All the experimental protocols were approved by the Ethics Committee of Wuxi 9th Affiliated Hospital of Soochow University and were obtained with the informed consent of the patients. Normal nucleus pulposus tissues were obtained from five patients (2 females and 3 males, aged 18–49 years) who underwent spine surgery of burst thoracolumbar fracture and degenerative nucleus pulposus, and the annular fibers tissues were gently obtained from 11 patients (4 females and 7 males, aged 37–61 years) who underwent surgery of disc excision for lumbar degenerative disease. Nucleus pulposus tissues were washed three times with PBS, then cut into pieces, and digested with 1 mg/ml of type II collagenase (Solarbio, China) for 4 h at 37°C. After being filtered through a 70-μm filter, the suspension was centrifuged at 300 × g for 5 min, and the isolated cells were cultured in Complete Culture Medium of Mesenchymal Stem Cell (Cyagen, China) containing 88% basal medium, 10% MSC cell-qualified fetal bovine, 1% penicillin-streptomycin, and 1% glutamine. Annular fibers tissues were washed three times with PBS, then cut into pieces, and digested with 1 mg/ml of type I collagenase (Solarbio, China) for 2 h at 37°C. After being filtered through a 70-μm filter, the suspension was centrifuged at 300 × g for 5 min, and the isolated cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco, United States) containing 10% fetal bovine serum (FBS) (Gemini, China) and 1% penicillin–streptomycin (Gibco, USA). Finally, all the isolated NPSCs and AFCs were incubated at 37°C in a humidified atmosphere of 5% CO₂ with the culture medium replaced every 3 days.

The NPSCs were identified by flow cytometry analysis and multipotential differentiation. The multipotential differentiation of NPSCs was determined using an MSCs Adipogenic Differentiation Kit (Cyagen, China) and an MSCs Osteogenic Differentiation Kit (Cyagen, China), respectively. Oil red O staining and Alizarin red staining were used to assess adipogenic and osteogenic differentiation. For the detection of surface markers by flow cytometry, NPSCs were stained for 30 min with fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated anti-human CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR monoclonal antibodies, and characterized using flow cytometry (BD Biosciences, USA). The monoclonal antibodies were all purchased from BioLegend, Inc.

The AFCs were identified by immunofluorescence analysis and toluidine blue staining. For the detection of type II collagen immunohistochemical staining, AFCs were incubated with primary antibody type II collagen (1:200, Abcam, USA) at 4°C overnight. After PBS washing three times, cells were incubated with FITC-conjugated goat-anti-rabbit secondary antibody (1:1,000, Beyotime, China) at room temperature for 1 h before counterstaining with DAPI (Solarbio, China). Images were obtained using a fluorescence microscopy (Olympus, Japan). For toluidine blue staining, AFCs stained with 1% toluidine blue for 30 min at room temperature before fixing in 4% paraformaldehyde for 30 min, then briefly rinsed in ethanol and observed using an inverted biological microscope (Olympus, Japan).

N-NPSCs and D-NPSCs were cultured in exosome-free medium for 2 days. NPSC-exo was isolated from supernatant by ultracentrifugation. The culture medium was centrifuged at 1,000 × g for 10 min at 4°C to remove dead cells and centrifuged at 10,000 × g for 30 min at 4°C to remove cell debris. Next, exosomes were centrifuged by ultracentrifugation at 100,000 × g for 70 min after filtering through 0.22-μm membrane filters. Moreover, exosomes were purified by a commercial kit using ExoJuice (WeinaBio, China) according to the instruction of the manufacturer. Briefly, exosome samples were transferred to 5-ml ultracentrifuge tubes and 500 µl of Exojuice was added to the bottom. The tube was then centrifuged at 100,000 × g for 70 min at 4°C, carefully recovered, and fractionated from the bottom. The first 200 µl of the liquid from the bottom of the tube was discarded, then the next 200 µl fraction of solution from the bottom was collected, which contained the purified exosomes.

After purification, morphology was observed by transmission electron microscopy, particle diameter and concentration were analyzed by flow nano analysis, and the exosomal markers, such as CD9, CD81, and TSG101 (Affinity, USA) were detected by Western blotting assay.

Cell counting kit-8 (CCK-8) assay was used to value the proliferation capacity of AFCs. Briefly, AFCs were plated in 96-well plates at a density of 5 × 10³ cells per well with 100 μl of complete culture medium. After 24 h, cells were treated with exosomes (or miRNA mimic/inhibitor) and incubated for 48 h, respectively. Finally, 10 μl of CCK-8 reagents (APExBIO, USA) was added to each well and then incubated for another 2 h at 37°C. The absorbance value at 450 nm was detected by a microplate reader (Biorad imark, USA).

