (A-C) EDU staining (red) and CCK-8 assays to observe the cell proliferation activity. Scale bar: 100 μm. (D-G) transwell assay and scratch test to measure the cell migration ability. Data are presented as the mean ± SD (n = 3). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

Fig. 5.

Hypo-Exos inhibit the apoptosis and extracellular matrix catabolism of chondrocytes in vitro. (A-D) TUNEL staining (green) and Annexin V/FITC/PI double-staining flow cytometry to analyze the cell apoptosis rates. Scale bar: 100 μm. (E-H) Immunofluorescence and western blotting to detect the expression levels of proteins related to extracellular matrix anabolism (COL2) and catabolism (MMP13). Scale bar = 50 μm. Data are presented as the mean ± SD (n = 3). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

Next, the effects of Hypo-Exos on the anabolic or catabolic processes of the ECM were investigated. The immunofluorescence analysis revealed that introducing Nor-Exos and Hypo-Exos increased the expression of COL2 and decreased the expression of MMP13. Moreover, compared to Nor-Exos, Hypo-Exos exhibited greater efficacy in promoting ECM synthesis and inhibiting ECM degradation (Fig. 5E and H). The western blotting analysis were similar to those of the immunofluorescence (Fig. 5F and G). Overall, we found that Hypo-Exos had a stronger chondroprotective effect.

The miR-124-3p is upregulated in Hypo-Exos and delivered to chondrocytes by exosomes

The findings of functional experiments showed that Hypo-Exos had better therapeutic outcomes than Nor-Exos in vivo and in vitro. Therefore, further investigation into why therapeutic efficacy differs between these two groups is essential. The miRNAs represent a significant functional component of exosomes and play a pivotal role in cellular communication. Therefore, we collected and extracted miRNAs from Nor-Exos and Hypo-Exos for microarray profiling, and then, compared the differences between them.

The results of miRNA microarray profile analysis revealed that 72 miRNAs were upregulated and 104 were downregulated in Hypo-Exos compared to their relative levels in Nor-Exos. A heat map and volcano plot were used to illustrate the miRNAs whose expression was upregulated and downregulated, respectively (Fig. 6A and B). The five most significantly upregulated miRNAs, including miR-124-3p, miR-210 -3p, miR-18a-3p, miR-130b-5p, and miR-877-3p, were selected and analyzed by RT-qPCR for further confirmation of their expression (Fig. 6C). Among the identified miRNAs, miR-124-3p showed a considerably greater level of expression in Hypo-Exos than in Nor-Exos. Previous studies have also shown that miR-124-3p has a beneficial effect on chondrocytes. Based on these findings, we focused on miR-124-3p to determine whether Hypo-Exos can regulate the behaviors of chondrocytes and inhibit cartilage degeneration by transferring miR-124-3p.

Fig. 6.

The miR-124-3p is upregulated in Hypo-Exos and transferred to chondrocytes via exosomes. (A) Heat map of the differentially expressed miRNAs between Nor-Exos and Hypo-Exos. (B) Volcano map of the differentially expressed miRNAs between Nor-Exos and Hypo-Exos. (C) Verification of the top five elevated miRNAs in Hypo-Exos by RT-qPCR. (D) The expression level of miR-124-3p in hypoxia M2 macrophages after transfected with miR-124-3p inhabitor. (E) The expression level of miR-124-3p in exosomes derived from hypoxia M2 macrophages transfected with miR-124-3p inhabitor. (F) The expression level of miR-124-3p in chondrocytes treated with exosomes derived from hypoxia M2 macrophages transfected with miR-124-3p inhabitor. Data are presented as the mean ± SD (n = 3). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

To demonstrate that Hypo-Exos can deliver miR-124-3p to chondrocytes, the expression of miR-124-3p was knocked down by transfecting a miR-124-3p inhibitor into hypoxia-cultured M2 macrophages (miR^KD-Hypo-macrophages); cells transfected with the corresponding negative control were used as a control (miR-NC^KD-Hypo-macrophage). The transfection efficiency was validated through RT-qPCR assays (Fig. 6D). The exosomes derived from miR^KD-Hypo-macrophages and miR-NC^KD-Hypo-macrophages were designated miR^KD-Hypo-Exos and miR-NC^KD-Hypo-Exos, respectively. The expression of miR-124-3p was considerably lower in the miR^KD-Hypo-Exos than in the miR-NC^KD-Hypo-Exos (Fig. 6E). We also determined the expression of miR-124-3p in chondrocytes cocultured with exosomes. Our findings demonstrated that the capacity of miR^KD-Hypo-Exos to deliver miR-124-3p into chondrocytes was lower (Fig. 6F). These results suggested that miR-124-3p derived from hypoxic M2 macrophages can be transferred to target chondrocytes through exosomes.

