Effects and mechanisms of synovial mesenchymal stem cell–derived extracellular vesicular LncRNA-SNHG14 on chondrocyte injury

Abstract

Objective

To investigate the effect of synovial mesenchymal stem cell (SMSC) derived extracellular vesicles (EVs) long non-coding ribonucleic acid (LncRNA) nuclear small RNA host gene 14 (SNHG14) on chondrocyte injury and its potential mechanism.

Methods

EVs of SMSC were isolated and identified by transmission electron microscopy, nanoparticle tracking analysis, Western blot and EVs uptake test. Chondrocytes induced by interleukin-1β (IL-1β) were treated with EVs isolated from SMSCs transfected with sh-NC or sh-SNHG14. The proliferation and apoptosis of rat chondrocytes were detected by CCK-8 test and flow cytometry. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) were detected by ELISA. Western blot was used to detect the expression of apoptosis-related proteins [Bcl-2-related X protein (Bax), cysteine aspartate protease-3 (caspase-3), cytochrome C (Cyt c)], chondrocyte proliferation and regeneration-related proteins [sex-determining region Y-box protein 9 (SOX9), matrix metalloproteinase-13 (MMP-13), collagen type II alpha 1 (COL2A1), basic fibroblast growth factor (bFGF)]. The effect of LncRNA-SNHG14 on high mobility group protein B1 (HMGB1)/Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling pathway was analyzed. By activating the pathway, its effect on the expression of LncRNA-SNHG14 and its effect on the above indicators were analyzed.

Results

LncRNA-SNHG14 in SMSC could be enriched and secreted in EVs, and chondrocytes can successfully uptake EVs. The expression level of LncRNA-SNHG14 in rat chondrocytes induced by IL-1β was increased, the cell survival rate was decreased, the apoptosis rate, TNF-α, IL-6 levels and Bax, caspase-3, Cytc, MMP-13 protein levels were increased, IGF-1 level and SOX9, COL2A1, bFGF protein levels were decreased, HMGB1, TLR4, NF-κB mRNA and protein levels were increased (P < 0.05). SMSC-EVs silencing LncRNA-SNHG14 increased cell survival rate, decreased apoptosis rate and Bax, caspase-3, Cytc, MMP-13 protein levels, and decreased TNF-α, IL-6 levels and HMGB1, TLR4, NF-κB expression levels, increased IGF-1 level and SOX9, COL2A1, bFGF protein levels (P < 0.05); after HMGB1 overexpression, the expression level of LncRNA-SNHG14 increased, the cell survival rate decreased, the apoptosis rate and the protein levels of Bax, caspase-3, Cytc and MMP-13 increased, the levels of IGF-1, SOX9, COL2A1 and bFGF decreased, and the levels of TNF-α and IL-6 increased (P < 0.05).

Conclusion

SMSC-EVs silencing LncRNA-SNHG14 can promote chondrocyte proliferation, inhibit chondrocyte apoptosis and inflammatory response in an in vitro model of IL-1β-induced chondrocyte injury. These effects are associated with reduced expression of HMGB1/TLR4/NF-κB pathway components, suggesting the involvement of this pathway.

1. Introduction

Osteoarthritis (OA) is characterised by progressive cartilage degeneration, synovial inflammation, and osteophyte formation. Articular cartilage is a highly differentiated tissue with very limited self-repair capacity. Currently, no disease-modifying therapies are capable of curing osteoarthritis (OA); most available treatments primarily relieve pain rather than restore normal cartilage function. Therefore, effective cartilage repair remains a central goal in OA management [1]. Synovial mesenchymal stem cells (SMSCs), which possess self-renewal and multilineage differentiation potential, have emerged as a promising cell-based strategy for tissue repair. Their therapeutic effects are largely mediated by extracellular vesicles (EVs), which deliver bioactive molecules to recipient cells, thereby promoting chondrocyte proliferation, inhibiting cartilage degeneration, and coordinating subsequent regenerative processes; SMSC-derived EVs are closely associated with OA and have been shown to enhance chondrocyte regeneration and improve extracellular matrix homeostasis [2].Cartilage injury and degeneration represent the core pathological basis of OA, and identifying key regulatory genes involved in chondrocyte damage is therefore critical for the development of effective therapeutic strategies. EVs contain a wide range of functional non-coding RNAs, among which long non-coding RNAs (lncRNAs) play pivotal roles in OA pathogenesis and participate directly in the process of chondrocyte injury [3,4].Long non-coding RNA small nucleolar RNA host gene 14 (LncRNA-SNHG14) is involved in chromatin remodelling and transcriptional regulation, modulates inflammatory responses, and has been implicated in neuronal injury, as well as in fundamental cellular processes such as proliferation and apoptosis [5,6]. Previous studies have shown that LncRNA-SNHG14 expression is significantly upregulated in sodium iodoacetate-induced rat chondrocyte injury, and that its suppression can attenuate chondrocyte damage [7]. However, it remains unclear whether LncRNA-SNHG14 exerts its effects in OA through transport via SMSC-derived EVs. Against this background, the present study aims to investigate the role of SMSC-derived EVs LncRNA-SNHG14 in chondrocyte injury and to elucidate its underlying molecular mechanisms. This work seeks to provide new insights into the pathogenesis of osteoarthritis and to offer potential directions for its clinical diagnosis and treatment.

