Apoptotic extracellular vesicles derived from MSCs exposed to hypoxic and inflammatory environments slow intervertebral disc degeneration by enhancing cell activity and regulating immunity microenvironment
Weiqi Zhang, Xiaowei Ma, Han Yin, Dazhuang Miao, Tianhao Guo, Wei Chen, Zhiyong Hou, Yingze Zhang, Xianda Gao, Di Zhang
TL;DR
This study shows that extracellular vesicles from stem cells, treated with a hypoxic and inflammatory environment, can slow disc degeneration by boosting cell activity and reducing inflammation.
Contribution
The novel approach uses hypoxia and inflammation-pretreated stem cell-derived apoptotic vesicles to target IVDD.
Findings
I-ApoEVs significantly inhibited NPCs senescence and promoted ECM synthesis.
I-ApoEVs regulated the STAT6 pathway and induced M2 macrophage polarization.
The treatment alleviated inflammation-mediated degenerative damage in intervertebral discs.
Abstract
Intervertebral disc degeneration (IVDD) is characterized by the senescence and apoptosis of nucleus pulposus cells (NPCs), metabolic imbalance of the extracellular matrix (ECM), and local chronic inflammation, presenting a long-standing challenge in clinical treatment. Recent studies have confirmed that transplanted stem cells are prone to apoptosis in vivo, and the apoptotic extracellular vesicles (ApoEVs) they produce are key mediators of tissue repair. Given that both the physiological state of mesenchymal stem cells (MSCs) and the IVDD microenvironment exhibit hypoxic and inflammatory features, this study investigated the therapeutic effect and mechanism of MSCs-derived apoptotic bodies (I-ApoEVs) pretreated with a hypoxic-inflammatory composite microenvironment on IVDD. The results showed that compared with ApoEVs under conventional hypoxic conditions, I-ApoEVs more significantly…
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Taxonomy
TopicsSpine and Intervertebral Disc Pathology · Extracellular vesicles in disease · Spondyloarthritis Studies and Treatments
Introduction
1
Low back pain is a major global public health issue, with 26%-42% of chronic cases caused by intervertebral disc degeneration (IVDD) [1]. This disease also imposes a heavy economic and social burden [2]. The intervertebral disc (IVD) is composed of the nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplates. A healthy NP relies on an extracellular matrix (ECM) rich in type II collagen (COL II) to buffer spinal pressure. The core characteristics of IVDD include dysfunction and reduced quantity of NP cells [3], as well as pathological changes such as ECM metabolic imbalance, cell apoptosis, and inflammatory responses [4]. Currently, non-surgical and surgical treatments for IVD-related low back pain only address symptoms, highlighting an urgent need for more effective therapeutic strategies.
Some scholars have proposed an exogenous cell transplantation strategy, which aims to achieve cell supplementation by inducing the differentiation of cells from different sources into intervertebral disc cells [5]. Among these, bone marrow mesenchymal stem cells have become commonly used stem cells in the fields of cell therapy and tissue engineering due to their low immunogenicity. And these cells are widely present in the bone marrow microenvironment and possess both self-renewal ability and the potential to differentiate into various tissues. However, studies have shown that BMSCs undergo apoptosis shortly after transplantation [6]. Liu et al. confirmed that apoptosis is an essential prerequisite for BMSCs to exert their therapeutic effects [7], and further research has demonstrated that administration of apoptotic BMSCs can reduce disease severity while retaining repair capacity in vivo [8]. During cell apoptosis, extracellular vesicles (EVs) called apoptotic EVs (ApoEVs) are released. ApoEVs were once classified as harmful debris in early studies, but recent research has revealed that they can promote intercellular communication through signal molecule transduction, exhibiting tremendous application potential in fields such as immunotherapy [9], tissue regeneration [10] and drug delivery [11]. These EVs are phagocytosed by target cells and mediate intercellular communication by delivering bioactive molecules [12]. Compared with direct BMSCs transplantation, BMSCs-derived apoptotic extracellular vesicles can avoid potential risks such as immune rejection, decreased stem cell regenerative capacity, tumorigenicity, and ethical controversies [13]. To further enhance the repair capacity of BMSC-derived extracellular vesicles in IVDD, researchers have attempted various strategies. Notably, the 21% O_2_ normoxic environment commonly used in in vitro experiments differs significantly from the hypoxic microenvironment in vivo (2%∼8% O_2_ or lower) [14]. ApoEVs derived from hypoxia-preconditioned mesenchymal stem cells promote osteochondral regeneration by enhancing stem cell activity and regulating immunity [9]. Additionally, preconditioning with inflammatory factors (e.g., TNF-α) can increase relevant components within EVs, thereby improving their repair ability and immunomodulatory effects [15,16]. Notably, inflammatory mediators and reactive oxygen species formed during IVDD induce the apoptosis of transplanted stem cells [6,17]. However, no previous studies have clarified whether ApoEVs generated by BMSCs cultured in a hypoxic-inflammatory composite microenvironment exhibit enhanced therapeutic efficacy.
During the progression of IVDD, the abnormal expression of inflammatory factors is closely associated with macrophage polarization. The expression of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) gradually increases with the advancement of degeneration [18]. These inflammatory cytokines not only directly reduce the number and impair function of NPCs [19], but also induce the upregulated expression of chemokines such as CCL2, CCL3, and CCL4, which recruit and activate immune cells. As the only inflammatory cell capable of penetrating IVD [20], changes in the number of macrophages are highly correlated with the degenerative process. As key effector cells in the immune response, macrophages can switch their functions through phenotypic plasticity, thereby regulating inflammatory responses and tissue remodeling [21]. Previous studies have confirmed that degenerative myeloid cells induce macrophage infiltration and subsequent M1 polarization via the CCL2/7-CCR2 signaling axis [22]. Single-cell sequencing data have also demonstrated that M1-dominated macrophage infiltration is a characteristic feature of IVDD [23]. Additionally, magnoflorine exerts a clear protective effect by intervening IVDD induced by M1-polarized macrophages [24]. M1 macrophages promote the apoptosis, pyroptosis and ferroptosis of NPCs by regulating the inflammatory microenvironment [25]. In contrast, M2 macrophages play a protective role: their conditioned medium can inhibit the upregulation of pro-inflammatory factors such as IL-1β and IL-6, as well as MMP-13, while significantly promoting the synthesis of ECM components including COL II and ACAN [26]. Furthermore, M2-type macrophages alleviate IVDD both in vivo and in vitro by inhibiting Rspo2 production, reducing NPCs apoptosis and suppressing ECM catabolism [27]. STAT6 serves as a core signaling node for M2 polarization regulated by Th2 cytokines (IL-4/IL-13). M2 polarization signaling mediated by IL-4/IL-13 depends entirely on the JAK-STAT6 pathway, and as the only critical node in this pathway, the phosphorylation and nuclear translocation of STAT6 are essential for the initiation of M2 polarization [[28], [29], [30], [31]]. Meanwhile, phosphorylated STAT6 translocates into the nucleus and directly binds to the promoter regions of genes encoding M2 phenotypic markers such as Arg1, CD206, and IL-10 to initiate their transcription, acting as a direct driver of M2 phenotypic differentiation [[30], [31], [32]]. Furthermore, STAT6 amplifies the M2 polarization effect by activating downstream target pathways and molecules including PPARγ and Nrf2 to form a cascade regulatory network [[33], [34], [35]]. The polarization status of macrophages can directly affect the survival of NPCs and the metabolic balance of ECM. However, to date, no study has explored the repair capacity of ApoEVs in the context of IVDD treatment through macrophage polarization regulation.