The miR-124-3p is involved in Hypo-Exos-mediated inhibition of chondrocytes injury and cartilage degeneration in vitro and in vivo

A series of in vitro experiments were conducted to confirm that Hypo-Exos regulate the behavior of chondrocytes, primarily by delivering miR-124-3p. Specifically, miR^KD-Hypo-Exos and miR-NC^KD-Hypo-Exos were introduced during IL-1β-induced chondrocytes injury to observe alterations in the corresponding evaluation indices. The number of EDU-positive cells of the miR^KD-Hypo-Exos group was consistently lower than that of the miR-NC^KD-Hypo-Exos group (Fig. 7A and B). Moreover, introducing miR^KD-Hypo-Exos considerably decreased the OD value, confirming the significant inhibitory effect on proliferation (Fig. 7C). In terms of migration, the miR^KD-Hypo-Exos inhibited the migration capacity to a greater extent than the miR-NC^KD-Hypo-Exos, as determined by the transwell and scratch assays (Fig. 7D-G). TUNEL staining and Annexin V-FITC/PI revealed that compared to the miR-NC^KD-Hypo-Exos, the introduction of the miR^KD-Hypo-Exos significantly decreased the ability to inhibit IL-1β-induced apoptosis (Fig. 7H-K). Besides, compared to the immunofluorescence analysis observed with miR-NC^KD-Hypo-Exos, the miR^KD-Hypo-Exos substantially downregulated COL2 expression and upregulated MMP13 expression (Fig. 7L and P). The western blotting analysis confirmed these findings (Fig. 7N and O).

Fig. 7.

Knockdown of miR-124-3p in hypoxia M2 macrophages weakens the Hypo-Exos-mediated positive effect of chondrocytes in vitro. (A-C) EDU staining (red) and CCK-8 assays to observe cell proliferation activity. Scale bar: 100 μm. (D-G) Transwell and scratch test to measure the cell migration ability. (H-K) TUNEL staining (green) and Annexin V/FITC/PI double-staining flow cytometry to analyze the cell apoptosis rates. Scale bar: 100 μm. (L-P) Immunofluorescence and western blotting to detect the expression levels of proteins related to extracellular matrix anabolism (COL2) and catabolism (MMP13). Scale bar: 50 μm. Data are presented as the mean ± SD (n = 3). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

To determine the function of miR-124-3p in Hypo-Exos-mediated KOA repair, a series of in vivo experiments were performed. The ELISA revealed that the levels of proinflammatory mediators were higher in the joint fluid of the miR^KD-Hypo-Exos group than the miR-NC^KD-Hypo-Exos group (Fig. 8A). The results of the hot plate and CatWalk tests indicated that the ability of the miR^KD-Hypo-Exos group to correct aberrant pain and gait was weaker than that of the miR-NC^KD-Hypo-Exos group (Fig. 8B-D). Through imaging, we found that the performance of the miR^KD-Hypo-Exos group was slightly lower than that of the miR-NC^KD-Hypo-Exos group (Fig. 8E), particularly in the quantitative analysis of the ROI in the subchondral bone (Fig. 8F-I). Pathological examination and immunochemical staining indicated that treatment with miR^KD-Hypo-Exos resulted in inferior outcomes, which included more prominent synovial inflammation, cartilage degeneration, and ECM catabolism (Fig. 8J-O).

Fig. 8.

Knockdown of miR-124-3p in hypoxia M2 macrophages attenuates the Hypo-Exos-mediated protective effect in vivo. (A) ELISA to quantify inflammatory factors (IL-6, IL-1β, and TNF-α) in joint fluid. (B) Hot plate to measure paw withdrawal latency. (C) Representative images of Catwalk. (D) Quantitative analysis of gait indexes. (E) Representative images of X-ray and 3D reconstruction based on micro-CT. Scale bar: 1 mm. (F-I) Quantitative analysis of the region of interest in the subchondral bone of tibia. (J) H&E staining of synovium and articular cartilage. Scale bar: 100 μm (10×), 50 μm (20×), 20 μm (40×). (K) S&F staining and immunohistochemical analysis in articular cartilage. Scale bar: 100 μm (10×), 20 μm (40×). (L) Quantitative analysis of the synovial score. (M) Quantitative analysis of the HC/CC ration. (N) Quantitative analysis of the OARIS score. (O) Quantitative analysis of COL2 and MMP13. Data are presented as the mean ± SD (n = 5). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

Overall, these results showed the crucial role of miR-124-3p, which may function as a biomarker involved in several functional changes mediated by Hypo-Exos.

