Human umbilical cord mesenchymal stem cell-derived exosomal miR-199a-3p inhibits the MAPK4/NF-κB signaling pathway to relieve osteoarthritis

Abstract

BACKGROUND

There is currently no effective treatment for osteoarthritis (OA), which is the most common joint disorder leading to disability. Although human umbilical cord mesenchymal stem cells (hUC-MSCs) are promising OA treatments, their use is limited by the condition itself, and understanding of the underlying mechanisms of OA is lacking.

AIM

To explore the specific molecular mechanism by which hUC-MSC-derived exosomal miR-199a-3p improves OA.

METHODS

Sodium iodoacetate was injected into rat articulations to construct an animal model of OA. Interleukin (IL)-1β was used to induce human chondrocytes (CHON-001) to construct an OA chondrocyte model. Exosomes in hUC-MSCs were isolated using Ribo^™ Exosome Isolation Reagent. Real-time reverse transcriptase-polymerase chain reaction and western blotting were used to detect the expression of related genes and proteins, and damage to CHON-001 cells and rat articular cartilage tissue was evaluated by enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling staining and hematoxylin and eosin staining.

RESULTS

hUC-MSC-derived exosomes (hUC-MSC-Exos) inhibited the expression of IL-1β-induced inflammatory cytokines, namely, IL-6, IL-8 and tumor necrosis factor-α. hUC-MSC-Exos also improved the viability but inhibited the apoptosis of CHON-001 cells, improved the pathological condition of articular cartilage tissue and alleviated the development of OA in vivo. Mechanistically, hUC-MSC-Exos downregulated the expression of mitogen-activated protein kinase 4 by delivering miR-199a-3p, thereby inhibiting the activation of the nuclear factor-kappaB signaling pathway, alleviating IL-1β-induced chondrocyte inflammation and apoptosis, and ultimately improving the development of OA.

CONCLUSION

hUC-MSC-derived exosomal miR-199a-3p alleviates OA by inhibiting the mitogen-activated protein kinase 4/nuclear factor-kappaB signaling pathway. The present findings suggest that miR-199a-3p delivery by hUC-MSC-Exos may be a novel strategy for the treatment of OA.

Key Words: Osteoarthritis; Human umbilical cord mesenchymal stem cells; Exosomes; MiR-199a-3p; Mitogen-activated protein kinase 4; Nuclear factor-kappaB

Core Tip: Osteoarthritis (OA), a debilitating joint disorder, currently lacks effective treatment options. This study reveals that exosomal miR-199a-3p derived from human umbilical cord mesenchymal stem cells offers a novel therapeutic strategy for OA. By targeting the mitogen-activated protein kinase 4/nuclear factor-kappaB signaling pathway, miR-199a-3p significantly reduces inflammation and inhibits apoptosis in chondrocytes. These findings highlight the potential of exosomal miR-199a-3p delivery as a promising approach for managing OA, providing a foundation for future clinical applications.

INTRODUCTION

Osteoarthritis (OA) is a whole-joint disease involving all joint tissues, including meniscal degeneration[1], inflammation and fibrosis of the infrapatellar fat pad[2,3], cartilage degeneration and subchondral bone remodeling. OA is associated with sex, age, obesity, and poor prognosis after trauma[4]. OA cartilage is characterized by changes in biomechanical and biochemical behavior, which are manifested mainly by chondrocyte apoptosis, articular cartilage degeneration, osteophyte formation, subchondral osteosclerosis and synovial inflammation[5]. Previous studies have reported that increased apoptosis of chondrocytes is crucial in OA and serves as the primary inducer in the development of this condition[6,7]. A positive relationship exists between the extent of cartilage injury and the degree of chondrocyte apoptosis[8]. On the one hand, increased chondrocyte apoptosis causes cartilage matrix degradation, cartilage matrix degeneration, cartilage structure disorders and secondary pathophysiological changes; on the other hand, reduced numbers of chondrocytes lead to local environmental disorders and structural incompleteness of cartilage, which further leads to the occurrence and development of OA[9,10]. Therefore, investigating the processes that govern the prevention of chondrocyte apoptosis is crucial for managing OA.

Mesenchymal stem cells (MSCs) are pluripotent stem cell populations that are promising options for OA treatment, but the optimal cell source for the OA treatment of MSCs is currently being debated[11]. Human umbilical cord MSCs (hUC-MSCs) not only have typical stem cell characteristics but are also abundant and easy to extract[12]. Therefore, hUC-MSCs have broad application prospects in the treatment of OA. hUC-MSCs often play a therapeutic role through a paracrine mechanism, and exosomes are paracrine factors that not only have similar biological functions to those of hUC-MSCs but also have the unique advantage of being more stable under different pathophysiological conditions[13]. Owing to the unique advantages of exosomes, we have focused on the effects of hUC-MSC-derived exosomes (hUC-MSC-Exos) on the progression of OA. Recent studies have shown that hUC-MSC-Exos have anti-inflammatory effects on OA chondrocytes[14]. Moreover, hUC-MSC-Exos promote cartilage regeneration in OA rats[15]. Although previous studies have investigated the therapeutic effects of hUC-MSC-Exos on OA, most have focused on the macroscopic level of cell function, and in-depth exploration of the specific mediating mechanism of exosomes and their interactions with other key molecules is lacking. MicroRNAs (miRNAs) are important components of exosomes and are key contributors to the overall biological function of exosomes[16]. Therefore, this study focused on exosome-specific miRNAs at the molecular level of intracellular signaling and explored the core role of hUC-MSC-Exos in the treatment of OA in more detail.

The literature revealed that the top 23 miRNAs expressed in MSC-Exos accounted for 79.1% of all miRNAs, and the top five were miR-1246, miR-23a-3p, miR-451a, miR-125b-5p, and miR-199a-3p[17]. In addition, miR-199a-3p was found to be highly expressed in hUC-MSC-Exos[18], and miR-199a was also implicated in the chondrogenesis and differentiation of chondrocytes[19]. Through bioinformatics analysis, we further revealed that miR-199a-3p was significantly under expressed in OA. Therefore, we speculate that the deletion of miR-199a-3p in OA can be restored by exosome delivery of miR-199a-3p by hUC-MSCs, thereby improving the biological behavior of chondrocytes. Therefore, miR-199a-3p was specifically selected as the key research object in this study to study the specific mechanism by which hUC-MSC-Exos improve OA.