This study found that BMSCs-derived apoptotic extracellular vesicles pretreated with a hypoxic and inflammatory composite microenvironment (I-ApoEVs) can more significantly inhibit the senescence of NPCs and promote the synthesis of ECM. Regarding the specific mechanism, I-ApoEVs can target and regulate the STAT6 signaling pathway through the functional molecules they carry, efficiently inducing the polarization of macrophages towards the M2 anti-inflammatory phenotype. In conclusion, relying on the dual synergistic effects of “inhibiting NPCs senescence ” and “regulating macrophage M2 polarization ”, I-ApoEVs provide a novel and highly translatable therapeutic strategy for the clinical treatment of IVDD. An overview of the study design is shown in Scheme 1.Scheme 1. Schematic diagram of apoptotic extracellular vesicles (ApoEVs) in relieving intervertebral disc degeneration. ApoEVs derived from hypoxic and inflammatory composite microenvironment preconditioned BMSCs enhance NPCs activity and regulate immunity to promote IVD repair.Scheme 1
Materials and methods
2
Culture for rat NPCs and BMSCs
2.1
In this study, 12-week-old Sprague–Dawley rats were purchased from the Hebei Laboratory Animal Center (China). Briefly, nucleus pulposus (NP) tissues were isolated, minced, and digested with 0.2% type II collagenase (Solarbio, China) for 6 h in the incubator (at 37 °C in 5% CO_2_). The digested NP tissue was washed with PBS, centrifuged at 1000 rpm for 5 min, and cultured in DMEM/F12 supplemented with 10% FBS and 1% penicillin–streptomycin in the incubator. Primary rat BMSCs were sourced from the femoral bone marrow cavity when rats were euthanized and cultured in α-MEM (Gibco, America) supplemented with 10% FBS and 1% penicillin–streptomycin in the incubator. Cell culture medium was changed every 2–3 days. Cells were passaged when reaching 70%–80% confluence, and cells from passages 2–3 were used for experiments.
BMSCs were then incubated at 37 °C in a low-oxygen environment comprising 1% O_2_ and 5% CO_2_ for 24 h. ApoEVs and I-ApoEVs were derived from BMSCs pretreated with staurosporine (STS, 200 nM) (MCE, America) for 12 h or inflammatory factor TNF-α (20 ng/ml) (PeproTech, America) for 12 h, and the details were listed as follows: (1) ApoEVs were derived from hypoxia-preconditioned BMSCs cultured with STS; (2) I-ApoEVs were derived from hypoxia-preconditioned BMSCs cultured with STS and TNF-α. For ApoEVs interventions in cell experiments, the ApoEVs were added to culture dishes to maintain the concentration of 20 μg/ml [36].
Isolation, characterization and uptake of ApoEVs and I-ApoEVs
2.2
For in vitro ApoEVs isolation, BMSCs were cultured in different medium. Conditioned medium was collected and sequentially centrifuged: 800×g for 10 min to remove suspended cells, 2000×g for 10 min to discard apoptotic bodies and cell debris, and further centrifuged at 16,000×g for 30 min at 4 °C to isolate ApoEVs [6,9]. The pellet was washed twice, resuspended in filtered PBS. Both EVs were quantified using the BCA Protein Assay Kit to measure the protein concentrations. The isolated and purified ApoEVs and I-ApoEVs were resuspended in PBS to an appropriate concentration, and their particle size distribution and particle concentration were detected using a Flow Nano Analyzer (Nanofcm, China). ApoEVs and I-ApoEVs morphology were examined via transmission electron microscopy (TEM, Hitachi, Japan), and ApoEV markers were detected by Western blot (WB; Bio-Rad Laboratories, USA). Purified ApoEVs were stored at −80 °C. NPCs and macrophages were seeded in confocal dish at a density of 2 × 104 cells and cultured for 24 h. To evaluate endocytosis efficiency, both EVs were labeled with the 10 μM DiR dye (MCE, America) and then were co-cultured with NPCs and macrophages for 12 and 24 h, respectively. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA), stained with cytoskeleton staining (Beyotime, China) and DAPI solution (Solarbio, China), and examined using a fluorescence microscope (Olympus, Japan).
Effect of ApoEVs on macrophages immunomodulation
2.3
Macrophage RAW264.7 cells were cultured in DMEM with addition of 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO_2_. When reaching 70%–80% confluence, the cells in Control group were not given any special treatment. M1 group cells were treated with interferonγ (IFN‐γ) (20 ng/ml, Sigma-Aldrich, America) and lipopolysaccharide (LPS, 100 ng/ml, Sigma-Aldrich, America) for 24 h. The treatment groups were also added into ApoEVs and I-ApoEVs. The usage of STAT6 inhibitor AS1517499 in this study strictly follows the standardized literature protocols: 10 mg/kg via intraperitoneal injection in vivo (pre-treatment + maintenance every 2 days) and 1 μM pre-treatment followed by co-culture in vitro. The specificity was confirmed through a system of “STAT6 detection + target gene verification + blank control” [28,29,31,33].
Senescence model of NPCs
2.4
A cellular oxidative stress-induced senescence model was established using passage 2–3 NPCs. Hydrogen peroxide (H_2_O_2_) stock solution was diluted to a final concentration of 100 μΜ using complete medium. Four experimental groups were prepared: complete medium (control group), H_2_O_2_ group, H_2_O_2_ + ApoEVs group and H_2_O_2_ + I-ApoEVs group. When reaching 70% confluency, cells were treated with the respective medium for 24 h. Following treatment, cultures were terminated, and samples were collected for subsequent experiments.
Cell cytotoxicity and proliferation assessments
2.5
NPCs were seeded into 96-well plates at a density of 3000 cells/well to evaluate cell proliferation using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan). The experiment comprised four groups: control group (DMEM/F12 medium only), H_2_O_2_ group, ApoEVs group and I-ApoEVs group. At days 1, 3, 5, and 7 of culture, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. Subsequently, the medium was aspirated and replaced with treatment media from different groups. Optical density (OD) values at 450 nm were then measured using a microplate reader. On day 2, a live/dead staining assay was performed using a live-dead staining kit (Beyotime, China) to evaluate the cytotoxicity of various EVs following the manufacturer's instructions. The cell cycle was assessed via flow cytometry. In brief, we used trypsin solution to digest and collect treated cells and then used 70% ethanol to fix the cells at 4 °C for 24 h. After PBS washes, 500 μL of propidium iodide (PI) staining (Beyotime, China) solution was added to each sample at 37 °C for 30 min. Subsequently, the percentages of G1-, S-, and G2-phase cells were measured using a flow cytometer (BD FACSCelesta, America) and analyzed by FlowJo software.
Cell apoptosis assay
2.6
The NPCs apoptosis was detected by V-FITC/PI according to the instructions (Beyotime, China). Briefly, samples were harvested and washed with PBS for three times, and then resuspended in the binding buffer. A total of 5 μL AnnexinV-FITC and 5 μL PI were added into the suspension. After incubation in the dark for 15 min, mixtures were analyzed using a fluorescence microscope or flow cytometry.
EdU staining assay
2.7
EdU staining kit (Beyotime, China) was used to examine the proliferation status of NPCs. NPCs were seeded in a 48-well plate at a density of 2 × 10^4^ cells per well and co-cultured with different groups medium. EdU staining dye was added to the culture medium. The subsequent steps were conducted according to the manufacturer's protocol. Finally, the results were observed using a fluorescence microscope (Olympus, Japan). The results were analyzed via ImageJ software.