Transwell assay was used to value the migration capacity of AFCs. Briefly, AFCs (5 × 10⁴ cells per well) in 100 μl of serum-free medium was transferred to the upper chambers of the Transwell, then 600 μl of medium treated with exosomes (or miRNA mimic/inhibitor) was added to the bottom chambers, as previously described, respectively. After 24 h, cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Then the cells were observed and the stained migrated cells were counted by using a microscope.

Exosomes or cells were lysed with RIPA buffer, then the protein concentration was measured using the BCA protein detection kit (Beyotime, China). Each sample was mixed with protein loading buffer (5×) and heated at 95°C for 10 min to denature. Then protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Germany). PVDF membranes were blocked with 5% skim milk for 1 h, and primary antibody (MMP-13, collagenase II, IGFR1, p-PI3K, p-AKT, GAPDH, and β-ACTIN) (Bioss, China) was added in for incubating overnight at 4°C, then the secondary antibody conjugated with horseradish peroxidase was incubated for 2 h at room temperature. Fluorescence was analyzed using the enhanced chemiluminescence kit (Tanon, China) and imaged by the luminescent image analyzer (Tanon, China), and the absorbance value of each band was calculated using the ImageJ software.

Apoptosis rates were evaluated using an Annexin V/PI apoptosis detection kit (BD Biosciences, USA) according to the instruction of the manufacturer. Annexin V-FITC (5 μl) and PI (5 μl) solutions were added into 100 μl of cell suspension, respectively. The cells were incubated at room temperature for 15 min in the dark, cells were detected, and analyzed by flow cytometry (Beckman, USA).

Exosomal miRNAs were sequenced in N-NPSC-exo and D-NPSC-exo. The total RNA was isolated from the exosomes using TRIzol reagent (Life Technologies, United States) according to the instructions of the manufacturer. The extracted RNA was then quantified using the OneDrop (WuYi, China). The sample quality control, library preparation, and sequencing were performed by HuaYing, China.

According to the results of the miRNA sequencing of exosomes, the mimics and inhibitors of let-7b-5p and the control were synthesized by WeinaBio (Foshan, China).

AFCs were transfected with miRNA mimics or control at 100 nM with Lipofectamine 3,000 reagent (Invitrogen, USA) and then cultured for 48 h. To further confirm the effects of miRNAs in exosomes, AFCs were cultured in medium with D-NPSC-exo (50 μg/ml) and then were transfected with miRNA inhibitors (100 nM) or control for 48 h. Cell proliferation, migration, and apoptosis was detected by CCK-8 assay, Transwell assay, and flow cytometry analysis, and the relative proteins were detected by Western blotting.

The total RNA was isolated from the exosomes and cells by using TRIzol reagent (Life Technologies, USA). For miRNA, the first-strand cDNA was synthesized by using miRNA 1st Strand cDNA Synthesis Kit (Vazyme, China), then real-time PCR was performed by miRNA Universal SYBR qPCR Master Mix (Vazyme, China) under LightCycler480 Real-Time PCR Detection System (Roche, Switzerland). For mRNA, the first-strand cDNA was synthesized by using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China), then real-time PCR was performed by Universal SYBR qPCR Master Mix (Vazyme, China). U6 was used as an internal reference for miRNAs, and beta-actin was for mRNAs. Comparative quantification was determined using the 2^−ΔΔCt method. The primer sequences used are summarized in Supplementary Table S1.

GraphPad Prism 8 software was used for statistical analysis. Data are expressed as mean ± standard deviation. The differences between the two groups were analyzed with the unpaired t-test. A p-value < 0.05 was considered statistically significant.

Isolated NPSCs displayed fibroblast-like morphology and vortex-styled adherent growth in culture (Figure 1A). The results of induced differentiation in vitro showed visible calcium deposits formed and stained bright red by Alizarin red staining after osteogenic differentiation, and lipid droplets appeared and stained red by Oil red O staining after adipogenic differentiation (Figure 1B). Based on the flow cytometry analysis, NPSCs were positive for surface markers CD29, CD44, CD73, and CD105 (>90%), and negative for surface markers CD34, CD45, and HLA-DR (<2%) (Figure 1C). AFCs displayed a long spindle or polygon shape (Figure 1D), and expressed type II collagen by immunofluorescence staining (Figure 1E left panel). The toluidine blue staining showed AFCs were purple and exhibited metachromatic granules (Figure 1E right panel).