Nuclear factor-kappaB (NF-κB) is a transcription factor that influences inflammation and immunity, and it regulates various genes associated with cell growth, programmed cell death, development, and tumor progression[20,21]. A growing body of research indicates that NF-κB regulates inflammatory cytokines and plays a role in cartilage differentiation and degradation[22]. In OA cartilage, NF-κB directly or indirectly promotes the apoptosis of OA chondrocytes and exacerbates the inflammatory response of cartilage tissue by inducing the synthesis of nitric oxide, cyclooxygenase-2, inducible nitric oxide synthase, and prostaglandin E2[23]. In addition, studies have shown that a reduction in miR-199a-3p is involved in the NF-κB activation pathway, which leads to inflammation and progressive atheromatosis[24]. Moreover, the overexpression of miR-199a-3p negatively regulates the activation of the phosphatidylinositol 3-kinase/protein kinase B/NF-κB signaling pathway and inhibits lipopolysaccharide-induced inflammation in a bovine mammary epithelial cell line[25]. We also identified a targeted binding site between miR-199a-3p and the NF-κB inducer mitogen-activated protein kinase 4 (MAPK4) via the StarBase website. Whether miR-199a-3p can also inhibit the progression of inflammatory OA by targeting the MAPK4-mediated NF-κB signaling pathway remains to be further confirmed and studied.

In conclusion, this study aimed to reveal the intrinsic link between miR-199a-3p and the MAPK4/NF-κB signaling pathway in hUC-MSC-Exos and to elaborate on how exosomal miR-199a-3p regulates this signaling pathway, thereby affecting the occurrence and development of OA through cell and animal experiments. These findings are helpful for further understanding the pathogenesis of OA and identifying potential targets for OA treatment.

MATERIALS AND METHODS

Culture and transfection of hUC-MSCs

hUC-MSCs were obtained from the Shenzhen Otwo Biotech Co., Ltd. (HTX2318) and were grown in alpha minimal essential medium (HyClone, UT, United States) supplemented with 10% fetal bovine serum (10100147, Gibco, NY, United States) and 1% penicillin-streptomycin (15070063, Gibco, NY, United States). The cells were incubated at 37 °C with 5% CO₂. hUC-MSCs were plated in 24-well plates and incubated overnight. When the cell density reached 60%-70%, Lipofectamine 3000 reagent (Invitrogen, Grand Island, NY, United States) was used to transfect the NC mimic (sense: 5’-UUGUACUACACAAAAGUACUG-3’; antisense: 5’-GUACUUUUGUGUAGUACAAUU-3’), miR-199a-3p mimic (5’-ACAGUAGUCUGCACAUUGGUUAGU-3’; 3’- UAUGUCAUCAGACGUGUAACCAAU-5’), NC inhibitor (5’-CAGUACUUUUGUGUAGUACAA-3’), and miR-199a-3p inhibitor (5’-UAACCAAUGUGCAGACUACUGU-3’) into the cells, respectively. The transfection efficiency was measured after the cells were cultured in a 5% CO₂ incubator at 37 °C for 48 hours.

Isolation of hUC-MSC-Exos

We seeded 1 × 10⁶ hUC-MSCs in a T75 flask and cultured them until they reached 80%-90% confluence. Then, RiboTM exosome isolation reagent (C10130-1, RiboBio, Guangzhou, China) was used to isolate exosomes from normal, miR-199a-3p-overexpressing or miR-199a-3p-deficient hUC-MSCs. Briefly, the cell culture supernatant (45 mL) was collected, the mixture was centrifuged at 350 × g for 10 minutes, and the supernatant was collected. The mixture was centrifuged at 2000 × g for 30 minutes, and the supernatant was collected. The mixture was centrifuged at 10000 × g for 30 minutes, and the supernatant was collected. A 1/3 volume of RiboM Exosome lsolation Reagent was added, and the mixture was allowed to stand overnight at 4 °C. Two milliliters of the mixture was transferred to a 2 mL centrifuge tube and centrifuged at 15000 × g for 30 minutes at 4 °C, after which the supernatant was carefully aspirated with a pipette. In the same manner, the mixture was transferred to the same tube and centrifuged until all the mixture had been transferred and centrifuged, and the exosomes were contained in the pellet.

Identification of hUC-MSC-Exos

Transmission electron microscopy: The morphology and structure of the exosomes were observed by transmission electron microscopy (TEM). The exosome inclusions were first suspended in phosphate buffered saline (PBS) (20 μL). A drop (5 μL) of the resuspended exosome mixture was placed on a carbon-coated copper grid and allowed to adhere for 5 minutes. Then, the excess liquid was blotted off using filter paper. The grid was stained with 2% uranyl acetate for 1 minute, and then the grid was air-dried for 10 minutes. The morphology and size of the exosomes were observed via TEM.

Nanoparticle tracking analysis: The particle size of the exosomes was analyzed via a NanoSight system (Malvern Instruments, United States). Exosomes were suspended in PBS at a concentration of approximately 2 μg/μL and diluted by a factor of 100 before analysis. The quantification of exosomes was performed using the built-in software of the NanoSight system. A light microscope with a perpendicular beam axis was also used to inspect the exosomes before NanoSight analysis to ensure that the sample was homogenous and free of large aggregates. The diluted exosome sample was injected into the NanoSight sample chamber, and the instrument was set to capture a 60-second video. Nanoparticle tracking analysis software was used to analyze the videos to assess light scattering, which provided information on the size and concentration of the exosomes.

Uptake of hUC-MSC-Exos by human chondrocytes

For the uptake experiment, 5 × 10⁴ human chondrocytes (CHON-001) were seeded in a 24-well plate. Exosomes were labeled with a PKH67 fluorescence labeling kit (Sigma-Aldrich, MO, United States) according to the manufacturer’s instructions. Briefly, an appropriate volume of PKH67 dye solution was added to the exosome suspension, and the mixture was incubated for 5 minutes at room temperature. The labeled exosomes were then added to the wells containing the CHON-001 chondrocytes at a ratio of 10 μg of exosomes per well. After the labeled exosomes were incubated with CHON-001 cells for 12 hours, the cells were fixed with 4% paraformaldehyde. The cells were incubated with 4% paraformaldehyde for 15 minutes at room temperature. Then, the paraformaldehyde solution was removed, and the cells were washed three times with PBS before being imaged using a fluorescence microscope to observe the uptake of the labeled exosomes.