2. Materials and methods

2.1. Experimental cells and major reagents

Primary rat chondrocytes (Catalog No. CP-R087, 5 × 10⁵Cells/T25) and synovial mesenchymal stem cells (SMSC, Catalog No. CP-R304, 5 × 10⁵Cells/T25) were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Both cell types were authenticated by the supplier using short tandem repeat (STR) profiling and tested negative for mycoplasma contamination via PCR-based assay. Cells were used at passage 3 for all experiments to maintain consistency and reproducibility. EVs isolation kits were obtained from Guangzhou Gesea Biotechnology, while HMGB1 overexpression plasmids and corresponding empty vectors were purchased from Shandong Weizhen Biotechnology. Transfection reagents, CCK-8 kits, recombinant human interleukin-1β (IL-1β), and apoptosis detection kits were supplied by Beijing Solarbio Science & Technology Co., Ltd.Enzyme-linked immunosorbent assay (ELISA) kits for tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and insulin-like growth factor-1 (IGF-1) were purchased from Shanghai Enzyme-linked Biotechnology. Primary and secondary antibodies against Bax, caspase-3, cytochrome c (Cyt c), SOX9, MMP-13, collagen type II alpha 1 (COL2A1), and basic fibroblast growth factor (bFGF) were obtained from Abcam (USA), whereas antibodies against HMGB1, Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and the internal control GAPDH were purchased from Santa Cruz Biotechnology (USA).Total RNA extraction kits, protein quantification kits, and enhanced chemiluminescence (ECL) reagents were obtained from Beijing TransGen Biotech. Reverse transcription kits and quantitative real-time PCR (qRT-PCR) kits were purchased from Dalian Takara Bio Inc., and PCR primers were synthesised by Shanghai Sangon Biotech. Short hairpin RNAs targeting SNHG14 (sh-SNHG14) and negative control sequences (sh-NC) were obtained from Shanghai GenePharma Co., Ltd.A SpectraMAX Plus 384 microplate reader was purchased from Molecular Devices (USA), and a PIKORed 96 real-time fluorescence quantitative PCR system was obtained from Thermo Fisher Scientific (USA). Fluorescence inverted microscopy was performed using an Olympus microscope (Japan), nanoparticle tracking analysis was conducted with a ZetaView instrument (Particle Metrix, Germany), and flow cytometry was carried out using a Beckman Coulter flow cytometer.

2.2. Methods

2.2.1. Morphological observation and identification of SMSCs

Synovial mesenchymal stem cells (SMSCs, passage 3) were seeded in culture flasks and maintained in fresh medium, which was replaced every 3 days. When cell confluence reached approximately 70–80%, the cells were digested with 0.25% trypsin and passaged, after which morphological characteristics were examined under an inverted phase-contrast microscope. Flow cytometry was performed to identify SMSCs based on the expression of surface markers CD29, CD34, and CD45.

2.2.2. Isolation of EVs

SMSCs were seeded into six-well plates at a density of 2 × 10⁵ cells per well and cultured in EVs-depleted serum for 48 h. The culture supernatants were collected, and EVs were isolated according to the manufacturer's instructions for the ExoQuick-TC EVs isolation kit. The obtained EVs were resuspended in phosphate-buffered saline (PBS), centrifuged at 3000 r/min for 30 min at 4 °C, and the supernatant was discarded before final resuspension in buffer. The expression of extracellular vesicular marker proteins CD63 and CD81 was analyzed by Western blotting.

2.2.3. Identification of EVs

EVs were diluted in PBS to a concentration of 1 × 10⁷ particles/mL and loaded into the sample chamber of the ZetaView instrument for nanoparticle tracking analysis, where Brownian motion of the particles was recorded. For transmission electron microscopy, a drop of the EVs suspension was placed onto a copper grid and air-dried at room temperature, followed by negative staining with 3% phosphotungstic acid for 2 h and counterstaining with 2% uranyl acetate for 30 s. The grids were then examined and imaged using a transmission electron microscope.

2.2.4. Tracing of EVs uptake by chondrocytes

SMSC-derived EVs were fluorescently labelled using the Exo-Green tracer. Briefly, EVs were resuspended in 500 μL PBS, mixed thoroughly with 60 μL Exo-Green, and incubated at 37 °C for 10 min; the reaction was terminated by adding 90 μL ExoQuick-TC reagent, followed by incubation on ice for 30 min. The mixture was centrifuged at 12,000 r/min for 5 min, the pellet was collected, and the labelled EVs were resuspended in 500 μL PBS. The Exo-Green–labelled EVs were then added to cultured rat chondrocytes, and EVs uptake was observed using a fluorescence imaging system.

2.2.5. Cell treatment and experimental grouping

Chondrocytes cultured under normal conditions for 48 h were designated as the control (Con) group. To establish an in vitro injury model, recombinant human IL-1β was added to chondrocytes to a final concentration of 10 ng/mL [8], followed by incubation for 48 h (IL-1β group).

To evaluate the paracrine effects of SMSCs, IL-1β–stimulated chondrocytes were co-cultured with SMSCs using a Transwell system (0.4 μm pore size) for 48 h, allowing exchange of soluble factors without direct cell–cell contact. This group was defined as the SMSC group.

For EV intervention experiments, SMSCs were first seeded into six-well plates at a density of 1 × 10⁵ cells per well. When cell confluence reached approximately 80%, cells were transfected with sh-NC or sh-SNHG14 using Lipofectamine 3000. After 6 h, the medium was replaced with EV-depleted culture medium (prepared by ultracentrifugation at 120,000×g for 18 h), and cells were cultured for an additional 48 h. The culture supernatants were then collected, and EVs were isolated using the ExoQuick-TC kit according to the manufacturer's protocol. The isolated EVs were resuspended in PBS and quantified for protein concentration using a BCA assay. For treatment, IL-1β–pretreated chondrocytes (10 ng/mL, 48 h) were incubated with 50 μg/mL of EVs (based on total protein) for 48 h. These groups were defined as the IL-1β + sh–NC–EV group and the IL-1β + sh-SNHG14-EV group.