Cell migration
2.8
Transwell assays and wound healing were performed to assess cell migration. For the Transwell assay, the lower chamber received 600 μL of medium containing ApoEVs or I-ApoEVs. 1 × 10^5^ NPCs were seeded into the upper chamber (Corning, America). After 24 h of incubation, cells were fixed, and non-migrated cells were gently removed with a cotton swab. Cells were stained with 1% crystal violet for 15 min. The numbers of migrated cells were quantified. For the wound healing assay, NPCs were seeded in 6-well plates at a density of 5 × 10^5^ cells per well. Linear wounds were made via a yellow tip, and washed with PBS for three times. Cells were then incubated for an additional 12 and 24 h in different groups medium. The wound closure was examined using an optical microscope and quantified using ImageJ software.
TUNEL assay
2.9
The level of BMSCs and NPCs apoptosis was detected by using TUNEL staining kit (Beyotime, China) following the manufacturer's instructions. After treatment with different group medium, cells were fixed with 4% PFA and permeabilized. Samples were incubated with TUNEL reaction mix in the dark for 60 min at 37 °C. After counterstained with DAPI, TUNEL-positive cells were observed under a fluorescence microscope.
Senescence-associated β-galactosidase (SA-β-gal) staining
2.10
SA-β-gal kit (Beyotime, China) was conducted following the recommendations of manufacturer. NPCs were incubated in 6-well plate for 24 h with different groups medium and washed with PBS, fixed with 1 mL of SA-β-gal fixative for 15 min at room temperature, and subsequently incubated with SA-β-gal working solution at 37 °C in the absence of CO_2_ overnight. And then, the stained cells were captured by using a light microscope. Images were analyzed by calculating the positive area ratio using ImageJ software.
NPCs extracellular matrix (ECM) staining
2.11
NPCs were seeded in a 6-well plate at a density of 3 × 10^5^ cells per well. When reaching 70%–80% confluency, the plate was replaced with the same senescence-inducing medium and different groups medium. Cells were cultured for 1 and 3 days. After stimulation, cells were fixed with 4% PFA for 30 min, followed by staining with Alcian blue solution (Servicebio, China) for 15 min and Safranin O staining solution (Servicebio, China) for 5 min. The stained cells were captured under an optical microscope.
Quantitative real-time polymerase chain reaction (qRT‒PCR)
2.12
Cells were cultured in 6-well plates, and RNA of different treatment groups was obtained using the RNA Isolation Kit (Vazyme, China). Complementary DNA (cDNA) was synthesized using the cDNA Synthesis Kit (Vazyme, China) according to the manufacturer's instructions. Amplification was performed using a RT-PCR system (Light Cycler 96, Switzerland). GAPDH served as the housekeeping gene. Relative expression levels of the target gene, compared with the control group, were calculated using the 2^–ΔΔCt^ method. The primers used for qRT‒PCR were listed in Table S1 (Supporting Data).
Western Blotting (WB) analysis
2.13
The corresponding samples were collected, washed with PBS and lysed using RIPA lysis (Servicebio, China). After lysis, samples were centrifuged at 12000 rpm and 4 °C for 10 min, then collect the supernatant. The protein concentration was normalized using a BCA protein assay kit. After added loading buffer at a volume of 1/4 that of the protein solution (Servicebio, China), the lysates were heated at 95 °C for 10 min. Proteins were separated on a 12% gel (Servicebio, China) and transferred to polyvinylidene fluoride membranes (Millipore, America) via a Bio-Rad device (Bio-Rad, America). Membranes were blocked in PBST (PBS with 5% skim milk and 1% Tween-20) for 2 h at room temperature, then incubated with primary antibodies at 4 °C overnight. After three PBST washes, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Target protein was visualized via chemiluminescent detection solution, and data were quantified using ImageJ software.
Immunofluorescence (IF) assay
2.14
Plates containing cells were rinsed three times with PBS and fixed with 4% PFA at room temperature for 30 min. Subsequently, cells were permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific, America) for 15 min, followed by blocking with immunostaining blocking solution (Beyotime, China) for 1 h. Primary antibodies targeting P53 (Beyotime, China), MMP13 (Bioss, China), COL II (Bioss, China), ACAN (Bioss, China), CD86 (proteintech, China), CD206 (proteintech, China), BCL-2 (Beyotime, China) and γ-H2AX (Beyotime, China) were applied and incubated overnight at 4 °C. Afterward, the cells were incubated with Alexa Fluor 594- or 488-conjugated Goat AntiRabbit IgG (H + L) antibodies (ZS Bio, China) for 1 h at room temperature in the dark. Cells were then stained with DAPI for 15 min in the dark at room temperature. Finally, plates were washed three times with PBS for 5 min each. A fluorescence microscope was used to obtain the stained result.
IF staining of β-galactosidase staining in cells
2.15
Fluorescein diβ-D-galactopyranoside (FDG, MCE, America) was used to detect SA-β-gal activity. Cells were seeded into 48-well plate and cultured with different group mediums. After incubation, an aliquot of the reaction buffer is added into each well and then added 2 mmol/L FDG. The plate was incubated in the dark at 37 °C for 24 h without adding carbon dioxide to the incubator. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Then nuclei were stained using DAPI (Beyotime, China) for 15 min in the dark at room temperature. The stained result was captured under a fluorescence microscope.
Animal model
2.16
Sprague–Dawley rats (250–300 g) were supplied by Hebei Laboratory Animal Center (China). All experiments were approved by the Laboratory Animal Ethics Committee. An IVDD model was established via 22G needle puncture at the center of C5–10 IVDs, as previously reported [37]. Next, 5 μL of EVs (from different treatment groups) or PBS was injected into the model using a microinjector, with the same dosage repeated once a week. Details of animal modeling and EVs injection are available in Fig. S3 (Supporting Data). To avoid cross-interference between adjacent segments and ensure independent therapeutic effect evaluation, treated IVDs were spaced, and each rat tail was assigned to one treatment group.
Evaluation of treatment efficacy for IVDD
2.17
In vivo experimental outcomes were assessed via radiological and histological analyses. At 4- and 8-weeks post-surgery, the rats were positioned prone for MRI scans and x-rays to observe signal and structural changes in the IVDs. Disc Height Index (DHI) was then calculated with ImageJ software as presented in Fig. S2 (Supporting Data). The Pfirrmann grade was applied to determine the severity of IVDD in each group, detailed in Table S2 (Supporting Data). For histological assessment, intervertebral disc tissues were collected from rat tails. These samples were fixed in 4% paraformaldehyde, decalcified in 10% ethylenediamine tetraacetic acid solution, and embedded in paraffin before being sectioned into 5 μm slices. The sections were stained using hematoxylin and eosin (HE), Safranin-O/Fast Green (SF), and immunofluorescence protocols. Histological grading of rat IVDs was performed based on Table S3 (Supporting Data).