Human chondrocyte culture and transfection

Dulbecco’s modified Eagle medium (11050147, Gibco, NY, United States) supplemented with 10% fetal bovine serum (10100147, Gibco, NY, United States) and 1% penicillin-streptomycin was used to culture CHON-001 chondrocytes (American Type Culture Collection) at 37 °C with 5% CO₂. To establish the OA chondrocyte model, CHON-001 cells were treated for 24 hours with 10 ng/mL interleukin (IL)-1β (Sigma-Aldrich, MO, United States). To evaluate the effect of hUC-MSC-Exos on CHON-001 cells, 1 μg/mL hUC-MSC-Exos that were normal, overexpressed, or knocked down with miR-199a-3p was cocultured with CHON-001 cells for 24 hours prior to IL-1β treatment. To evaluate the impact of the NF-κB signaling pathway, CHON-001 cells were incubated with 50 μM NF-κB activator phorbol 12-myristate 13-acetate (PMA, HY-18739, MedChemExpress, NJ, United States) for 24 hours. After the cell density reached 60%-70%, the NC mimic, miR-199a-3p mimic, NC inhibitor, miR-199a-3p inhibitor, OE-NC, and OE-MAPK4 were transfected into CHON-001 cells according to the instructions of the Lipofectamine 3000 reagent (Invitrogen, Grand Island, NY, United States), and the cells were cultured in a 5% CO₂ incubator at 37 °C for 48 hours, after which the transfection efficiency was tested.

Construction of the animal model

Fifty male Sprague-Dawley (SD) rats aged 8 weeks and weighing 200 to 220 g were obtained from the Animal Experiment Center of Kunming Medical University. Upon arrival, the rats were acclimatized for one week. The following five distinct experimental groups were used (n = 10/group): The control group, OA group, OA + Exos, OA + miR-199a-3p mimic-Exos, and OA + miR-199a-3p inhibitor-Exos. The rats were anesthetized with 2.5% isoflurane and injected with 50 μL of 20 mg/mL sodium iodoacetate (MIA; Sigma-Aldrich, MO, United States) through the patellar ligament into the internal space of the right knee joint using a 26.5 G needle to induce OA. The control group received injections of physiological saline. For the OA + Exos, OA + miR-199a-3p mimic-Exos, and OA + miR-199a-3p inhibitor-Exos groups, OA group rats received intra-articular injections of hUC-MSC-Exos (40 μg/100 μL) weekly starting one week after MIA injection[26]. The rats were sacrificed via cervical dislocation 6 weeks after the operation. The articular cartilage tissues of the rats were collected for the determination of related indices.

Western blot analysis

To obtain total protein, hUC-MSCs, CHON-001 cells, and rat articular cartilage tissues were lysed with RIPA buffer (Sigma-Aldrich, MO, United States) containing 1% protease and phosphatase inhibitors. The concentration of the extracted proteins was determined using a bicinchoninic acid assay kit (Thermo Scientific, NY, United States) according to the manufacturer’s instructions. Following protein quantification, the isolated total proteins were separated via 12% sodium-dodecyl sulfate gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, MA, United States). To prevent nonspecific binding, the membranes were blocked at room temperature for 1.5 hours with a 5% solution of skim milk powder. When using phosphoantibodies, the membranes were blocked with 5% skim milk in Tris-borate-sodium Tween-20 buffer for to reduce non-specific binding. The membranes were incubated overnight at 4 °C with the following diluted primary antibodies: CD81 (1:2000), CD63 (1:1000), Bcl-2 (1:1000), Bax (1:1000), p-NF-κB p65 (1:1000), MAPK4 (1:1000), and β-actin (1:1000). The membranes were then incubated with a secondary antibody diluted (1:4000, ab97051, Abcam, United Kingdom) for 1 hour. The membranes were subsequently developed via an enhanced chemiluminescence kit (Millipore, MA, United States). Finally, the resulting protein bands were semiquantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).

Quantitative real-time polymerase chain reaction

TRIzol reagent (Invitrogen, Grand Island, NY, United States, catalog number 15596026) was used to extract total RNA from hUC-MSCs, CHON-001 cells, and rat articular cartilage tissues. To produce cDNA, the RNA was reverse transcribed with a first-strand cDNA synthesis kit (Genenode, China). A SYBR Green Real-Time polymerase chain reaction Kit (Solarbio, China) was used for real-time polymerase chain reaction analysis. The results were determined using the 2^-ΔΔCt method, with U6 and β-actin used as the internal controls. The primer sequences are shown in Table 1.

Table 1 Primer sequences.

Genes	Sequence
miR-199a-3p (Hum & Mmu)	F: 5’-GCGCGACAGTAGTCTGCACAT-3’
	R: 5’-AGTGCAGGGTCCGAGGTATT-3’
MAPK4	F: 5’-TGAGAAGGGTGACTGCATCG-3’
	R: 5’-ACCAAACCATTGACACCGAAG-3’
U6	F: 5’-CTCGCTTCGGCAGCACA-3’
	R: 5’-AACGCTTCACGAATTTGCGT-3’
β-actin	F: 5’-CATGTACGTTGCTATCCAGGC-3’
	R: 5’-CTCCTTAATGTCACGCACGAT-3’

F: Forward primer; R: Reversed primer; MAPK4: Mitogen-activated protein kinase 4.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor (TNF)-α (SEKH-0047 or SEKR-0009, Solarbio), IL-8 (SEKH-0016 or SEKR-0014, Solarbio), and IL-6 (SEKH-0013 or SEKR-0005, Solarbio) were utilized in the present study. Briefly, CHON-001 cell culture medium was transferred to a sterile centrifuge tube and centrifuged at 1000 × g for 10 minutes at 4 °C. An equal volume of the supernatant was subsequently transferred into small EP tubes designated for testing. Rat articular cartilage tissues were minced, and an appropriate volume of PBS (weight-to-volume ratio of 1:9) was added to a glass homogenizer for thorough grinding on ice. To increase tissue and cell lysis, the homogenate was subjected to multiple freeze-thaw cycles or ultrasonic disruption. The homogenate was centrifuged at 5000 × g for 10 minutes at 4 °C, and the supernatant was carefully collected. The working solutions were incorporated according to the manufacturer’s instructions, and the levels of cytokines (TNF-α, IL-8, and IL-6) were measured using a microplate reader (BioTek, Biotek Winooski, VT, United States) at an optical density of 450 nm.