To investigate the role of HMGB1 in chondrocyte injury, SMSCs were seeded into six-well plates (1 × 10⁵cells per well) and transfected at 80% confluence with either empty vector (20 μmol/L) or HMGB1 overexpression plasmid (20 μmol/L) using Lipofectamine 3000. The transfected SMSCs were then co-cultured with IL-1β–induced chondrocytes (10 ng/mL) using a Transwell system for 48 h. These groups were designated as the IL-1β + empty vector group and the IL-1β + HMGB1 overexpression group, respectively.

2.2.6. Quantitative real-time PCR analysis of LncRNA-SNHG14, HMGB1, TLR4, and NF-κB mRNA expression

Chondrocytes from each group described in Section 1.2.5 were harvested, and total RNA was extracted using a commercial RNA isolation kit. Reverse transcription was performed in a 20 μL reaction containing 2 μL RNA, 2 μL 10 × RT buffer, 0.4 μL dNTPs, 1 μL MultiScribe reverse transcriptase, 2 μL 10 × random primers, and RNase-free ddH₂O to volume, followed by cDNA synthesis.Quantitative real-time PCR was conducted according to the manufacturer's instructions using a SYBR Green detection system. Each reaction mixture (20 μL) contained 10 μL SYBR Green Master Mix, 0.8 μL of forward and reverse primers, 1 μL cDNA template, and ddH₂O to volume. The amplification protocol consisted of an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Relative mRNA expression levels of LncRNA-SNHG14, HMGB1, TLR4, and NF-κB were calculated using the 2^−ΔΔCt method, with GAPDH serving as the internal reference.The primer sequences were as follows: LncRNA-SNHG14 forward, 5′-GGGTGTTTACGTAGACCAGAACC-3′, and reverse, 5′-CTTCCAAAAGCCTTCTGCCTTAG-3’; GAPDH forward, 5′-ATCTGAGCAGGTTGCTCCAC-3′, and reverse, 5′-GGCCCTTTACACTGTGAGCC-3’; HMGB1 forward, 5′-AAAGGAGATCCTAAGAAGCCGA-3′, and reverse, 5′-TCATAACGAGCCTTGTCAGCC-3’; TLR4 forward, 5′-CCAGGTGTGAAATTGAGACAATTG-3′, and reverse, 5′-AAGCTGTCCAATATGGAAACCC-3’; and NF-κB forward, 5′-CAGATACCACTAAGACGCACCC-3′, and reverse, 5′-CTCCAGGTCTCGCTTCTTCACA-3’.

2.2.7. Cell proliferation assay by CCK-8

Chondrocytes from each group described in Section 1.2.5 were seeded into 96-well plates at a density of 8 × 10³ cells per well and incubated at 37 °C for 24 h. Fresh culture medium (100 μL) and CCK-8 solution (10 μL) were then added to each well, followed by incubation at 37 °C for 2 h, after which absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated as follows: experimental group OD value/control group OD value × 100%.

2.2.8. Flow cytometric analysis of apoptosis

Chondrocytes from each group were seeded into six-well plates at a density of 1 × 10⁵ cells per well. Cells were stained with 5 μL Annexin V-FITC and incubated at room temperature for 15 min in the dark, followed by incubation with 10 μL propidium iodide (PI) staining solution for an additional 10 min under light-protected conditions. The proportion of apoptotic cells was then determined by flow cytometry.

2.2.9. Measurement of inflammatory cytokines by ELISA

Culture supernatants from chondrocytes in each group were collected and added to ELISA plates at 100 μL per well. After sealing, the plates were incubated at room temperature for 2 h, followed by incubation with biotinylated antibody solution (100 μL per well) for 1 h at room temperature. The plates were then washed, incubated with horseradish peroxidase–conjugated streptavidin for 20 min, and developed with tetramethylbenzidine (TMB) substrate for 20 min before the reaction was terminated. Absorbance was measured at 450 nm, and the concentrations of TNF-α, IL-6, and IGF-1 were calculated according to the standard curves.

2.2.10. Western blot analysis

Chondrocytes from each group were lysed to extract total cellular protein, and protein concentrations were determined prior to analysis. Proteins were separated by SDS-PAGE at 80 V for 30 min followed by 120 V for 90 min, then transferred onto polyvinylidene difluoride (PVDF) membranes using a wet transfer system at 300 mA for 90 min.After transfer, membranes were blocked at room temperature for 1 h and incubated at 4 °C for 24 h with primary antibodies against Bax, caspase-3, cytochrome c (Cyt c), SOX9, MMP-13, COL2A1, bFGF, HMGB1, TLR4, and NF-κB (all diluted 1:1000), with GAPDH used as an internal loading control (1:1000). Membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies (1:5000) at room temperature for 1 h, and protein bands were visualised using an enhanced chemiluminescence (ECL) detection system. Band intensities were quantified by densitometric analysis using a gel imaging system.

2.3. Statistical analysis

Statistical analyses were performed using SPSS version 25.0. All quantitative data are presented as the mean ± standard deviation (±s). Normality of data distribution was assessed using the Shapiro–Wilk test, and all data in this study conformed to a normal distribution (P > 0.05). Comparisons between two groups were conducted using the independent-samples t-test, while comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) t-test for pairwise comparisons. The LSD test was chosen because it is appropriate for pre-specified comparisons with equal group sizes and homogeneous variances, and its use is consistent with established practice in exploratory mechanistic studies. All experiments were performed with nine independent biological replicates (n = 9), meaning that each replicate represented an independently cultured and treated sample. A P value < 0.05 was considered statistically significant.