Morphology, diameter, and surface markers of NPSC-exo were characterized by transmission electron microscopy, flow nano analysis, and Western blotting, respectively. NPSC-exo was displayed as cup-shaped vesicles using transmission electron microscopy (Figure 1F). The mean diameter of the exosomes was 66.64 nm, and the concentration of exosomes was 8.8 × 10⁹ particles/ml using flow nano analysis (Figure 1G). Western blotting results showed that NPSC-exo were positive for exosomal surface markers TSG101, CD81, and CD9 (Figure 1H).

To explore the effect of the NPSC-exo in AFCs, N-NPSC-exo and D-NPSC-exo in concentrations of 10 and 50 μg/ml were incubated with AFCs, respectively. Then cell proliferation, migration, apoptosis, and extracellular matrix metabolism were analyzed using CCK-8 assay, Transwell migration assay, flow cytometry, and Western blotting, respectively. Our results showed that D-NPSC-exo in low and high concentrations also significantly inhibited cell proliferation (Figure 2A), suppressed cell migration (Figure 2B), and promoted the apoptosis of AFCs (Figure 2C). On the contrary, N-NPSC-exo on high concentration tended to promote cell proliferation and migration, and inhibit cell apoptosis. In order to detect the effect of NPSC-exo on extracellular matrix metabolism in AFCs, protein expression levels of Col II, and MMP13 were detected by Western blotting. D-NPSC-exo in high (50 μg/ml) concentrations significantly promoted the expression of MMP13 and inhibits the expression of Col II. However, the N-NPSC-exo had no effect on extracellular matrix secretion in AFCs (Figure 2D).

To detect the different miRNA constituents of N-NPSC-exo and D-NPSC-exo, miRNA sequencing was conducted. A clustered heat map (Figure 3A) shows the upregulated and downregulated miRNA expressions, and a volcano plot (Figure 3B) shows log 2 (fold change) (N-NPSC-exo vs. D-NPSC-exo) on the x-axis and −log 10 (p-value) on the y-axis. Ten significantly up-/downregulated miRNAs in N-NPSC-exo compared with D-NPSC-exo were listed (Figure 3C), and let-7b-5p was upregulated in D-NPSC-exo on the microarray data. Consistent with the sequencing results, the expression of let-7b-5p was upregulated in D-NPSC-exo compared with N-NPSC-exo by real-time PCR analysis (Figure 3D).

The results of CCK-8 assay and transwell assay showed that overexpression of let-7b-5p by transfecting with let-7b-5p mimic significantly inhibited proliferation (Figure 4A) and migration (Figure 4B) of AFCs. The transfection of let-7b-5p mimic significantly promoted the apoptotic ratio compared with the NC mimic by Annexin V/PI apoptosis detection kit (Figure 4C). Moreover, the decreased protein level of Col II and elevated MMP13 expression demonstrated that overexpression of let-7b-5p mimic affected the synthesis of the extracellular matrix (Figure 4D).

To further confirm that D-NPSC-exo inhibited proliferation, migration, and extracellular matrix synthesis, and promoted apoptosis of AFCs by delivering let-7b-5p, the let-7b-5p inhibitor was applied to transfect with D-NPSC-exo-treated AFCs. The overall effects of D-NPSC-exo on AFCs were abolished by the let-7b-5p inhibitor. Compared with the NC inhibitor groups, the cell proliferation (Figure 5A) and migration (Figure 5B) of AFCs were promoted, and cell apoptosis (Figure 5C) of AFCs was reduced. Meanwhile, the protein expression of MMP13 was reduced, and the protein expression of Col II was promoted (Figure 5D). Collectively, exosomal let-7b-5p plays important roles in cell degeneration of AFCs.

Finally, we explored the mechanism by which exosomal let-7b-5p aggravated cell degeneration of AFCs. A binding site at 3′-UTR of IGF1R was predicted on let-7b-5p by TargetScan (Figure 6A). The result of real-time PCR confirmed that the mRNA expression of IGF1R was significantly decreased in AFCs by transfecting with let-7b-5p (Figure 6B). Similarly, this decrease in the mRNA expression of IGF1R appeared in D-NPSC-exo-treated AFCs (Figure 6C). To test whether exosomal let-7b-5p modulates IGF1R and the PI3K/Akt signal pathway, protein expression levels of IGF1R, p-PI3K, and p-AKT in AFCs transfected with let-7b-5p mimic were detected by Western blotting. The result of Western blotting confirmed that the protein expressions of IGF1R, p-PI3K, and p-AKT were decreased in AFCs by transfecting with let-7b-5p (Figure 6D). Similarly, this decrease in the protein expressions of IGF1R, p-PI3K, and p-AKT appeared in D-NPSC-exo-treated AFCs (Figure 6E). Collectively, exosomal let-7b-5p leads to targeted inhibition of IGF1R followed by inhibiting the activation of the PI3K/Akt pathway in AFCs.