CCK-8

For the CCK-8 assay, 5 × 10³ cells per well were seeded into 96-well plates and incubated at 37 °C for 24 hours in a 5% CO₂. After this initial incubation period, the cells were subjected to treatments as indicated. A total of 10 μL of CCK-8 reagent (C0037, Beyotime, China) was added to each well after treatment, followed by incubation for 2 hours. An automatic microplate reader was used to measure the absorbance of each well.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling staining

Following the manufacturer’s protocol, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL) staining was performed using a detection kit from Beyotime (China). The paraffin-embedded sections of articular cartilage tissue were dewaxed with xylene and then washed with ethanol. CHON-001 cells were fixed for 30 minutes in a 4% paraformaldehyde solution. The samples were treated with proteinase K at 100 g/mL, washed, and incubated with 3% hydrogen peroxide. The sections were permeabilized with 0.5% Triton X-100, washed, incubated with the TUNEL reaction mixture, and analyzed via fluorescence microscopy (400857, NiKon, Tokyo, Japan).

Dual-luciferase assay

A bioinformatics platform (http://starbase.sysu.edu.cn/) was used to identify miR-199a-3p-binding sites and MAPK4-binding sites. The 3’-untranslated region of MAPK4, which contains a miR-199a-3p-binding site, was cloned and inserted into the pGL3 vector (Promega, WI, United States), resulting in a wild-type (WT) MAPK4 construct. The MAPK4 vector mutants (MUTs) were generated utilizing a site-directed mutagenesis kit (Stratagene, CA, United States). Using Lipofectamine 3000, 293T cells (CL-0005, Procell, Wuhan, China) were cotransfected with the WT or MUT vector and the miR-199a-3p mimic or NC mimic. After 48 hours, the luciferase activity was evaluated via a dual-luciferase reporter assay (Promega, WI, United States).

RNA pull-down assay

GenePharma generated a biotinylated Bio-miR-199a-3p probe using a biotin RNA labeling solution. The probe was mixed with streptavidin agarose beads (PierceTM) and incubated at 4 °C overnight. Next, an RNase inhibitor was added to the cell lysate, followed by incubation on ice for 1 hour. The eluted proteins were analyzed via western blot analysis.

Histological analysis

The articular cartilage tissues of rats were placed in 4% paraformaldehyde for one week, then decalcified in a room temperature shaking table for one month. The tissue was then embedded in paraffin and cut into 5 μm slices. The sections were then deparaffinized in xylene and rehydrated with successive ethanol washes. The sections were then stained with hematoxylin and eosin (Solarbio, China), toluidine blue (Dingguo, China), and Safranin O/Fast Green (Solarbio, China). Finally, a light microscope (Eclipse 80i, Nikon, NY, United States) was used to observe the samples.

Immunohistochemistry

Paraffin-embedded articular cartilage tissues were deparaffinized and rehydrated. An antigen retrieval procedure was then performed using 0.01 M citrate buffer adjusted to a pH of 6.0. The slides were incubated with a MAPK4 antibody (1:200, 18738-1-AP, Proteintech, China) overnight at 4 °C. Immunodetection was then performed with an HRP secondary antibody in combination with a DAB chromogenic reagent. A fluorescence microscope (400857, Nikon, Japan) was used to examine the tissues.

Bioinformatic analysis

The limma algorithm was used to analyze the miRA gene expression data of the GSE213070 dataset for OA. The linear model was then established using the lmFit and eBayes functions, and the statistics were calculated. For visualization of the data, we utilized the ggplot2 package to plot the volcano plot to show the significance and magnitude of the change in miRNA expression. Moreover, pandas was used in Python to import data, and the seaborn library was used for heatmap generation.

Statistical analysis

GraphPad Prism software (Version 8, San Diego, CA, United States) was used to analyze all the data. Each experiment was repeated at least three times. Analysis of variance (ANOVA) and t tests were used to determine the statistical significance. A P value less than 0.05 was considered statistically significant.

RESULTS

hUC-MSC-Exos relieve IL-1β-induced inflammation and apoptosis in chondrocytes

To investigate the effects of the hUC-MSC-Exos on inflammation and apoptosis in chondrocytes, the hUC-MSC-Exos were isolated. TEM revealed that the purified hUC-MSC-Exos exhibited a typical exosomal bilayer ultrastructure with a complete structure and round vesicles (Figure 1A). The particle sizes of the hUC-MSC-Exos ranged from 50 nm to 150 nm according to nanoparticle tracking analysis (Figure 1B). Moreover, the levels of the CD63 and CD81 surface markers were significantly greater in the exosomes than in the hUC-MSCs (Figure 1C). The hUC-MSC-Exos were also labeled with PKH67. The results revealed that CHON-001 cells internalized the hUC-MSC-Exos (Figure 1D). After successful exosome extraction, we cocultured hUC-MSC-Exos with IL-1β-treated CHON-001 cells to observe their effects on IL-1β-induced chondrocyte inflammation and apoptosis. The ELISA results revealed that IL-1β treatment significantly promoted the expression of IL-6, IL-8, and TNF-α, and further addition of the hUC-MSC-Exos inhibited the expression of IL-6, IL-8, and TNF-α (Figure 1E). Compared with the control, IL-1β treatment significantly reduced CHON-001 viability and increased apoptosis; nonetheless, treatment with hUC-MSC-Exos alleviated the effects of IL-1β (Figure 1F and G). Compared with the control, treatment with IL-1β significantly decreased Bcl-2 levels while simultaneously increasing Bax levels. Furthermore, the subsequent addition of hUC-MSC-Exos resulted in an increase in Bcl-2 expression and a reduction in Bax expression (Figure 1H). These results indicate that hUC-MSC-Exos alleviate IL-1β-induced inflammation and apoptosis in CHON-001 cells. After the therapeutic effect of the hUC-MSC-Exos was clarified, the specific mechanism of action was further studied.