3. Results

3.1. Identification of EVs and verification of the SMSC phenotype

Morphological observation showed that the cultured cells were closely arranged, exhibited a uniform appearance, and displayed a typical spindle-shaped morphology (Fig. 1A). Flow cytometric analysis demonstrated that the isolated cells were highly positive for the MSC marker CD29 (99.2% ± 0.5%), and negative for the hematopoietic lineage markers CD34 (1.1% ± 0.3%) and CD45 (0.9% ± 0.4%). This immunophenotype (CD29⁺CD34–CD45–) confirms the mesenchymal stromal/stem cell identity of the isolated population, consistent with ISCT guidelines (Fig. 1B). Under transmission electron microscopy, SMSC-derived EVs (SMSC-Exo) exhibited a characteristic hemispherical morphology with a concave side (Fig. 2A). ZetaView nanoparticle tracking analysis revealed that the mean diameter of the EVs was approximately 130 nm (Fig. 2B). The protein expression levels of CD63 were (0.46 ± 0.08) in SMSCs and (0.92 ± 0.11) in SMSC-EVs, and those of CD81 were (0.50 ± 0.09) in SMSCs and (0.88 ± 0.13) in SMSC-EVs, respectively. The levels of LncRNA-SNHG14, CD63, and CD81 were significantly higher in SMSC-Exo than in SMSCs (P < 0.05), confirming successful isolation of SMSC-derived EVs and indicating that LncRNA-SNHG14 is enriched and secreted via EVs (Fig. 2C).

Fig. 1.

A: Morphological characteristics of SMSCs observed under an inverted microscope. Representative images from three independent experiments are shown (n = 3) (scale bar = 100 μm). B: Flow cytometric analysis confirming the SMSC phenotype. The cells were highly positive for the mesenchymal marker CD29, and negative for the hematopoietic markers CD34 and CD45. This profile (CD29⁺CD34–CD45–) is consistent with the ISCT criteria for mesenchymal stem cells (n = 3).

Fig. 2.

A: Morphology of SMSC-derived exosomes observed by transmission electron microscopy. Representative image from three independent experiments is shown (n = 3) (scale bar = 100 nm). B: Size distribution and concentration of exosomes analyzed by nanoparticle tracking analysis (n = 3). Data represent mean ± SD from three independent experiments. C: Expression of extracellular vesicular surface marker proteins (n = 3). Molecular weight markers: CD63 ∼26 kDa, CD81 ∼22 kDa, GAPDH ∼37 kDa.

3.2. Effects of LncRNA-SNHG14 silencing on chondrocyte proliferation and apoptosis

Compared with the control (Con) group, the IL-1β group showed a significant increase in LncRNA-SNHG14 expression, accompanied by reduced cell viability and increased apoptosis, as well as elevated protein levels of Bax, caspase-3, and cytochrome c (Cyt c) (P < 0.05). In contrast, relative to the IL-1β group, the SMSC group exhibited lower LncRNA-SNHG14 expression, higher cell viability, and reduced apoptosis, together with decreased expression of Bax, caspase-3, and Cyt c (P < 0.05).When compared with both the SMSC group and the IL-1β + sh–NC–Exo group, the IL-1β + sh-SNHG14-Exo group demonstrated a further reduction in LncRNA-SNHG14 expression, enhanced cell viability, and markedly decreased apoptosis, along with lower levels of Bax, caspase-3, and Cyt c (P < 0.05). No significant differences were observed between the SMSC group and the IL-1β + sh–NC–Exo group across these indices (P > 0.05). These results are summarised in Fig. 3 and Table 1.

Fig. 3.

Effects of LncRNA-SNHG14 silencing on chondrocyte apoptosis. A: Apoptotic rate of chondrocytes. Representative dot plots from three independent experiments are shown (n = 3). B: Protein expression levels of Bax, caspase-3, and Cyt c. Representative blots from three independent experiments are shown (n = 3). Molecular weight markers: Bax ∼21 kDa, caspase-3 ∼35 kDa (pro-form) and ∼17 kDa (cleaved form), Cyt c ∼15 kDa, GAPDH ∼37 kDa.

Table 1.

Effects of LncRNA-SNHG14 silencing on chondrocyte proliferation and apoptosis ($\bar{x}$ ±s, n = 9).

Group	LncRNA-SNHG14	Cell survival rate(%)	Apoptosis rate(%)	Bax	caspase-3	Cyt c
Con Group	1.00 ± 0.02	100.01 ± 0.00	16.96 ± 2.63	0.28 ± 0.03	0.19 ± 0.02	0.21 ± 0.02
IL-1β Group	5.57 ± 1.03a	46.85 ± 9.46a	62.28 ± 7.76a	0.87 ± 0.10a	0.91 ± 0.09a	0.93 ± 0.08a
SMSC Group	3.96 ± 0.57ab	60.13 ± 8.15ab	40.11 ± 3.37ab	0.60 ± 0.05ab	0.62 ± 0.10ab	0.64 ± 0.10ab
IL-1β+sh–NC–Exo Group	3.88 ± 0.62ab	60.85 ± 7.63ab	40.05 ± 3.59ab	0.58 ± 0.06ab	0.61 ± 0.11ab	0.63 ± 0.12ab
IL-1β+sh-SNHG14-Exo Group	1.36 ± 0.18abcd	88.74 ± 6.69abcd	23.11 ± 2.18abcd	0.35 ± 0.04abcd	0.33 ± 0.03abcd	0.40 ± 0.05abcd
F	92.795	85.181	146.441	131.685	112.314	98.907
P	0.000	0.000	0.000	0.000	0.000	0.000

Notes: Compared with the Con group, ^aP < 0.05; compared with the IL-1β group, ^bP < 0.05; compared with the SMSC group, ^cP < 0.05; compared with the IL-1β + sh–NC–Exo group, ^dP < 0.05.