Statistical analysis
2.18
All the data were performed by using Graphpad Prism 10.1.2 (GraphPad Software, America) and presented as the means ± standard deviations (mean ± SD). To assess the statistical significance of any differences, statistical Two-tailed unpaired Student's t-test and one-way ANOVA followed by the Tukey–Kramer test were used in this study. All experiments were independently repeated at least three times. P < 0.05 was considered statistically significant while P > 0.05 was considered nonsignificant (ns) (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Results
3
Isolation and characterization of BMSC-derived apoEVs
3.1
Optical microscopy showed that after induction with STS or STS + TNF-α, BMSCs shrank into round or irregular shapes, with vesicular protrusions and budding structures emerging on their surface (Fig. 1A). In the 400 × images of the BMSCs + STS and BMSCs + STS + TNF-α groups, the characteristic vesicular protrusions (bulge-like membrane structures on the cell surface) and budding structures (small membrane-derived protrusions detaching from the cell body) of apoptotic cells can be observed more intuitively and clearly. These high-magnification images directly visualize the key morphological markers of BMSCs during apoptosis induction. These structures subsequently detached to form apoptotic extracellular vesicles (ApoEVs). Cell viability assay indicated that the cell viability of the apoptosis-induced groups decreased significantly over time (Fig. 1B). Annexin V fluorescence staining (Fig. 1C) and TUNEL staining strongly validated the efficiency of BMSCs apoptosis induction (Fig. 1D and E). Transmission electron microscopy (Fi. 1F) clearly showed the vesicular structures of ApoEVs and I-ApoEVs, with no obvious morphological differences. Hypoxia is a basic condition simulating the in vivo physiological microenvironment of the intervertebral disc, not an intervention variable”, and the only variable is “whether TNF-α inflammatory preconditioning is performed”. Particle size analysis (Fig. 1F) defined the size distribution characteristics of the two types of apoptotic bodies: the average diameter of ApoEVs was 309.91 nm, while that of I-ApoEVs was 321.70 nm. Concentration determination indicated that I-ApoEVs had a superior yield (Fig. S1, Supporting Data). Western blot detected the expression of apoptotic marker Cleaved Caspase-3, verifying the formation of ApoEVs and I-ApoEVs (Fig. 1G). These results suggest that I-ApoEVs may carry more cargo. The high purity of ApoEVs was fully verified from three dimensions: morphology, particle size, and specific markers. This indirectly excluded significant contamination by cell debris and macrovesicles, and the results were consistent with the purity criteria reported in the literature [6,9]. To further evaluate the endocytosis of apoptotic bodies, fluorescently labeled apoptotic extracellular vesicles were co-incubated with NPCs and macrophages. Fluorescence imaging combined with quantitative analysis showed that both cell types were capable of internalizing ApoEVs and I-ApoEVs, and the endocytic efficiency of I-ApoEVs exhibited a more significant advantage over time (Fig. 1H–K). Additionally, the fluorescently labeled ApoEVs and I-ApoEVs successfully entered the intervertebral disc and were phagocytosed by nucleus pulposus cells (Fig. 1L). No obvious changes were observed in all the harvested organs (Fig. S4, Supporting Data). In summary, this study systematically analyzed the biological characteristics of BMSCs-derived apoptotic extracellular vesicles and confirmed that they are successfully internalized by both NPCs and macrophages.Fig. 1A) Bright-field morphological images of bone mesenchymal stem cells (BMSCs) under normal and apoptotic states. The images are presented at two magnifications: 200 × (top row) and 400 × (bottom row), with corresponding scale bars of 200 μm and 200 μm, respectively. B) Cell viability of BMSCs during apoptosis induction was detected by the CCK-8 assay. C) Annexin V fluorescent staining of apoptotic BMSCs (scale bar = 100 μm). D) TUNEL staining (Red) shows the positive apoptotic BMSCs (scale bar = 100 μm). E) Quantitative analysis of TUNEL positive cells in different groups. F) Transmission electron microscopy (TEM) shows the morphology and size of the isolated BMSC-derived ApoEVs, scale bar (scale bar = 250 μm). And the size distribution of both ApoEVs. G) Western blot of apoptotic markers caspase 3 and cleaved caspase in BMSCs, apoptotic BMSC (Apo-BMSCs), ApoEVs and I-ApoEVs. J) Confocal microscopy images show NPCs internalizing ApoEVs (Red) at 12 and 24 h in vitro (scale bar = 50 μm) and I) corresponding quantitative analysis. K) Confocal microscopy images show macrophages internalizing ApoEVs (Red) at 12 and 24 h in vitro (scale bar = 50 μm) and H) corresponding quantitative analysis. L) Confocal microscopy images show the internalization of ApoEVs by NPCs after injection into the IVDs in vivo (scale bar = 200 μm). ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 1
Effect of both ApoEVs on the proliferation and migration ability of NPCs
3.2
Reduced proliferative capacity, a central feature of NPCs senescence, leads to insufficient synthesis of ECM in the intervertebral disc (e.g., COL II and ACAN), which in turn exacerbates disc degeneration. EdU staining assays (Fig. 2A and B) showed that the proportion of EdU-positive cells in the ApoEVs and I-ApoEVs treatment groups was significantly higher than that in the control group, with the I-ApoEVs group exhibiting a superior proliferation-promoting effect. Cell proliferation curves (Fig. 2C) further confirmed the I-ApoEVs group could significantly enhance the viability of proliferating cells. Live-cell imaging and density statistics (Fig. 2D and E, green indicating live cells) demonstrated that cells cultured with ApoEVs or I-ApoEVs all survived and both treatments promoted an increase in cell numbers, with the effect of I-ApoEVs being more pronounced. Cell cycle analysis (Fig. 2F and G) revealed that both could drive the cell cycle progression into the S phase, providing cyclin-dependent momentum for cell proliferation. At the molecular mechanism level, the detection of Cyclin D1 gene expression (Fig. 2J) showed that the relative expression of this gene was significantly upregulated in the I-ApoEVs group, corroborating its proliferation-promoting function at the molecular level.Fig. 2A) EdU assay to detect the cell proliferation (scale bar = 200 μm) and B) corresponding quantitative analysis in each group. C) Proliferation ability of NPCs cultured with ApoEVs and I-ApoEVs by using CCK-8 assay. D) Fluorescent morphological images of living nucleus pulposus cells (Green) in different treatment groups at various time points (scale bar = 200 μm) and E) corresponding quantitative analysis in each group. F) Cell cycle analysis was performed by flow cytometry and G) the interpretation of results in each group (1 for control, 2 for ApoEVs, 3 for I-ApoEVs). H) Transwell assay (scale bar = 40 μm) and I) corresponding quantitative analysis. J) The mRNA expression levels of Cyclin D1 in different groups of NPCs. K) Wound healing assay on cell migration of NPCs (scale bar = 250 μm) and L) corresponding quantitative analysis. ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 2
The effects of apoptotic extracellular vesicles on the migration ability of NPCs were detected by scratch assay and Transwell assay. According to statistics (Fig. 2H and I), the number of migrated cells in the I-ApoEVs group was higher than that in the ApoEVs group, indicating that the I-ApoEVs group improved cell motility. As shown in the figures (Fig. 2K and L), after 12 and 24 h of treatment, the scratch closure rate of NPCs treated with I-ApoEVs was significantly higher than that of ApoEVs. These results suggest that I-ApoEVs are more effective in preventing NPC senescence through dual effects: 1) activating key regulatory factors of the cell cycle to promote proliferation and delay replicative senescence; 2) improving cell-ECM interactions to enhance migration and maintain tissue repair potential.