Figure 1 Human umbilical cord mesenchymal stem cell-derived exosomes relieve interleukin-1β-induced inflammation and apoptosis in chondrocytes. A: Transmission electron microscopy images of the human umbilical cord mesenchymal stem cell-derived exosomes (scale bar = 100 nm); B: Particle size distribution of the purified human umbilical cord mesenchymal stem cell-derived exosomes determined by nanoparticle tracking analysis; C: Detection of CD63 and CD81 exosome surface markers by western blot analysis; D: PKH-67 tracking experiments verified the uptake of exosomes by CHON-001 cells (scale bar = 10 μm); E: Enzyme-linked immunosorbent assay was used to detect interleukin-6, interleukin-8, and tumor necrosis factor-α in CHON-001 cells; F: CCK-8 detection of CHON-001 cell viability; G: Apoptosis of CHON-001 cells detected via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling staining (scale bar = 100 μm); H: In CHON-001 cells, Bcl-2 and Bax expression levels were detected by western blot analysis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. hUC-MSCs: Human umbilical cord mesenchymal stem cells; Exos: Exosomes; DAPI: 4’,6-diamidino-2-phenylindole; IL: Interleukin; TNF: Tumor necrosis factor; OD: Optical density; TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling.

hUC-MSC-Exos relieve IL-1β-induced chondrocyte inflammation and apoptosis through the delivery of miR-199a-3p

Next, the effects of hUC-MSC-Exos on chondrocyte inflammation and apoptosis through the delivery of factors were investigated. We analyzed the differentially expressed miRNAs from the GSE213070 NCBI database OA miRA gene expression dataset and found that miR-199a-3p was under expressed in OA (Figure 2A). Real-time reverse transcriptase-polymerase chain reaction revealed that the expression of miR-199a-3p in CHON-001 cells was downregulated in the IL-1β group compared with that in the control group and that the expression of miR-199a-3p was restored by further addition of the hUC-MSC-Exos (Figure 2B). Moreover, compared with that in hUC-MSCs, a greater abundance of miR-199a-3p was observed in hUC-MSC-Exos (Figure 2C). We transfected the miR-199a-3p mimic or miR-199a-3p inhibitor into hUC-MSCs, and the expression of these genes was significantly upregulated in the miR-199a-3p mimic group and significantly downregulated in the miR-199a-3p inhibitor group, indicating successful transfection (Figure 2D). Next, exosomes from hUC-MSCs in which miR-199a-3p was overexpressed or knocked down were cocultured with CHON-001 cells. Compared with that in the IL-1β group, the addition of the hUC-MSC-Exos significantly promoted the expression of miR-199a-3p in the CHON-001 cells, the expression of miR-199a-3p was greater when it was added to the hUC-MSC-Exos overexpressing miR-199a-3p, and the expression of miR-199a-3p was reduced by the addition of the hUC-MSC-Exos with miR-199a-3p inhibitor (Figure 2E). The ELISA results revealed that, compared with those in the IL-1β group, the levels of IL-6, IL-8, and TNF-α were lower in the miR-199a-3p mimic-Exo group. Moreover, treatment with exosomes containing the miR-199a-3p inhibitor decreased the expression levels of IL-6, IL-8, and TNF-α, but this effect was not significant (Figure 2F). Additionally, compared with the effects in the IL-1β group, the addition of the miR-199a-3p mimic-Exos significantly increased the viability and inhibited the apoptosis of CHON-001 cells, whereas the effect of the miR-199a-3p inhibitor-Exos was weakened (Figure 2G and H). Compared with the IL-1β group, the miR-199a-3p mimic-Exo group presented increased Bcl-2 expression but reduced Bax levels. Conversely, treatment with miR-199a-3p inhibitor-Exos diminished this response (Figure 2I). These findings demonstrated that hUC-MSC-Exos containing miR-199a-3p attenuate the inflammation and apoptosis of CHON-001 cells induced by IL-1β.

Figure 2 Human umbilical cord mesenchymal stem cell-derived exosomes relieve interleukin-1β-induced chondrocyte inflammation and apoptosis through the delivery of miR-199a-3p. A: Differential expression of microRNAs (miRNAs) in osteoarthritis was analyzed by heat and volcano maps; B: The expression levels of miR-199a-3p in CHON-001 cells were assessed via quantitative real-time polymerase chain reaction (qRT-PCR); C: The expression levels of miR-199a-3p in human umbilical cord mesenchymal stem cells and exosomes was detected by qRT-PCR; D: The efficiency of miR-199a-3p transfection was assessed in human umbilical cord mesenchymal stem cells via qRT-PCR; E: QRT-PCR analysis was conducted to determine the expression levels of miR-199a-3p in CHON-001 cells; F: An enzyme-linked immunosorbent assay was used to measure the interleukin-6, interleukin-8, and tumor necrosis factor-α levels in CHON-001 cells; G: The survival rate of CHON-001 cells was evaluated via CCK-8; H: Apoptosis was assessed through terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling staining (scale bar = 100 μm); I: The levels of the Bcl-2 and Bax apoptosis-related proteins were detected in CHON-001 cells using western blot analysis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. hUC-MSCs: Human umbilical cord mesenchymal stem cells; Exos: Exosomes; NC: Normal control; NC mimic: Negative control mimic; NC inhibitor: Negative control inhibitor; DAPI: 4’,6-diamidino-2-phenylindole; OD: Optical density; IL: Interleukin; TNF: Tumor necrosis factor; TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling.

Activation of the NF-κB signaling pathway attenuates the protective effect of hUC-MSC-Exos on chondrocytes

The mechanism by which hUC-MSC-Exo-derived miR-199a-3p affects chondrocytes was next investigated. Notably, the NF-κB signaling pathway is associated with both the differentiation of chondrocytes and the degradation of cartilage[22], and it affects chondrocyte apoptosis and inflammation[23]. Therefore, the expression levels of NF-κB were evaluated. Compared with the control, treatment with IL-1β increased the levels of p-NF-κB p65. However, the expression of p-NF-κB p65 decreased after treatment with the hUC-MSC-Exos (Figure 3A). To further validate the role of the NF-κB signaling pathway, CHON-001 cells were treated with PMA, an activator of NF-κB. Compared with those in the group treated with IL-1β + Exos, the expression levels of IL-6, IL-8, and TNF-α were increased in the group treated with PMA (Figure 3B). In addition, compared with the IL-1β + Exos group, treatment with PMA decreased the viability and increased the apoptosis rates of CHON-001 cells (Figure 3C and D). Moreover, compared with treatment with IL-1β + Exos, treatment with PMA significantly decreased Bcl-2 levels and increased Bax expression (Figure 3E). These results indicate that exosomes derived from hUC-MSCs reduce the inflammation and apoptosis induced by IL-1β in CHON-001 cells through the inhibition of the NF-κB signaling pathway.