3.3. Effects of LncRNA-SNHG14 silencing on inflammatory responses and the expression of proteins related to chondrocyte proliferation and regeneration

Relative to the Con group, the IL-1β group displayed significantly increased levels of TNF-α and IL-6, as well as elevated MMP-13 protein expression, whereas IGF-1 levels and the expression of SOX9, COL2A1, and bFGF were markedly reduced (P < 0.05). Compared with the IL-1β group, the SMSC group showed attenuated inflammatory responses, as evidenced by decreased TNF-α and IL-6 levels and reduced MMP-13 expression, alongside increased IGF-1 levels and upregulated SOX9, COL2A1, and bFGF expression (P < 0.05).Furthermore, compared with both the SMSC group and the IL-1β + sh–NC–Exo group, the IL-1β + sh-SNHG14-Exo group exhibited lower TNF-α and IL-6 levels and reduced MMP-13 expression, together with higher IGF-1 levels and increased expression of SOX9, COL2A1, and bFGF (P < 0.05). No significant differences were observed between the SMSC group and the IL-1β + sh–NC–Exo group (P > 0.05). These findings are presented in Fig. 4 and Table 2.

Fig. 4.

Expression of proteins associated with chondrocyte proliferation and regeneration. Western blot analysis of SOX9, MMP-13, COL2A1, and bFGF protein expression. Representative blots from three independent experiments are shown (n = 3). Molecular weight markers: SOX9 ∼65 kDa, MMP-13 ∼60 kDa (latent form) and ∼48 kDa (active form), COL2A1 ∼140 kDa, bFGF ∼17.5 kDa, GAPDH ∼37 kDa.

Table 2.

Effects of LncRNA-SNHG14 silencing on inflammatory responses and the expression of proteins associated with chondrocyte proliferation and regeneration ($\bar{x}$ ±s, n = 9).

Group	TNF-α(pg/mL)	IL-6(pg/mL)	IGF-1(ng/mL)	SOX9 protein	MMP-13 protein	COL2A1 protein	bFGF protein
Con Group	63.58 ± 11.19	116.39 ± 28.79	63.29 ± 11.09	0.96 ± 0.05	0.22 ± 0.03	0.98 ± 0.07	0.97 ± 0.06
IL-1β Group	274.15 ± 31.38a	585.54 ± 115.18a	13.24 ± 2.41a	0.32 ± 0.03a	0.89 ± 0.10a	0.30 ± 0.02a	0.26 ± 0.04a
SMSC Group	186.30 ± 22.13ab	316.29 ± 85.43ab	30.06 ± 5.02ab	0.51 ± 0.04ab	0.70 ± 0.11ab	0.55 ± 0.09ab	0.48 ± 0.05ab
IL-1β+sh–NC–Exo Group	184.27 ± 30.61ab	312.27 ± 92.31ab	31.54 ± 8.51ab	0.53 ± 0.06ab	0.68 ± 0.12ab	0.57 ± 0.10ab	0.50 ± 0.07ab
IL-1β+sh-SNHG14-Exo Group	98.10 ± 20.71abcd	188.21 ± 22.73abcd	50.02 ± 7.45abcd	0.74 ± 0.07abcd	0.40 ± 0.08abcd	0.77 ± 0.08abcd	0.70 ± 0.10abcd
F	104.062	47.352	59.906	199.900	72.555	98.502	142.009
P	0.000	0.000	0.000	0.000	0.000	0.000	0.000

Notes: Compared with the Con group, ^aP < 0.05; compared with the IL-1β group, ^bP < 0.05; compared with the SMSC group, ^cP < 0.05; compared with the IL-1β + sh–NC–Exo group, ^dP < 0.05.

3.4. Effects of LncRNA-SNHG14 silencing on the HMGB1/TLR4/NF-κB signalling pathway

Compared with the control (Con) group, the IL-1β group exhibited significantly increased mRNA and protein expression levels of HMGB1, TLR4, and NF-κB (P < 0.05). In contrast, relative to the IL-1β group, the SMSC group showed marked reductions in both mRNA and protein levels of HMGB1, TLR4, and NF-κB (P < 0.05).Furthermore, when compared with the SMSC group and the IL-1β + sh–NC–Exo group, the IL-1β + sh-SNHG14-Exo group demonstrated significantly lower mRNA and protein expression of HMGB1, TLR4, and NF-κB (P < 0.05). No significant differences were observed between the SMSC group and the IL-1β + sh–NC–Exo group (P > 0.05). These results are presented in Fig. 5 and Table 3.

Fig. 5.

Protein expression associated with the HMGB1/TLR4/NF-κB signalling pathway. Western blot analysis of HMGB1, TLR4, and NF-κB protein expression. Representative blots from three independent experiments are shown (n = 3). Molecular weight markers: HMGB1 ∼30 kDa, TLR4 ∼92 kDa, NF-κB p65 ∼65 kDa, GAPDH ∼37 kDa.

Table 3.

Effects of LncRNA-SNHG14 silencing on the expression of proteins associated with the HMGB1/TLR4/NF-κB signalling pathway ($\bar{x}$ ±s, n = 9).

Group	mRNA			Protein
	HMGB1	TLR4	NF-κB	HMGB1	TLR4	NF-κB
Con Group	1.00 ± 0.01	1.01 ± 0.03	1.00 ± 0.03	0.20 ± 0.02	0.18 ± 0.01	0.22 ± 0.03
IL-1β Group	5.54 ± 1.16a	5.63 ± 1.12a	6.02 ± 1.08a	0.93 ± 0.03a	0.95 ± 0.04a	0.97 ± 0.05a
SMSC Group	3.13 ± 0.57ab	3.10 ± 0.62ab	3.25 ± 0.63ab	0.80 ± 0.06ab	0.76 ± 0.05ab	0.81 ± 0.04ab
IL-1β+sh–NC–Exo Group	3.11 ± 0.52ab	3.08 ± 0.55ab	3.21 ± 0.71ab	0.78 ± 0.07ab	0.74 ± 0.06ab	0.80 ± 0.06ab
IL-1β+sh-SNHG14-Exo Group	1.62 ± 0.13abcd	1.77 ± 0.14abcd	1.82 ± 0.83abcd	0.41 ± 0.05abcd	0.39 ± 0.03abcd	0.44 ± 0.07abcd
F	70.750	70.951	59.577	342.549	501.879	316.233
P	0.000	0.000	0.000	0.000	0.000	0.000

Notes: Compared with the Con group, ^aP < 0.05; compared with the IL-1β group, ^bP < 0.05; compared with the SMSC group, ^cP < 0.05; compared with the IL-1β + sh–NC–Exo group, ^dP < 0.05.