I-ApoEVs’ role in mitigating oxidative stress and senescence of NPCs
3.3
Hydrogen peroxide (H_2_O_2_) can stably induce the accumulation of reactive oxygen species (ROS) and trigger senescence-related phenotypes. The pathological progression of IVDD is closely associated with oxidative stress-driven cellular senescence [38]. SA-β-gal staining assay (Fig. 3A) combined with quantitative statistics (Fig. 3B) showed that the proportion of senescent cells in the H_2_O_2_-induced group was significantly increased, while the ApoEVs and I-ApoEVs treatment groups could significantly reduce the proportion of senescent cells, with I-ApoEVs exhibiting a more prominent anti-senescence effect. Malondialdehyde (MDA) impairs cell membrane integrity and damages matrix synthesis-related protein (Fig. 3C). Superoxide dismutase (SOD), as the core antioxidant enzyme for ROS scavenging in the organism, its activity directly reflects the cellular antioxidant capacity (Fig. 3D). I-ApoEVs achieve this by more efficiently enhancing SOD activity (strengthening the antioxidant defense system) and reducing MDA content (alleviating lipid peroxidation damage). A nucleus pulposus cell senescence model was successfully established via 24-h H_2_O_2_ treatment, and cell viability was evaluated using the CCK-8 assay (Fig. 3E). Results demonstrated that compared with the H_2_O_2_-induced senescent group, cell viability was significantly restored in both ApoEVs and I-ApoEVs treatment groups, with the I-ApoEVs group exhibiting a more prominent recovery effect. ROS fluorescence staining can directly reflect the intracellular ROS accumulation level (Fig. 3F). The fluorescence signal in the I-ApoEVs group was significantly reduced, and the fluorescence intensity was close to that of the normal group, indicating that I-ApoEVs were significantly more efficient than ApoEVs in scavenging excessive intracellular ROS.Fig. 3A) Bright-field morphological images of nucleus pulposus cells (NPCs) in different treatment groups. The images are presented at two magnifications: 100 × (top row) and 400 × (bottom row), with corresponding scale bars of 200 μm and 50 μm, respectively. B) Semi-quantitative analysis of SA-β-gal positive cells in different groups. C) levels of malondialdehyde (MDA) and D) Superoxide dismutase (SOD) activity were detected by corresponding kits in each group. E) Cell viability of NPCs during senescence induction was detected by the CCK-8 assay. F) Reactive Oxygen Species (ROS) staining of NPCs in different groups (scale bar = 100 μm) and H) corresponding quantitative analysis. G) P53 staining of NPCs in different groups (scale bar = 150 μm) and I) corresponding quantitative analysis. J-K) The mRNA expression levels of P16 and P21 in different groups of NPCs. ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Fig. 3
As a classic senescence-related tumor suppressor gene, fluorescence staining of P53 and quantitative analysis of fluorescence intensity directly demonstrated that I-ApoEVs could more effectively inhibit the accumulation of senescence-associated P53 protein, blocking or reversing the senescence process of NPCs at the protein level (Fig. 3G and I). Detection of P16 and P21 gene expression (Fig. 3J and K) showed that I-ApoEVs exerted a more prominent regulatory effect on the expression of senescence-related genes. P16 and P21 Western blot results showed that compared with the control group, H_2_O_2_ treatment significantly upregulated P16 and P21 protein expression, verifying the successful establishment of the H_2_O_2_-induced NPC senescence model (Fig. S8 in the Supplement Data). Notably, I-ApoEVs exerted a more significant downregulatory effect on P16 and P21 protein expression than ApoEVs (*p < 0.05), consistent with our previous qRT-PCR results at the mRNA level, further confirming the superior anti-senescence capacity of I-ApoEVs. The above results clarify the core advantage of I-ApoEVs in alleviating oxidative damage of NPCs and maintaining cellular homeostasis.
Impact of I-ApoEVs on the intervertebral disc ECM
3.4
The core function of NPCs is to synthesize and maintain the homeostasis of the extracellular matrix, which is mainly composed of type II collagen (COL II), aggrecan (ACAN), and glycosaminoglycans (GAGs). As the material basis for the intervertebral disc to maintain elasticity and load-bearing function, ECM homeostasis disruption—caused directly by NPC senescence—further exacerbates NPC senescence. The gene expression levels of COL II (Fig. 4A) and ACAN (Fig. 4B) reflect ECM synthesis capacity, while the expression of ADAMTS-5 (Fig. 4C) and MMP13 (Fig. 4D) indicates ECM degradation capacity. The results showed that I-ApoEVs had a more prominent regulatory effect on the expression of matrix synthesis genes, clarifying the bidirectional regulation of NPCs matrix synthesis and degradation-related genes.Fig. 4A-D) Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to evaluate the mRNA expression levels of extracellular matrix synthesis markers (COL II, ACAN) and extracellular matrix degradation markers (ADAMTs-5 and MMP13) in different groups of NPCs. E) Safranin O staining and Alcian Blue staining F) of NPCs in different media for 3 and 7 days (scale bar = 100 μm). G) Immunofluorescence staining of COL II and β-gal in each group (scale bar: 100 μm) and I-J) corresponding quantitative analysis. H) Immunofluorescence staining of ACAN and MMP13 in each group (scale bar = 100 μm) and K-L) corresponding quantitative analysis. ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 4
Safranin O staining (Fig. 4E) and Alcian blue staining (Fig. 4F) of NPCs intuitively presented the content and distribution of ECM in each group at the histological level. Dynamic results from day 3 to day 7 revealed that the I-ApoEVs group exhibited a uniform and dense staining pattern, which was closer to the normal state. This indicated that I-ApoEVs not only increased the total content of proteoglycans but also promoted the ordered deposition of PGs around cells, thereby restoring the spatial network structure of ECM. This structure can transmit normal mechanical signals through integrins, further inhibiting NPC senescence and breaking the vicious cycle of “senescence → ECM destruction → further senescence”. In contrast, the “patchy” staining in the ApoEVs group suggested that it could not fully restore ECM spatial homeostasis, resulting in limited repair effects.
In this study, multi-marker co-localization fluorescence staining (as shown in Fig. 4G and H) enabled simultaneous observation of the expression of multiple targets. The staining results revealed a spatiotemporal correlation between decreased COL II expression and increased β-gal expression in the H_2_O_2_ group, while treatment with ApoEVs and I-ApoEVs could synchronously reverse these two phenotypes. Fig. 4H further clarified the changes in the co-localization pattern of ACAN and MMP13, intuitively confirming the synergistic regulatory role of I-ApoEVs in inhibiting matrix degradation and promoting matrix synthesis. Combined with quantitative analysis of fluorescence intensity (Figures I-L), the improvement effect of I-ApoEVs showed more significant statistical differences across multi-dimensional detections. In summary, I-ApoEVs can maintain ECM homeostasis more persistently.