The miR-124-3p regulates STAT3 by directly targeting 3’ untranslated region (UTR)

To elucidate the pathways through which miR-124-3p in Hypo-Exos affects the biological behavior of chondrocytes, two online databases, including TargetScan and miRanda, were used to predict the target genes of miR-124-3p. Among the predicted target genes, STAT3 exerts deleterious effects on chondrocytes viability, apoptosis, and ECM degradation. Thus, STAT3 was selected for further investigation. RT-qPCR assays were performed to assess the relationship between miR-124-3p and STAT3. The miR^KD-Hypo-Exos considerably increased the expression level of STAT3 compared to treatment with miR-NC^KD-Hypo-Exos (Fig. S1A). Moreover, a luciferase reporter assay was conducted on transfected HEK293 cells to confirm the presence of the miR-124-3p-binding site in the STAT3 3’UTR. The relative luciferase activity increased when the downregulated miR-124-3p was cotransfected with the STAT3 WT luciferase construct but not when it was cotransfected with the MUT construct (Fig. S1B and C). These results supported the hypothesis that STAT3 is a target gene of miR-124-3p.

The miR-124-3p attenuates IL-1β-induced chondrocytes injury by targeting STAT3

Due to targeted regulatory effects, alterations in miR-124-3p expression led to changes in the expression of its downstream target gene. A rescue experiment was conducted to determine the function of STAT3 in the behavior of chondrocytes regulated by miR-124-3p. Specifically, siSTAT3 was transfected into chondrocytes treated with miR^KDHypo-Exos were used to detect the level of expression of STAT3, while cells transfected with siNC were used as the control group. RT-qPCR assays were performed to evaluate the transfection efficiency, and the results revealed that compared to siNC, siSTAT3 significantly decreased the level of expression of the STAT3 (Fig. S2A). This result indicated that siSTAT3 counteracted the increase in STAT3 expression levels induced by miR^KDHypo-Exos.

The influence of this counteracting effect on the behavior of chondrocytes was subsequently investigated. CCK-8 and EDU staining revealed that knocking down STAT3 facilitated cell proliferation during co-treatment with miR^KD-Hypo-Exos (Fig. 9A-C). The scratch assays and transwell showed that compared to the control, the inhibition of STAT3 increased the cell migration ability (Fig. 9D-G). TUNEL staining and Annexin V-FITC/PI showed that a decrease in STAT3 expression strongly prevented cell apoptosis (Fig. 9H-K). Immunofluorescence and western blotting analysis indicated that a reduction in STAT3 expression decreased ECM catabolism (Fig. 9L-P). In conclusion, the rescue experiments demonstrated that siSTAT3 partially reversed the detrimental effects of miR^KD-Hypo-Exos on chondrocytes, which confirmed the hypothesis that miR-124-3p regulates the behavior of chondrocytes by targeting STAT3.

Fig. 9.

The miR-124-3p-abundant Hypo-Exos play positive effect by targeting STAT3. (A-C) EDU staining (red) and CCK-8 assays to observe cell proliferation activity. Scale bar: 100 μm. (D-G) Transwell and scratch test to measure the cell migration ability. (H-K) TUNEL staining (green) and Annexin V/FITC/PI double-staining flow cytometry to analyze the cell apoptosis rates. Scale bar: 100 μm. (L-P) Immunofluorescence and western blotting to detect the expression levels of proteins related to extracellular matrix anabolism (COL2) and catabolism (MMP13). Scale bar: 50 μm. Data are presented as the mean ± SD (n = 3). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001