3.5. Effects of activation of the HMGB1/TLR4/NF-κB signalling pathway on chondrocyte proliferation and apoptosis

Compared with the IL-1β + empty vector group, the IL-1β + HMGB1 overexpression group showed significantly increased expression of LncRNA-SNHG14, along with elevated protein levels of HMGB1, TLR4, and NF-κB (P < 0.05). These changes were accompanied by reduced cell viability and increased apoptosis, as well as higher expression levels of Bax, caspase-3, and Cyt c (P < 0.05), as shown in Fig. 6 and Table 4.

Fig. 6.

Effects of HMGB1/TLR4/NF-κB pathway activation on chondrocyte apoptosis. A: Apoptotic rate of chondrocytes. Representative dot plots from three independent experiments are shown (n = 3). B: Protein expression levels of Bax, caspase-3, and Cyt c. Representative blots from three independent experiments are shown (n = 3). Molecular weight markers: Bax ∼21 kDa, caspase-3 ∼35 kDa (pro-form) and ∼17 kDa (cleaved form), Cyt c ∼15 kDa, GAPDH ∼37 kDa.

Table 4.

Effects of HMGB1 overexpression on chondrocyte proliferation and apoptosis ($\bar{x}$ ±s, n = 9).

Group	LncRNA-SNHG14	HMGB1 protein	TLR4 protein	NF-κB protein	Cell survival rate(%)	Apoptosis rate(%)	Bax	caspase-3	Cyt c
IL-1β+No load group	1.01 ± 0.03	0.63 ± 0.08	0.61 ± 0.09	0.57 ± 0.11	59.63 ± 9.25	40.16 ± 3.67	0.55 ± 0.04	0.61 ± 0.05	0.62 ± 0.09
IL-1β+HMGB1Overexpression group	2.57 ± 0.18	0.81 ± 0.17	0.85 ± 0.16	0.90 ± 0.12	47.01 ± 3.62	51.19 ± 4.16	0.68 ± 0.08	0.78 ± 0.16	0.81 ± 0.13
t	25.646	2.874	3.922	6.082	3.811	5.965	4.837	3.042	3.605
P	0.000	0.011	0.001	0.000	0.002	0.000	0.000	0.008	0.002

3.6. Effects of activation of the HMGB1/TLR4/NF-κB signalling pathway on inflammatory responses and the expression of proteins related to chondrocyte proliferation and regeneration

Relative to the IL-1β + empty vector group, the IL-1β + HMGB1 overexpression group exhibited significantly increased levels of TNF-α and IL-6, as well as elevated MMP-13 protein expression (P < 0.05). In parallel, IGF-1 levels and the expression of SOX9, COL2A1, and bFGF were markedly reduced (P < 0.05), as shown in Fig. 7 and Table 5.

Fig. 7.

Expression of proteins associated with chondrocyte proliferation and regeneration. Representative blots from three independent experiments are shown (n = 3). Molecular weight markers: SOX9 ∼65 kDa, MMP-13 ∼60 kDa (latent form) and ∼48 kDa (active form), COL2A1 ∼140 kDa, bFGF ∼17.5 kDa, GAPDH ∼37 kDa.

Table 5.

Effects of HMGB1/TLR4/NF-κB pathway activation on inflammatory responses and the expression of proteins associated with chondrocyte proliferation and regeneration ($\bar{x}$ ±s, n = 9).

Group	TNF-α(pg/mL)	IL-6(pg/mL)	IGF-1(ng/mL)	SOX9 protein	MMP-13 protein	COL2A1 protein	bFGF protein
IL-1β+No load group	187.41 ± 20.16	312.58 ± 86.31	30.11 ± 4.15	0.52 ± 0.05	0.71 ± 0.08	0.54 ± 0.08	0.47 ± 0.06
IL-1β+HMGB1Overexpression group	225.71 ± 20.31	508.54 ± 75.49	20.16 ± 3.05	0.40 ± 0.06	0.81 ± 0.07	0.41 ± 0.07	0.30 ± 0.04
t	4.015	5.127	5.796	4.609	2.822	3.669	7.072
P	0.001	0.000	0.000	0.000	0.012	0.002	0.000

4. Discussion

Articular cartilage has a very limited capacity for self-repair following injury, and cartilage damage represents a major risk factor for the development of osteoarthritis (OA). Effective restoration of injured cartilage is therefore critical for preventing disease progression or potentially reversing OA. Chondrocyte apoptosis, persistent inflammatory responses, oxidative stress, and reduced anabolic activity collectively compromise the integrity of the cartilage extracellular matrix, ultimately leading to degenerative joint changes.Synovial mesenchymal stem cells (SMSCs) have been shown to promote chondrocyte proliferation and extracellular matrix synthesis primarily through the paracrine actions of EVs, thereby contributing to the repair of knee cartilage defects. However, the specific bioactive components within SMSC-derived EVs that mediate these protective effects remain poorly defined [9]. Increasing evidence indicates that long non-coding RNAs (lncRNAs) play important regulatory roles in chondrocyte proliferation, apoptosis, and matrix metabolism, suggesting that they may represent promising targets for the development of novel therapeutic strategies [10].Interleukin-1β (IL-1β) is a key pro-inflammatory cytokine involved in the progression of OA, as it regulates the expression of inflammatory mediators and directly influences chondrocyte apoptosis. Accordingly, IL-1β–induced chondrocyte injury is widely used as an in vitro model to recapitulate key pathological features of OA [11]. In the present study, primary rat chondrocytes were exposed to IL-1β to establish an in vitro OA model, and the results demonstrated increased apoptosis, reduced cell proliferation, and enhanced secretion of inflammatory cytokines, leading to inflammatory chondrocyte injury. These findings confirm the successful establishment of a cellular model that reliably reflects OA-related pathological changes and provide a basis for investigating potential protective strategies against chondrocyte injury.