I-ApoEVs protect NPCs from apoptosis
3.5
The above results indicate that senescent NPCs exhibit reduced ability to repair oxidative damage. Oxidative stress essentially arises from an imbalance between intracellular ROS generation and scavenging. Excessive ROS can trigger apoptotic signaling pathways through multiple routes, serving as the “initiating factor” of apoptosis. TUNEL staining assay (Fig. 5A) combined with quantitative statistics (Fig. 5C) showed that the proportion of TUNEL-positive cells in the H_2_O_2_ group was significantly increased, while the ApoEVs and I-ApoEVs treatment groups could significantly reduce the proportion of apoptotic cells, with I-ApoEVs exhibiting a more prominent anti-apoptotic effect. Apoptosis flow cytometry further confirmed that I-ApoEVs could more efficiently reduce the loss of functional cells (Fig. 5E and F). As a classic anti-apoptotic protein, fluorescence staining and fluorescence intensity quantification of BCL-2 demonstrated that I-ApoEVs could block apoptosis by upregulating BCL-2 expression (Fig. 5B and D). As shown in the WB data, we validated the expression of γ-H2AX, BAX, and BCL-2: H_2_O_2_ treatment significantly increased the levels of γ-H2AX and BAX while decreasing BCL-2 expression, whereas I-ApoEVs exhibiting a more pronounced regulatory effect (Fig. S7 in the Supplement Data). These protein expression patterns were consistent with the mRNA expression trends observed in our previous quantitative real-time polymerase chain reaction (qRT-PCR) analysis (Fig. 5H–J), further confirming that ApoEVs (especially I-ApoEVs) effectively inhibit H_2_O_2_-induced cell apoptosis and DNA damage, thereby enhancing the reliability of our study conclusions.Fig. 5A) TUNEL staining (Green) shows the positive apoptotic NPCs in each group (scale bar = 200 μm). B) Immunofluorescence staining was performed to evaluate the expression of anti-apoptotic marker (BCL-2) in NPCs with different groups (scale bar = 100 μm) and D) corresponding quantitative analysis. C) Quantitative analysis of TUNEL positive cells in different groups. F) Cell apoptosis analysis was performed by flow cytometry and E) the interpretation of results in each group. G) The expression levels of γ-H2AX (DNA damage marker) in NPCs from different groups were detected by fluorescent staining (scale bar = 100 μm) and H) corresponding quantitative analysis. I-K) qRT-PCR was performed to evaluate the expression of anti-apoptotic marker (BCL-2) and apoptotic markers (Bax and Cleaved Caspase-3) in NPCs from different groups. ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 5
During apoptosis, the endogenous endonuclease of cells is activated, specifically cleaving DNA strands at linker regions. γ-H2AX fluorescence staining showed that the expression of γ-H2AX, a DNA damage marker, was significantly increased in the H_2_O_2_ group, while ApoEVs and I-ApoEVs could effectively inhibit its expression (Fig. 5G and H). Detection of Bcl-2, Bax, and Cleaved Caspase-3 gene expression revealed that ApoEVs and I-ApoEVs could significantly reverse this change in gene expression profile, with I-ApoEVs exerting a more prominent regulatory effect on gene expression (Fig. 5I–K). I-ApoEVs maintain cell numbers by inhibiting apoptosis, providing a “cellular basis” for extracellular matrix (ECM) synthesis.
Regulation of immune microenvironment of I-ApoEVs in vitro and vivo
3.6
Mounting evidence suggests an increased proportion of macrophages is detected in IVDD of humans and rodents, as well as in the surrounding muscle and adipose tissues [39]. Studies have reported that M1-type macrophages can exacerbate IVDD [40], while M2-type macrophages promote tissue repair and exert certain anti-inflammatory effects [9]. Regulating macrophage polarization can alleviate intervertebral disc degeneration. Immunofluorescence staining of CD86 and CD206 showed that I-ApoEVs significantly downregulated the expression of M1 marker CD86 and increased the expression of M2 marker CD206 (Fig. 6A and B). These results verify the function of I-ApoEVs in regulating macrophage polarization: they can inhibit M1 polarization and promote M2 polarization, exhibiting stronger anti-inflammatory and repair capabilities. Flow cytometry detecting M2 polarization marker CD206 showed: ∼0.6% positive rate in Control group, ∼51.1% in M2 group (model validated), and ∼25% in ApoEVs group (mild polarization promotion). In I-ApoEVs group, CD206 positive rate rose to ∼35%, with better M2 polarization-promoting effect than ApoEVs group (Fig. S6 in the Supplement Data). The M1 control was to clarify the “polarization direction” (verifying that macrophages do not polarize toward the pro-inflammatory M1 phenotype after I-ApoEVs treatment), while the newly added M2 positive control focused on the “polarization extent”. It could be clearly shown that I-ApoEVs did not merely upregulate M2 markers, but truly induced macrophages to polarize toward the functionally mature M2 phenotype, thus avoiding the concern of false-positive polarization. In contrast, the regulatory effect of ApoEVs on macrophage polarization is relatively weak. Cells were collected for qRT-PCR to detect the mRNA expression levels of M1-and M2-related inflammatory factors (Fig. 6C–G and I). These results showed that after treatment, the expression of M2 macrophage-related genes (Arg-1, CD163, and IL-10) was significantly increased, while the expression of M1 macrophage-related genes (iNOS, IL-1β, and TNF-α) was significantly decreased in both the ApoEVs and I-ApoEVs groups. These results were consistent with the aforementioned staining results.Fig. 6A) Immunofluorescence staining of macrophage polarization markers CD86 and CD206 in macrophages treated with different ApoEVs in vitro (scale bar = 25 μm) and B) corresponding quantitative analysis. C-E) qRT-PCR analysis was performed to evaluate the gene expression of M1 macrophage surface markers (iNOS, TNF-a and IL-1β) in macrophages cultured with different ApoEVs. F-G, I) qRT-PCR analysis was performed to evaluate the gene expression of M2 macrophage surface markers (Arg-1, IL-10 and CD163) in macrophages cultured with different ApoEVs. H) Immunofluorescence staining of M1 macrophage surface markers (CD86) and M2 macrophage surface markers (CD206) in IVD tissues in vivo (scale bar = 1 mm). J) Quantitative analysis of immunofluorescence staining for CD206 and CD86 in IVD tissues in vivo. ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Fig. 6
The inflammatory microenvironment of the intervertebral disc is closely related to the repair and remodeling of the injured site. Through immunofluorescence double staining of intervertebral disc tissue sections, the infiltration and polarization phenotype of macrophages in the degenerated intervertebral disc could be directly observed (Fig. 6H and J). In the I-ApoEVs group, the infiltration of CD86^+^ M1-type cells in the intervertebral disc were significantly reduced, while CD206^+^ M2-type cells were widely distributed in the nucleus pulposus area and injured site (uniform coverage of red fluorescence). The above results indicate that both ApoEVs and I-ApoEVs can regulate the inflammatory environment of the intervertebral disc and promote tissue regeneration by inducing M2 macrophage polarization, with I-ApoEVs exhibiting an overall superior effect compared to ApoEVs.