Figure 3 Activation of the nuclear factor-kappaB signaling pathway attenuates the protective effect of human umbilical cord mesenchymal stem cell-derived exosomes on chondrocytes. A: P-nuclear factor-kappaB p65 expression in CHON-001 cells was determined using western blot analysis; B: Interleukin-6, interleukin-8, and tumor necrosis factor-α levels in CHON-001 cells were determined via enzyme-linked immunosorbent assay; C: A CCK-8 assay was used to assess the viability of CHON-001 cells; D: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling staining was used to detect apoptosis in CHON-001 cells (scale bar = 100 μm); E: Bcl-2 and Bax apoptosis-related proteins in CHON-001 cells were detected via western blot analysis. ^bP < 0.01, ^cP < 0.001. NF-κB: Nuclear factor-kappaB; IL: Interleukin; TNF: Tumor necrosis factor; Exos: Exosomes; PMA: Phorbol 12-myristate 13-acetate; DAPI: 4’,6-diamidino-2-phenylindole; OD: Optical density; TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling.

MiR-199a-3p inhibits the activation of the NF-κB signaling pathway by targeting MAPK4

To better understand miR-199a-3p and NF-κB signaling, the downstream targets associated with both miR-199a-3p and molecules linked to NF-κB were screened. First, a literature review revealed that MAPK4 is connected to these two pathways[27,28]. We further examined the expression of MAPK4 mRNA and protein in CHON-001 cells and found that, compared with control conditions, IL-1β treatment significantly promoted the expression of MAPK4 mRNA and protein, whereas hUC-MSCs-Exo treatment reversed this phenomenon (Figure 4A and B). Next, StarBase analysis revealed that miR-199a-3p has a targeted binding site for the NF-κB signaling pathway-related protein MAPK4 (Figure 4C). Dual-luciferase reporter assays and RNA pull-down assays verified that miR-199a-3p can target and modulate the expression levels of MAPK4 (Figure 4D and E). In CHON-001 cells, miR-199a-3p was then overexpressed and knocked down (Figure 4F); overexpression of miR-199a-3p inhibited the expression of MAPK4 mRNA and protein and p-NF-κB p65, whereas knockdown of miR-199a-3p promoted the expression of MAPK4 mRNA and protein and p-NF-κB p65 (Figure 4G and H). Finally, MAPK4 was overexpressed in CHON-001 cells (Figure 4I), and compared with the IL-1β + miR-199a-3p mimic, the overexpression of MAPK4 significantly promoted the expression of p-NF-κB p65 (Figure 4J). These findings suggest that miR-199a-3p suppresses the NF-κB signaling pathway by reducing the expression of MAPK4.

Figure 4 MiR-199a-3p inhibits the activation of the nuclear factor-kappaB signaling pathway by targeting mitogen-activated protein kinase 4. A: The level of mitogen-activated protein kinase 4 (MAPK4) mRNA in CHON-001 cells was measured via quantitative real-time polymerase chain reaction; B: Western blot analysis was used to evaluate MAPK4 protein expression in CHON-001 cells; C: StarBase (http://starbase.sysu.edu.cn/) was used to predict the specific binding sites of miR-199a-3p and MAPK4; D: Dual-luciferase reporter assays validated the interaction between miR-199a-3p and MAPK4; E: The interaction of miR-199a-3p with MAPK4 was confirmed by RNA pull-down assays; F: Quantitative real-time polymerase chain reaction was used to assess the miR-199a-3p transfection efficiency in CHON-001 cells; G: The expression levels of MAPK4 mRNA in CHON-001 cells were evaluated via quantitative real-time polymerase chain reaction; H: Western blot analysis was conducted to evaluate the expression levels of MAPK4 and p-nuclear factor-kappaB p65 in CHON-001 cells; I: Western blot analysis was used to assess the effectiveness of MAPK4 transfection in CHON-001 cells; J: Western blot analysis was used to examine the expression levels of p-nuclear factor-kappaB p65 in CHON-001 cells. ^bP < 0.01, ^cP < 0.001. MAPK4: Mitogen-activated protein kinase 4; IL: Interleukin; Exos: Exosomes; NC: Normal control; NC mimic: Negative control mimic; NC inhibitor: Negative control inhibitor; OE-NC: Overexpression negative control; OE-MAPK4: Overexpression mitogen-activated protein kinase 4; NF-κB: Nuclear factor-kappaB.

Overexpression of MAPK4 attenuates the effects of miR-199a-3p on chondrocyte inflammation and apoptosis

To further confirm whether miR-199a-3p affects chondrocyte inflammation and apoptosis by regulating MAPK4 expression. First, western blot analysis of MAPK4 expression revealed that miR-199a-3p mimic transfection significantly inhibited the expression of MAPK4, but this effect was reversed by the overexpression of MAPK4 (Figure 5A). ELISA revealed that the levels of IL-6, IL-8, and TNF-α were lower in the IL-1β + miR-199a-3p mimic group than in the IL-1β group. Moreover, overexpression of MAPK4 significantly increased the levels of IL-6, IL-8, and TNF-α (Figure 5B). Compared with the IL-1β group, the overexpression of miR-199a-3p increased the survival rate but reduced the apoptosis rate of CHON-001 cells. Meanwhile, overexpression of MAPK4 partially alleviated the impact of miR-199a-3p overexpression (Figure 5C and D). Compared with the IL-1β group, the miR-199a-3p-overexpressing group increased Bcl-2 expression but reduced Bax expression. Furthermore, MAPK4 overexpression partially attenuated the effects of miR-199a-3p overexpression (Figure 5E). These findings suggest that miR-199a-3p inhibits MAPK4 expression, which subsequently decreases the inflammation and apoptosis triggered by IL-1β in CHON-001 cells.