Discussion

Role of M2φ-Exos in tissue injury repair

M2φ-Exos have demonstrated tissue repair-promoting effects in various disease models. One study showed that M2φ-Exos could suppress bone loss in a mouse periodontitis model by activating the IL-10/IL-10R signaling pathway, exhibiting significant bone protective effects [53]. Additionally, Wang et al. injected M2φ-Exos into the femoral fracture site of diabetic mice and found that they could accelerate fracture healing by activating the PI3K/AKT pathway and inducing macrophage polarization from M1 to M2, providing new perspectives and potential strategies for treating diabetic fractures [54]. In a recent study, Guo et al. discovered that M2φ-Exos could reduce infarction size and promote angiogenesis in rats with myocardial infarction, thereby improving cardiac function. This effect was related to the delivery of miR-132-3p and the inhibition of THBS1 expression in endothelial cells [55]. Similarly, Luo et al. explored the angiogenic potential of M2φ-Exos in a flap transplantation model and found that M2φ-Exos could enhance angiogenesis by activating the HIF1AN/HIF-1α/VEGFA pathway, thereby improving the survival rate of transplanted flaps [57].

In OA treatment, related studies have also demonstrated that M2φ-Exos could delay OA progression by exerting anti-inflammatory and reparative effects. For example, Da-Wa et al. analyzed the therapeutic effects of M2φ-Exos in an OA rat model and found that M2φ-Exos significantly alleviated joint cartilage inflammation and pathological damage in rats by activating the PI3K/AKT/mTOR pathway. Additionally, they increased the expression of key anabolic proteins in cartilage tissue [52]. Qian et al. reported that M2φ-Exos protected OA mouse cartilage and improved abnormal gait by targeting TLR3 and COL10A1 through miR-26b-5p [60].

In our study, the injection of M2φ-Exos into the knee joint cavity of OA rats similarly showed that M2φ-Exos effectively inhibited OA progression, evidenced by reduced joint inflammation, improvement in behavioral and imaging assessments, and alleviation of cartilage and synovial pathological damage. This further corroborates the significant role of M2φ-Exos in OA treatment.

Advantages of hypoxic preconditioning in cell-derived exosome applications

Various strategies have been developed to enhance the therapeutic efficacy of cells or exosomes. Although the mechanisms behind these strategies may differ, they share a common strategic goal. For cellular therapies, recent studies have optimized cell therapy based on chimeric antigen receptor (CAR) T-cell engineering [61]. CAR-T cell engineering involves genetically modifying T cells to activate immune responses, thereby enhancing the antitumor function of the immune system and improving therapeutic efficacy [62, 63]. For exosomes, exosomal molecular engineering is commonly used for the design and modification of exosomes. In addition, hypoxic preconditioning has also been frequently applied. Hypoxic preconditioning regulates the therapeutic efficacy of exosomes by altering the composition and quantity of their cargo. This method has been widely used to enhance the biological effects of stem cell-derived exosomes. For example, Liu et al. compared the therapeutic effects of exosomes derived from human umbilical mesenchymal stem cells under normoxic and hypoxic conditions in a mouse femoral fracture model. They found that, compared to normoxic exosomes, hypoxic exosomes could better promote angiogenesis and accelerate fracture healing by transporting miR-126 to endothelial cells through the SPRED1/Ras/Erk signaling pathway [64]. Guo et al. established a muscle atrophy model in elderly rats to compare the effects of exosomes derived from bone marrow mesenchymal stem cells under normoxic and hypoxic conditions on skeletal muscle regeneration and muscle function recovery. They found that, under hypoxic conditions, exosomes were more effective than normoxic ones in enhancing the proliferation and differentiation abilities of muscle satellite cells through the miR-210-3p/KLF7 signaling pathway [65].

This study applied hypoxic preconditioning to the culture of M2 macrophages, focusing on the impact of hypoxic preconditioning on the biological effects of M2φ-Exos. The findings suggest that, similar to stem cell hypoxic preconditioning, hypoxic preconditioning enhances the biological effects of M2φ-Exos, thereby improving their therapeutic efficacy both in vitro and in vivo.

The promoting role of miR-124-3p in OA cartilage injury repair

miR-124-3p is a widely expressed microRNA in organisms and has been reported to play a positive role in OA cartilage injury repair [66–70]. Studies have found that miR-124-3p is downregulated in a mouse OA model induced by medial meniscus instability surgery and in chondrocytes induced by LPS. Overexpression of miR-124-3p, however, could alleviate cartilage damage and inhibit chondrocyte pyroptosis [68]. Moreover, extracellular vesicles derived from mesenchymal stem cells could promote chondrocyte proliferation and migration and suppress apoptosis through the circHIPK3/miR-124-3p/MYH9 signaling pathway, thus effectively preventing OA progression [69]. In a recent study, Dong et al. discovered that exosomes derived from bone marrow mesenchymal stem cells treated with quercetin significantly inhibited the decline in chondrocyte viability by upregulating miR-124-3p expression. This also reversed the IL-1β-induced upregulation of MMP13 and ADAMTS5 expression and the downregulation of COL2A1 expression in vitro [70].