I-ApoEVs ameliorate IVDD development in rat in vivo
3.7
The signal intensity of MRI T2-weighted imaging is positively correlated with the hydration status of nucleus pulposus tissue—stronger signals indicate higher water content in the nucleus pulposus. MRI results combined with statistical analysis of Pfirrmann grading intuitively showed that intervertebral disc degeneration in the IVDD group progressed significantly over time, with a gradual loss of nucleus pulposus signal intensity (Fig. 7A and E). In contrast, the ApoEVs and I-ApoEVs treatment groups significantly delayed the degenerative process, with I-ApoEVs exhibiting a more prominent improvement effect. X-ray examination combined with statistical analysis of disc height index (DHI) further confirmed at the anatomical structure level that ApoEVs and I-ApoEVs effectively maintained disc height, providing a morphological basis for subsequent studies (Fig. 7C and F).Fig. 7A) Representative T2-weighted MRI images of rat coccygeal vertebrae at 4 and 8weeks after surgery and different treatment. B) Hematoxylin and eosin (HE) staining images of rat coccygeal vertebrae 4 and 8 weeks after treatment (upper scale bar = 1 mm, lower scale bar = 500 μm). C) X-ray images of the intervertebral disc of rat coccygeal at 4 and 8 weeks after treatment. D) Safranin-O/Fast Green (SF) staining images of rat coccygeal vertebrae 4 and 8 weeks after treatment (upper scale bar = 1 mm, lower scale bar = 500 μm). E) Pfirrmann grade analysis was used to quantitatively evaluate the disc degeneration degree. F) The changes of Disc Height Index (DHI) in each group were calculated by X-ray images. G) Histological score of different groups. I, K) Immunofluorescence of COL II in each group 4 and 8 weeks after treatment in IVD tissues in vivo (scale bar = 1 mm) and H, J) corresponding quantitative analysis of relative fluorescence intensity of COL II in different groups. ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 7
Hematoxylin-eosin (HE) staining (Fig. 7B) and Safranin O-fast green staining (Fig. 7D) collectively demonstrated that the IVDD group had significant loss of intervertebral disc matrix components, a marked reduction in nucleus pulposus area, and significant fibrous tissue infiltration, ultimately leading to near-fusion of intervertebral spaces and an increase in histological scores (Fig. 7G). The ApoEVs and I-ApoEVs treatment groups significantly improved these degenerative indicators; notably, the extracellular matrix staining intensity, nucleus pulposus structural integrity, and tissue layers in the I-ApoEVs group were closer to those of the normal group, confirming the superior therapeutic effect of I-ApoEVs at the histological level in vivo.
COL II fluorescence staining combined with fluorescence intensity quantification (Fig. 7H–K) showed that ApoEVs and I-ApoEVs significantly upregulated its expression. I-ApoEVs had a more prominent regulatory effect: it exerted positive regulation on COL II metabolism in the early stage, with fluorescence intensity close to that of the normal group, and continuously promoted COL II synthesis and matrix repair in the long term, showing a significant advantage in COL II fluorescence at 8 weeks.
I-ApoEVs improve NPCs viability and regulate immune microenvironment by activating STAT6
3.8
STAT6 (signal transducer and activator of transcription 6) is a member of the STAT family and is associated with the inflammatory phenotypes of various diseases [41]. Studies have shown that the expression level of STAT6 in IVDD affects the polarization status of M2 macrophages [42]. However, no relevant research has clarified how STAT6 and I-ApoEVs regulate intervertebral disc degeneration.
First, STAT6 fluorescence staining and quantification revealed that the expression level of STAT6 in the I-ApoEVs group was significantly higher than that in the ApoEVs group, indicating that I-ApoEVs can efficiently activate the STAT6 pathway (Fig. 8A and B). In contrast, after adding AS1517499, a specific inhibitor of STAT6, the STAT6 activation effect of I-ApoEVs was significantly blocked (Fig. 8C and D). SA-β-gal staining showed that I-ApoEVs could significantly reduce the positive rate of H_2_O_2_-induced nucleus pulposus cell (NPCs) senescence, while AS1517499 could reverse this effect (Fig. 8E and F). Cellular ROS fluorescence staining (Fig. 8G and Fig. S5 in Supporting Data)) and viability assay (Fig. 8H) further confirmed that I-ApoEVs inhibit oxidative stress and cellular damage by activating STAT6. Regarding the regulation of macrophage polarization, qPCR detection (Fig. 8I–L) indicated that I-ApoEVs could significantly upregulate the gene expression of M1 markers (iNOS, TNF-α) and simultaneously downregulate the expression of M2 markers (Arg-1, IL-10) after adding AS1517499. CD86/CD206 double staining and quantification intuitively showed that I-ApoEVs can inhibit M1 polarization and promote M2 polarization, and this effect can be blocked by AS1517499 (Fig. 8M and P). Finally, Safranin O staining of NPCs (Fig. 8N) and TNF-α/COL II double staining (Fig. 8O–Q and R) demonstrated that I-ApoEVs reduce the release of the pro-inflammatory factor TNF-α and promote COL II synthesis by activating STAT6, while AS1517499 can reverse these effects. In summary, these results support the hypothesis that I-ApoEVs’ therapeutic effects in NPCs are mediated through a STAT6-dependent mechanism, regulating macrophage polarization, inhibiting nucleus pulposus cell senescence and promoting ECM repair.Fig. 8A) Immunofluorescence staining of STAT6 (signal transducer and activator of transcription 6) in macrophages (scale bar = 50 μm) and B) corresponding quantitative analysis. C) Immunofluorescence staining and D) corresponding quantitative analysis of STAT6 in macrophages treated with or without specific STAT6 antagonist (AS1517499). E) β-gal staining of NPCs in different groups (scale bar = 100 μm). F) Semi-quantitative analysis of SA-β-gal positive cells in different groups. G) Immunofluorescence staining of ROS in NPCs (scale bar = 50 μm). H) Cell viability of NPCs during senescence induction was detected by the CCK-8 assay. I, J) qRT-PCR analysis of iNOS and TNF-α in macrophages cultured with different ApoEVs. K, L) qRT-PCR analysis of Arg-1 and IL-10 in macrophages. M) Immunofluorescence staining of CD86 and CD206 in macrophages (scale bar = 25 μm) and P) corresponding quantitative analysis. N) Safranin O staining of NPCs in different media for 3 days (scale bar = 100 μm). O) Immunofluorescence staining of COL II and TNF-α in each group (scale bar = 100 μm) and Q, R) corresponding quantitative analysis. The AS1517499 group means the I-ApoEVs treatment group with the addition of a specific STAT6 antagonist (AS1517499). ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Fig. 8
Inhibition of STAT6 can exacerbate the progress of IVDD
3.9
MRI (Fig. 9A) and Pfirrmann grading (Fig. 9B) showed that the degree of intervertebral disc degeneration in the I-ApoEVs group was significantly milder than that in the model group, and the disc height index (Fig. 9C) was also closer to that of the normal group. X-ray imaging further confirmed this repair effect at the gross structural level (Fig. 9D), while the STAT6 inhibitor AS1517499 could reverse the above effects. Regarding the regulation of the intervertebral disc inflammatory microenvironment, CD86/CD206 double staining of nucleus pulposus tissue indicated that the infiltration of CD86^+^ M1-type macrophages was significantly reduced and the proportion of CD206^+^ M2-type macrophages was increased in the I-ApoEVs group, and this polarization regulatory effect could be reversed by the STAT6 inhibitor AS1517499 (Fig. 9E and F). At the level of matrix repair and inflammation inhibition, Fig. 9G and H showed that the expression of the pro-inflammatory factor TNF-α was significantly decreased and COL II synthesis was significantly increased in the I-ApoEVs group. This effect was blocked after AS1517499 intervention, confirming that I-ApoEVs achieved the dual effects of “inhibiting inflammation and promoting matrix repair” in vivo by activating the STAT6 pathway. I-ApoEVs can effectively alleviate intervertebral disc degeneration by regulating the STAT6 signaling pathway, and its therapeutic effect can be reversed by a STAT6 inhibitor.Fig. 9A) Representative T2-weighted MRI images of rat coccygeal vertebrae at 4 weeks after surgery and different treatment. B) Pfirrmann grade analysis was used to quantitatively evaluate the disc degeneration degree. C) The changes of DHI in each group were calculated by X-ray images. D) X-ray images of the intervertebral disc of rat coccygeal at 4 weeks after treatment. E) Immunofluorescence staining of CD86 and CD206 in IVD tissues in vivo (scale bar = 500 μm) and F) corresponding quantitative analysis. G) TNF-α and COL II co-immunofluorescence staining of nucleus pulposus tissue in the intervertebral disc in vivo (scale bar = 500 μm) and H) corresponding quantitative analysis. The AS1517499 group means the I-ApoEVs treatment group with the addition of a specific STAT6 antagonist (AS1517499). ns, no significance (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Fig. 9
Discussion
4
During intervertebral disc (IVD) degeneration, nucleus pulposus (NP) cells undergo senescence, accompanied by reduced cell numbers and decreased synthesis of the extracellular matrix (ECM). Stem cell transplantation can exert therapeutic effects through cell differentiation and the release of active factors, but transplanted stem cells often undergo massive and rapid apoptosis [7,43]. This “paradoxical phenomenon” between high apoptosis rate and therapeutic efficacy has not been clarified. Notably, existing studies have confirmed that stem cell apoptosis is not a simple cell death process; it can also participate in tissue repair and inflammation regulation [7]. A special type of extracellular vesicle called apoptotic extracellular vesicles (ApoEVs) is released during cell apoptosis. Studies have found that ApoEVs are not merely waste products of cell apoptosis but can regulate bone homeostasis [44], metabolic homeostasis [45] during tissue regeneration in vivo. However, there are no clear research reports or conclusions on whether ApoEVs secreted by BMSCs can effectively repair degenerated intervertebral discs.