LncRNA-SNHG14 has been reported to promote the release of inflammatory mediators by acting as a competing endogenous RNA, thereby suppressing miR-223-3p and upregulating forkhead box protein O3α (Foxo3a) expression, which aggravates lipopolysaccharide-induced injury in alveolar type II epithelial cells [12]. In neuronal models, LncRNA-SNHG14 can inhibit miR-22-3p and activate the NF-κB signalling pathway, ultimately leading to enhanced neuronal apoptosis [13].In addition, LncRNA-SNHG14 has been shown to function as a molecular sponge for miR-30e-5p, thereby regulating SOX4 expression and relieving its inhibitory effects on downstream inflammation- and oxidative stress–related genes. Silencing LncRNA-SNHG14 increases miR-30e-5p expression, suppresses NF-κB pathway activation, enhances antioxidant enzyme expression, and attenuates cellular injury [14]. In a mouse model of ischaemic stroke, LncRNA-SNHG14 expression is markedly upregulated, and its knockdown indirectly regulates aquaporin 4 (AQP4) expression by sequestering miR-199b, leading to reduced inflammatory and oxidative stress responses and alleviation of ischaemic brain injury [15].Li Ming et al. [16] demonstrated that SMSC-derived exosomes overexpressing LncRNA-SNHG14 promote cartilage repair and alleviate inflammatory responses in a rat model of osteoarthritis. While both studies investigate SMSC-derived EVs and LncRNA-SNHG14 in the context of osteoarthritis, the present study differs in several key aspects. First, Li et al. focused on the effects of overexpressing LncRNA-SNHG14, whereas our study investigates the impact of silencing LncRNA-SNHG14, providing complementary evidence for its role in chondrocyte injury. Second, our study specifically examines the mechanistic involvement of the HMGB1/TLR4/NF-κB pathway, which was not explored in the previous report. Third, we employ a distinct experimental approach using purified EVs at defined concentrations (50 μg/mL) rather than co-culture systems, allowing more precise attribution of observed effects to EV-delivered LncRNA-SNHG14. These differences collectively extend our understanding of LncRNA-SNHG14's role in osteoarthritis pathogenesis and its potential as a therapeutic target. Other studies have shown that LncRNA-SNHG14 can activate the Wnt/β-catenin signalling pathway by targeting miR-137, thereby regulating chondrocyte apoptosis, inducing mitochondrial dysfunction, and exacerbating inflammation and extracellular matrix degradation [17].Current EVs research has largely focused on microRNAs, whereas proteomic analyses indicate that EVs can also exert biological effects through their protein cargo, either by releasing proteins to neighbouring cells or by delivering them through membrane fusion. Given its consistently high expression in osteoarthritic tissues, LncRNA-SNHG14 may represent a promising therapeutic target for osteoarthritis.In the present study, IL-1β stimulation markedly increased LncRNA-SNHG14 expression in chondrocytes, consistent with previous reports [17]. Notably, our findings extend existing knowledge by demonstrating that SMSC-derived EVs participate in chondrocyte injury through the transfer of LncRNA-SNHG14; this lncRNA is enriched within SMSC-derived EVs, can be efficiently secreted, and is readily taken up by chondrocytes.Further analyses revealed that, in the SMSC group, reduced LncRNA-SNHG14 expression was associated with enhanced cell viability and decreased apoptosis, along with lower expression levels of Bax, caspase-3, and cytochrome c (Cyt c). Importantly, silencing LncRNA-SNHG14 exerted more pronounced protective effects on chondrocyte proliferation and apoptosis. As key apoptosis-related factors, Bax, caspase-3, and Cyt c promote chondrocyte apoptosis, impair cartilage function, and contribute to the pathogenesis of osteoarthritis [18,19]. Collectively, these findings suggest that silencing LncRNA-SNHG14 may modulate chondrocyte function through the competing endogenous RNA(ceRNA) -mediated miRNA–mRNA regulatory network, influence autophagic activity, and inhibit apoptosis. These multifaceted regulatory effects suggest that targeting LncRNA-SNHG14 could provide a novel and versatile strategy for intervening in osteoarthritis.

Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) can damage chondrocytes by reducing their proliferative capacity and promoting the expression of matrix-degrading enzymes, thereby delaying cartilage repair. In contrast, insulin-like growth factor-1 (IGF-1) exerts anti-inflammatory effects, attenuates chondrocyte apoptosis, and mitigates inflammatory injury [20,21].Basic fibroblast growth factor (bFGF) enhances chondrocyte proliferation, accelerates cartilage tissue repair, and induces the differentiation of mesenchymal stem cells into chondrocytes, supporting cartilage regeneration. SOX9 serves as an early marker of chondrogenic differentiation and promotes COL2A1 expression, facilitating the differentiation of SMSCs toward a chondrogenic lineage. By contrast, matrix metalloproteinase-13 (MMP-13) degrades collagen and other matrix components, leading to cartilage destruction and chondrocyte injury [[22], [23], [24]].In the present study, silencing SMSC-Exo–derived LncRNA-SNHG14 resulted in decreased levels of TNF-α, IL-6, and MMP-13, alongside increased expression of IGF-1, SOX9, COL2A1, and bFGF, suggesting that LncRNA-SNHG14 knockdown suppresses inflammatory responses and promotes chondrocyte proliferation and regeneration. Mechanistically, silencing LncRNA-SNHG14 may act as a competing endogenous RNA (ceRNA) by sponging miRNAs such as miR-223-3p, thereby indirectly regulating downstream genes including Foxo3a and TLR4, inhibiting NF-κB pathway activation, and reducing the expression of inflammatory mediators such as TNF-α and IL-6. This regulatory mechanism may also drive transcription of type II collagen and aggrecan genes, restore chondrocyte anabolic activity, suppress apoptosis, and rebalance catabolic and anabolic processes in cartilage.Activation of the HMGB1/TLR4/NF-κB signalling pathway contributes to chondrocyte injury and plays a key role in osteoarthritis pathogenesis. Blocking this pathway may therefore represent a viable therapeutic strategy for OA [25]. Pathway activation exacerbates inflammation and oxidative stress, promotes chondrocyte apoptosis, and accelerates pathological changes in cartilage. TLR4, the primary receptor of HMGB1, interacts with HMGB1 to activate downstream NF-κB signalling, inducing nuclear translocation and upregulating leukocyte adhesion molecules. Activated NF-κB further modulates the expression of SOX9, COL2A1, and bFGF, thereby aggravating cartilage degeneration in osteoarthritic rats [[26], [27], [28], [29]].Qi et al. [30] demonstrated that activation of the HMGB1/TLR4/NF-κB signalling pathway can promote chondrocyte apoptosis and exacerbate inflammatory responses, suggesting that this pathway may serve as a potential therapeutic target for osteoarthritis.

In the present study, IL-1β–induced chondrocytes exhibited elevated expression levels of HMGB1, TLR4, and NF-κB, whereas silencing LncRNA-SNHG14 resulted in a significant reduction in their expression.

These findings indicate that LncRNA-SNHG14 silencing is associated with reduced expression of key molecules within the HMGB1/TLR4/NF-κB signaling cascade. However, two important limitations should be acknowledged. First, these data demonstrate changes in component expression rather than direct evidence of pathway activation or inhibition; definitive assessment of NF-κB pathway activity would require additional functional readouts such as p65 nuclear translocation, phosphorylation of p65, or IκBα degradation. Second, in the HMGB1 overexpression experiment, HMGB1 was overexpressed in SMSCs rather than directly in recipient chondrocytes; therefore, the current data cannot definitively establish whether HMGB1 acts downstream in chondrocytes, upstream in SMSCs, or indirectly through other secreted factors. Future studies should employ direct HMGB1 manipulation in recipient chondrocytes combined with functional pathway assays to conclusively determine the mechanistic hierarchy of the HMGB1/TLR4/NF-κB pathway in mediating the effects of SMSC-EV-derived LncRNA-SNHG14. Further supporting this mechanism, HMGB1 overexpression increased LncRNA-SNHG14 levels, upregulated HMGB1, TLR4, and NF-κB protein expression, decreased chondrocyte viability, and increased apoptosis, while antagonizing the regulatory effects of LncRNA-SNHG14 silencing on inflammatory responses and chondrocyte proliferation- and regeneration-associated proteins. These findings suggest that the protective role of silencing LncRNA-SNHG14 in chondrocyte injury may be mediated through inhibition of HMGB1/TLR4/NF-κB signalling.

It should be noted that many of the studies cited in this manuscript use the term "exosomes" to describe small extracellular vesicles isolated from mesenchymal stem cells. In accordance with the MISEV guidelines, we have adopted the more conservative term "extracellular vesicles (EVs)" for our own isolates, as the polymer precipitation method used does not definitively distinguish between exosome subtypes and other small extracellular vesicles. This terminological distinction should be considered when comparing our findings with those of previous reports.

Furthermore, several methodological limitations regarding EV isolation and characterization should be acknowledged. In this study, EVs were isolated using a polymer precipitation kit (ExoQuick-TC). Although this method efficiently recovers vesicles from culture supernatants, it may co-precipitate non-EV components such as protein aggregates, lipoproteins, and other macromolecular complexes, which can affect EV purity. Characterization of the isolated EVs was primarily based on transmission electron microscopy, nanoparticle tracking analysis, and Western blot detection of CD63 and CD81. Density gradient ultracentrifugation was not performed, and systematic exclusion of common non-EV contaminants via lipoproteins or RNA-binding protein markers was not carried out. Therefore, the possibility that some of the observed biological effects are partially attributable to co-isolated non-EV materials cannot be completely excluded. Future studies employing more rigorous purification methods, such as density gradient centrifugation or size-exclusion chromatography, are warranted to confirm the specific role of EV-delivered LncRNA-SNHG14 in chondrocyte injury.

In summary, in an IL-1β-induced chondrocyte injury model, SMSC-EVs with silenced LncRNA-SNHG14 enhance chondrocyte proliferation, suppress apoptosis, and attenuate inflammatory responses. These protective effects against chondrocyte injury are associated with reduced expression of HMGB1/TLR4/NF-κB pathway components, suggesting that this pathway may be involved in the underlying mechanism. These results provide a foundation for further in vivo studies to validate the therapeutic potential of targeting LncRNA-SNHG14 in articular cartilage injury and to confirm the functional involvement of HMGB1/TLR4/NF-κB signaling using direct pathway activation assays.

Ethical statement

All primary rat chondrocytes and synovial mesenchymal stem cells used in this study were commercially obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The use of this commercially available cell line does not require ethical approval, as it does not involve human or animal subjects directly.

Funding

No fundings.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.