In this study, exosome sequencing revealed that, compared to Nor-Exos, miR-124-3p expression was significantly elevated in Hypo-Exos, indicating that hypoxic preconditioning promoted the expression of miR-124-3p in M2φ-Exos. Further functional validation showed that the expression level of miR-124-3p was closely related to the biological effects of M2φ-Exos. Reducing miR-124-3p expression led to a decrease in the biological effects of Hypo-Exos, suggesting that miR-124-3p plays a crucial role in inhibiting chondrocyte inflammatory responses and delaying OA cartilage degeneration in Hypo-Exos. These results are consistent with the reported effects of miR-124-3p in the literature.

Role of STAT3 in OA progression and targeted therapeutic strategies

STAT3 is an important transcription factor in cellular signaling that plays a crucial role in various biological processes [71]. Previous studies have shown that STAT3 is closely associated with the development of OA, and inhibiting STAT3 expression can help improve cartilage damage caused by OA [72]. For instance, Li et al. found that the activation of STAT3 in endothelial cells is a critical trigger for OA development. Inhibiting STAT3 expression can reverse the proliferation of subchondral bone H-type blood vessels and significantly downregulate the volume and number of vessels, thus alleviating the degeneration of cartilage and subchondral bone [73].

Selective STAT3 inhibitors can serve as a potential targeted therapeutic strategy for OA. Lu et al. discovered that the small molecule compound STX-0119 specifically inhibits the phosphorylation of STAT3 and induces upregulation of peroxisome proliferator-activated receptor γ expression, thereby suppressing inflammation and promoting anabolic metabolism in an IL-1β-induced chondrocyte inflammation model [74]. Another study demonstrated that Alantolactone could reduce IL-1β-induced inflammation and alleviate cartilage degeneration in a mouse OA model by inhibiting both STAT3 and NF-κB signaling pathways [75]. Additionally, Xanthatin has been shown to specifically target and inhibit STAT3 expression, thereby exerting a protective effect on cartilage in OA [76].

Regulating STAT3-related signaling pathways offers another targeted strategy for OA treatment. Su et al. investigated the antifibrotic effects of fibroblast growth factor 10 (FGF10) in OA synovial tissue and confirmed that FGF10 inhibits synovial fibrosis in mice by modulating the IL-6/JAK2/STAT3 pathway, thus improving OA symptoms [77]. In another study, researchers assessed the effects of parathyroid hormone (PTH) on bone and cartilage metabolism in a collagenase-induced mouse OA model. They found that PTH exerts protective effects on cartilage and subchondral bone by downregulating JAK2/STAT3 and WNT5A/ROR2 expression [78]. Furthermore, various compounds such as Angelicin [79], M13 from Morinda officinalis [80], Toll-like receptor 7 [81], leptin [82], angiotensinogen [83], miR-216a-5p [84], and Bergenin [85] have been shown to influence OA progression by modulating STAT3-related signaling pathways.

In this study, we found that miR-124-3p, highly expressed in Hypo-Exos, is delivered to chondrocytes via M2φ-Exos. It subsequently binds to the 3’-UTR of STAT3 in chondrocytes, inhibiting post-transcriptional expression of STAT3 (Fig. 10). Further cellular experiments showed that Hypo-Exos exert a protective effect on chondrocytes through the miR-124-3p/STAT3 axis. This not only highlights the significant role of the miR-124-3p/STAT3 axis in enhancing the biological effects of M2φ-Exos in hypoxic preconditioning, but also provides supporting evidence for STAT3 signaling pathway modulation as a targeted therapeutic strategy for OA.

Fig. 10.