Apoptosis, a specific form of programmed cell death, is a crucial process for the development and homeostasis maintenance of multicellular organisms [46]. Apoptotic cells undergo a series of biological events, including cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA cleavage [47], followed by the formation of membrane-enclosed vesicles through membrane budding or blebbing—known as apoptotic extracellular vesicles (ApoEVs) [38]. Unlike cell debris or apoptotic byproducts, ApoEVs can mediate intercellular communication by transporting bioactive molecules [44]. Mesenchymal stem cell (MSC)-derived ApoEVs possess core functions similar to those of MSCs: they can exert immunomodulatory effects on various immune cells such as macrophages [48], and exhibit tissue regeneration-promoting efficacy in multiple disease models including myocardial infarction, osteoporosis, and muscle injury [17,44,49]. Compared with direct MSC transplantation, ApoEVs can also effectively avoid potential risks such as immune rejection and tumorigenicity. ApoEVs have emerged as highly promising therapeutic tools, attracting widespread attention for their application value in auxiliary diagnosis, inflammation regulation, regenerative therapy, and other fields. To further enhance the repair efficacy of MSC-derived extracellular vesicles in IVDD, researchers have attempted to pretreat MSCs through various strategies. Among these, hypoxic pretreatment is the most commonly used strategy in current MSC-derived EV research, as it significantly enhances the biological functions of EVs [50]. In vitro studies typically expose BMSCs to a normoxic environment of 21% O_2_, which is notably inconsistent with the physiological microenvironment in vivo—characterized by a hypoxic state of 2%–8% or lower. Compared with ApoEVs obtained under normoxic conditions, hypoxic-pretreated MSC-derived apoptotic bodies exhibit superior efficacy in promoting the proliferation and migration of BMSCs, as well as inducing chondrogenic differentiation [9].
During the progression of IVDD, NPCs produce large amounts of inflammatory factors and chemokines, which induce the migration of macrophages toward intervertebral disc [51]. Macrophage polarization plays a crucial role in regulating this core pathological process. M1-type macrophages induce NPC senescence by releasing pro-inflammatory factors to activate related signaling axes. Transmission electron microscopy observations have shown that NPCs in an M1-polarized microenvironment exhibit typical senescent morphological features such as nuclear membrane folding and lysosome proliferation [52]. In contrast, M2-type macrophages can effectively scavenge ROS and maintain mitochondrial homeostasis, exerting the opposite regulatory effect [53]. Single-cell sequencing data reveal that M2-type macrophage-conditioned medium increases the production of COL II and ACAN, while reducing the expression levels of senescence-related genes [54]. End-stage IVDD is characterized by ECM fibrotic remodeling, a process influenced by macrophage subsets through differential regulation of the TGF-β signaling pathway [55]. M1-type macrophages promote NPC transdifferentiation and abnormal collagen deposition via related pathways, whereas M2-type macrophages inhibit the process through multiple mechanisms [56]. Single-cell transcriptome studies indicate that the M2c subset significantly expresses the fibronectin ED-A isoform, which may be involved in regulating the dynamic balance of fibrotic microenvironment remodeling. In summary, macrophage polarization is a key regulatory factor in the pathological progression of IVDD, providing a highly promising target for treatment.
This study innovatively adopted hypoxic and inflammatory combined microenvironment to pretreat BMSCs, successfully preparing functionally enhanced I-ApoEVs. By regulating cell senescence-related signaling pathways, I-ApoEVs significantly delay the senescence and apoptosis of NPCs, while upregulating the synthesis of ECM. I-ApoEVs precisely target and activate the STAT6 signaling pathway, efficiently inducing the polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. This not only inhibits the release of pro-inflammatory factors but also promotes the secretion of anti-inflammatory factors, fundamentally remodeling the local inflammatory microenvironment and blocking the inflammatory-mediated degeneration. Other studies have also confirmed that the anti-inflammatory and reparative effects of mesenchymal stem cell-derived extracellular vesicles mostly depend on STAT6 pathway activation. Irisin-modified ones drive M2 polarization via the JAK2-STAT6 pathway to ameliorate sepsis-induced lung injury [57]. Chitosan-coated artesunate-modified extracellular vesicles alleviate ulcerative colitis by activating STAT6 to promote M2 polarization [30]. These studies corroborate that STAT6 is a conserved target for extracellular vesicles to exert anti-inflammatory and reparative effects, with its efficacy validated in multiple disease models. Endowed with advantages including simple preparation process, high biosafety, and clear mechanism of action, I-ApoEVs provide an innovative and practical new direction for the clinical translational treatment of IDD.
Conclusions
5
This study confirms that I-ApoEVs derived from BMSCs pretreated with a hypoxic-inflammatory combined microenvironment exhibit superior repair efficacy for IVDD compared to conventional ApoEVs. This finding not only clarifies the mechanism of action of I-ApoEVs but also suggests that “synergy between cell protection and immune regulation” may be a key entry point for IVDD treatment, providing a solid theoretical basis for the clinical translational application of I-ApoEVs.
Consent to participate and consent to publish declarations
All authors have made substantial contributions to the work, revised it critically, given final approval of the version to be published, agreed to be accountable for all aspects of the work, and consent to the submission and publication of this manuscript.
Clinical trial number
Not applicable.
Ethics approval
The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Hebei Medical University Third Hospital, with the approval number 2024-51. All efforts were made to minimize animal suffering.
Funding
This article is supported by the China National Natural Science Foundation (82072454).
CRediT authorship contribution statement
Weiqi Zhang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – original draft, Writing – review & editing. Xiaowei Ma: Data curation, Methodology, Software, Writing – original draft. Han Yin: Conceptualization, Investigation, Methodology, Software. Dazhuang Miao: Investigation, Methodology, Software, Validation. Tianhao Guo: Conceptualization, Formal analysis, Investigation, Software. Wei Chen: Conceptualization, Methodology, Resources. Zhiyong Hou: Formal analysis, Methodology, Project administration, Supervision. Yingze Zhang: Conceptualization, Project administration, Resources, Supervision, Writing – review & editing. Xianda Gao: Conceptualization, Investigation, Methodology, Software, Supervision, Writing – review & editing. Di Zhang: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare no conflict of interest in the study.
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