Figure 5 Overexpression of mitogen-activated protein kinase 4 attenuates the effects of miR-199a-3p on inflammation and apoptosis in chondrocytes. A: Mitogen-activated protein kinase 4 expression was detected by western blot; B: Interleukin-6, interleukin-8, and tumor necrosis factor-α expression in CHON-001 cells was detected via enzyme-linked immunosorbent assay; C: A CCK-8 assay was used to measure the viability of CHON-001 cells; D: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling staining was used to measure the degree of CHON-001 cell apoptosis (scale bar = 100 μm); E: Western blot analysis was used to detect Bcl-2 and Bax apoptosis-related proteins in CHON-001 cells. ^bP < 0.01, ^cP < 0.001. MAPK4: Mitogen-activated protein kinase 4; IL: Interleukin; OE-MAPK4: Overexpression mitogen-activated protein kinase 4; DAPI: 4’,6-diamidino-2-phenylindole; OD: Optical density; TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling.

hUC-MSC-Exos relieve OA progression in vivo through the delivery of miR-199a-3p

To verify the in vitro findings, the present study next investigated whether the delivery of miR-199a-3p by hUC-MSC-Exos mitigates the progression of OA in an animal model. Histological evaluations revealed that the joint architecture of the control rats remained intact, with a smooth surface, an orderly arrangement of chondrocytes, maintenance of chondrocyte numbers, and a matrix abundant in proteins. In contrast, the joint architecture of rats affected by OA was significantly disrupted, which was characterized by surface roughness and extensive areas devoid of chondrocytes. Furthermore, treatment with either normal or miR-199a-3p-overexpressing hUC-MSC-Exos improved the histopathological state of the rat articular cartilage, whereas the injection of miR-199a-3p-inhibitor hUC-MSC-Exos resulted in minimal improvement (Figure 6A-C). ELISA revealed that the levels of IL-6, IL-8, and TNF-α in the OA group were significantly greater than those in the control group. Furthermore, treatment with either the hUC-MSC-Exos or the miR-199a-3p mimic hUC-MSC-Exos significantly decreased the levels of inflammatory cytokines. Conversely, the injection of miR-199a-3p-inhibitor hUC-MSC-Exos diminished this inhibitory effect (Figure 6D). TUNEL staining revealed that rats with OA had significantly higher rates of apoptosis in their articular cartilage tissues than did normal rats. Furthermore, treatment with either hUC-MSC-Exos or miR-199a-3p mimic hUC-MSC-Exos significantly decreased cell apoptosis. Nevertheless, treatment with miR-199a-3p inhibitor hUC-MSC-Exos reduced the inhibitory effect (Figure 6E). Finally, compared with those in the control group, the expression levels of Bcl-2 and miR-199a-3p in the articular cartilage of OA rats were significantly lower, whereas the expression levels of Bax, MAPK4 and p-NF-κB p65 were significantly greater. Injection of the normal or miR-199a-3p mimic of the hUC-MSC-Exos reversed this phenomenon, whereas injection of the miR-199a-3p inhibitor of the hUC-MSC-Exos did not significantly reverse this phenomenon (Figure 6F-I). These findings suggest that hUC-MSC-Exos contribute to slowing the progression of OA in rats by delivering miR-199a-3p.

Figure 6 Human umbilical cord mesenchymal stem cell-derived exosomes relieve osteoarthritis progression in vivo through the delivery of miR-199a-3p. A: Hematoxylin and eosin staining was used to examine the pathological conditions of rat articular cartilage tissues (scale bar = 25 μm); B: Safranin O/fast green staining was used to assess the quantity of chondrocytes within rat articular cartilage tissues (scale bar = 25 μm); C: Examination of cartilage differentiation in rat articular cartilage tissues via toluidine blue staining (scale bar = 25 μm); D: Interleukin-6, interleukin-8, and tumor necrosis factor-α levels in rat articular cartilage tissue were detected via enzyme-linked immunosorbent assay; E: Evaluation of cell apoptosis in rat articular cartilage tissue via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling staining (scale bar = 100 μm); F: Western blot analysis was used to measure the expression of Bcl-2 and Bax apoptosis-related proteins; G: Quantitative real-time polymerase chain reaction was used to quantify miR-199a-3p expression in rat cartilage tissues; H: Immunohistochemistry was used to determine the presence of mitogen-activated protein kinase 4 in rat articular cartilage tissues (scale bar = 25 μm); I: P-nuclear factor-kappaB p65 expression in rat articular cartilage tissues was evaluated through western blot analysis. ^bP < 0.01, ^cP < 0.001. OA: Osteoarthritis; Exos: Exosomes; HE: Hematoxylin and eosin; TB: Toluidine blue; SOFG: Safranin O/fast green; NF-κB: Nuclear factor-kappaB; IL: Interleukin; TNF: Tumor necrosis factor; DAPI: 4’,6-diamidino-2-phenylindole; TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling.

DISCUSSION

OA represents the most common degenerative joint disorder, affecting approximately 250 million people[29,30]. In OA, chondrocyte homeostasis is disrupted, resulting in cartilage damage, and the depletion of chondrocytes is proposed as a potential mechanism contributing to the pathology of this condition[9]. The existing approaches for OA treatment are restricted and fail to halt the onset and progression of the condition. Consequently, devising new therapeutic strategies is essential and imperative. In 2003, Murphy et al[31] administered MSCs derived from goat bone marrow into an OA goat model and reported alleviation of both meniscus damage and the OA phenotype. This study prompted the incorporation of stem cell treatment into the field of OA research. Owing to the challenges posed by limited resources and the technical complexities involved in acquiring cells from bone marrow, researchers are progressively redirecting their attention toward specific alternatives, such as adipose-derived stem cells, which can be harvested before cultivation[32]. Adipose-derived stem cells harvested from other organs can safeguard cartilage and mitigate the progression of OA[33], primarily by increasing the survival rates of articular chondrocytes and improving joint functionality in individuals suffering from OA[34]. The umbilical cord serves as a remarkable source of MSCs because of its affordability, wide acceptability, and universal availability[35]. Compared with other types of MSCs, hUC-MSCs present certain biological advantages, including primordial qualities, proliferative capabilities, and immunosuppressive characteristics[36,37]. Exosomes released by MSCs are key molecules via which stem cells exert their therapeutic effects. These exosomes are actively involved in intercellular signaling processes, particularly influencing target cells, modulating the microenvironment, modulating immune responses related to inflammation, and facilitating the repair of injured tissues[38]. Jiang et al[39] reported that hUC-MSC-Exos may improve the efficacy of decellularized cartilage extracellular matrix scaffolds and stimulate osteochondral regeneration. Wang et al[14] suggested that hUC-MSC-Exos inhibit the inflammatory response in chondrocytes caused by IL-1β or M1 macrophages, thereby reducing the progression of OA. The present study revealed that CHON-001 cells effectively take up hUC-MSC-Exos, which subsequently decreases the inflammation and apoptotic processes induced by IL-1β in these cells. These results align with findings from earlier studies and further support the idea that hUC-MSC-Exos may have therapeutic potential for OA.