Schematic diagram of hypoxic M2 macrophage promotes proliferation and migration and inhabits apoptosis and ECM degradation in chondrocyte. Hypoxia mediates enhanced production of miR-124-3p, which is then transferred to chondrocyte where it exhibits its biological role and potential therapeutic effect in alleviating KOA via the miR124-3p/STATA3 axis

Strengths and limitations

This study has several strengths. First, it expands the application of hypoxic preconditioning at the cellular level, providing further evidence for the role and efficacy of hypoxic preconditioning. Second, through a comprehensive analysis using multiple methods, we examined the changes in the biological effects of M2φ-Exos after hypoxic preconditioning both in vitro and in vivo, offering new insights for future research and applications focused on enhancing the functionality of M2φ-Exos. Finally, by combining exosomal miRNA sequencing with functional validation, we uncovered potential molecular mechanisms, laying the foundation for related research and providing a novel approach for cell-free therapy in OA.

This study also has some limitations. First, although we found that miR-124-3p is highly expressed in M2φ-Exos after hypoxic preconditioning, we did not elucidate the mechanism by which hypoxic preconditioning increases its expression. Based on relevant literature, hypoxic preconditioning may influence miR-124-3p expression through its effect on HIF-1α. This process needs to be further validated in future studies. Second, while we used exosomal miRNA sequencing to identify miRNAs upregulated in M2φ-Exos following hypoxic preconditioning, we selected only the miRNA with the highest upregulation fold for further experiments, neglecting the potential roles of other miRNAs in this process. Future studies will continue to investigate whether other miRNAs contribute to this process. Third, in evaluating the in vivo therapeutic effects of exosomes, we constructed a post-traumatic OA model in rat knee joints through surgical intervention. However, this model may not fully replicate the pathological processes of human idiopathic OA. Therefore, it will be essential to employ more matched relevant models in future studies, such as spontaneous animal OA models. Finally, when validating the function of miR-124-3p, we only performed loss-of-function experiments by knocking down miR-124-3p expression in Hypo-Exos to observe the effects of reduced miR-124-3p expression on Hypo-Exos activity. Future research should include gain-of-function experiments, such as overexpressing miR-124-3p in Nor-Exos to assess the impact of increased miR-124-3p expression on Nor-Exos effects, in order to further validate the relationship between miR-124-3p and M2φ-Exos biological activity.

Conclusions

To summarize, our study demonstrated that M2φ-Exos prevent the development of KOA by promoting chondrocyte proliferation and migration and inhibiting chondrocyte apoptosis and ECM degradation. Additionally, hypoxic preconditioning is an extremely effective method for improving the therapeutic effects of M2φ-Exos, and the mechanism underlying such effects may be related to the miR-124-3p/STAT3 axis.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

KOA

Knee osteoarthritis

ECM

Extracellular matrix

MSCs

Mesenchymal stem cells

DMEM

Dulbecco’s minimum essential medium

FBS

Fetal bovine serum

DMEM/F12

Dulbecco’s minimum essential medium containing nutrient mixture F12

PBS

Phosphate buffered saline

TEM

Transmission electron microscopy

NTA

Nanoparticle tracking analysis

BCA

Bicinchoninic acid

DAPI

4,6-diamidino-2-phenylindole

PWL

Paw withdrawal latency

PWL

Paw withdrawal latency

RH

Right hind

LH

Left hind

RF

Right front

LF

Left front

Micro-CT

Micro-computed tomography

BV/TV

Bone volume fraction

Tb.Th

Trabecular thickness

Tb.N

Trabecular number

Tb.Sp

Trabecular separation

H&E

Hematoxylin and eosin

S&F

Safranin O-fast green

HC

Hyaline cartilage

CC

Calcified cartilage

OARSI

Osteoarthritis research society international

COL2

Collagen type II

MMP13

Matrix metalloproteinase-13

CCK-8

Cell Counting kit-8

EDU

5-ethynyl-2ʹ-deoxyuridine

TUNEL

TdT-mediated dUTP nick-end labeling

FITC

Fluorescein isothiocyanate

PI

Propidium iodide

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

TBST

Tris-buffered saline with Tween 20

Author contributions

GN and MZ designed the study; HL, YY and YG performed the experiments; HL drafted the manuscript; HL, BL and JY classified the figures; YY and PL analyzed the data; HL, YG and BL revised the manuscript. All authors read and approved the final manuscript.

Funding

This research was funded by the Research Project Supported by Shanxi Scholarship Council of China (2023 − 189), the Shanxi Provincial Basic Research Program (202203021221276) and the Shanxi Provincial Basic Research Program (202403021211110).

Data availability

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

Declarations

Ethics approval and consent to participate

The experimental procedures involving in animals was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (DW2024002).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.