Exosomes carry mRNAs, miRNAs, long noncoding RNAs (lncRNAs), and various other signaling molecules, which can be released into the extracellular environment through autocrine and paracrine mechanisms, triggering biological responses in target cells[40]. For example, Li et al[41] reported that hUC-MSC-Exos reduce apoptosis in human articular chondrocytes and diminish reactive oxygen species production by delivering miR-100-5p, thereby inhibiting articular chondrocyte damage and OA progression. The primary focus of the present study was the influence of miRNAs from hUC-MSC-Exos on chondrocyte regulation. Bioinformatics techniques and quantitative real-time polymerase chain reaction analysis revealed that miR-199a-3p expression was reduced in OA, whereas its levels were significantly increased in hUC-MSC-Exos. Gu and Xie[42] reported that the deletion of the lncRNA AC005165.1 increases IL-1β-induced apoptosis and inflammation in chondrocytes through the miR-199a-3p/TXNIP signaling pathway, thus exacerbating the progression of OA. Importantly, Zhao et al[43] reported that exosomes derived from subcutaneous adipose MSCs deliver miR-199a-3p to chondrocytes, leading to a reduction in chondrocyte damage and an increase in cartilage repair, which ultimately facilitates the management of OA. Therefore, we explored whether hUC-MSC-Exos function by delivering miR-199a-3p. We found that hUC-MSC-Exos overexpressing miR-199a-3p significantly inhibited the inflammation and apoptosis of CHON-001 cells and improved the pathology of articular cartilage tissue in rats in vivo, whereas the effect of hUC-MSC-Exos with miR-199a-3p knockdown was greatly reduced. On the basis of previous studies, we demonstrated for the first time that hUC-MSC-Exos alleviate IL-1β-induced chondrocyte inflammation and apoptosis by delivering miR-199a-3p.

Numerous studies on the regulatory mechanisms associated with miR-199a-3p have proposed that various signaling pathways, including the Wnt/β-catenin, phosphatidylinositol 3-kinase/protein kinase B, and NF-κB pathways, contribute to the onset and progression of OA[44,45]. By modulating cartilage matrix metabolism and maintaining chondrocyte phenotypes, the NF-κB signaling pathway plays a key role in the progression of OA[23,46]. Xue et al[47] reported that the inhibition of the IL-1β-induced activation of the NF-κB signaling pathway by LDC067 in chondrocytes offers a protective advantage against OA. Wu et al[48] reported that sinomenine inhibits NF-κB signaling in mouse chondrocytes induced by IL-1β, reducing inflammation and cartilage damage. Similarly, Wang et al[49] reported that by suppressing the NF-κB signaling pathway, curcumin affects collagen II and matrix metalloprotease 13 expression levels in chondrocytes, as well as IL-1β-induced cell proliferation. Moreover, miR-199a-3p is involved in regulating this pathway[25]. Currently research on how miR-199a-3p influences the NF-κB signaling pathway and its effects on OA progression is insufficient. The present study demonstrated that hUC-MSC-Exos reduce p-NF-κB p65 levels in CHON-001 cells and that PMA partially counteracts the inflammatory response and apoptosis caused by the hUC-MSC-Exos. MiR-199a-3p delivery through hUC-MSC-Exos reduces inflammation and apoptosis in CHON-001 cells induced by IL-1β.

Several studies examining the miR-199a-3p and NF-κB signaling pathways have reported that MAPK4 plays a key role. As an atypical MAPK, MAPK4 activates the NF-κB pathway[28], which contributes to the development of different types of tumors[50]. Therefore, the present study evaluated the expression of MAPK4 in OA, which revealed that MAPK4 was elevated both under OA conditions and in CHON-001 cells stimulated with IL-1β, indicating that MAPK4 is highly expressed in OA. In the present study, the StarBase database was used to predict binding sites for miR-199a-3p and MAPK4. Furthermore, miR-199a-3p inhibits the activation of the NF-κB signaling pathway by downregulating MAPK4 expression. Moreover, the overexpression of MAPK4 can partially weaken the inhibitory effect of miR-199a-3p on chondrocyte inflammation and apoptosis. However, Lu et al[27] reported that the inhibition of miR-199a-3p affects chondrocyte apoptosis and inflammation by increasing the expression of MAPK4, thereby worsening OA. This finding contradicts the present findings. This discrepancy may be due to variations in cell sources, culture conditions, and modeling strategies, as well as differences in the methods used to deliver miR-199a-3p.

CONCLUSION

In conclusion, our study revealed that hUC-MSC-Exos downregulated MAPK4 expression by delivering miR-199a-3p, thereby inhibiting the activation of the NF-κB signaling pathway, alleviating IL-1β-induced chondrocyte inflammation and apoptosis, and ultimately improving the development of OA. The present findings offer novel insights and potential targets for OA treatment. However, the present study has several limitations. First, the present study did not verify whether miR-199a-3p delivered by hUC-MSC-Exos alleviates OA symptoms by affecting the MAPK4/NF-κB signaling pathway in vivo, indicating that additional validation is necessary. Second, hUC-MSC-Exos are composed of various active molecules. Future research should explore other potential molecules, such as lncRNAs, that may aid in alleviating OA. Finally, the present discovery was made solely through cellular and animal studies, indicating a shortfall in clinical trials. The therapeutic use of hUC-MSC-derived exosomal miR-199a-3p in managing OA requires further investigation.