Exosomes from damaged disc cells carrying miR-27a-3p may worsen disc degeneration by triggering inflammatory immune cells

This study was conducted with the approval of the Ethical Review Committee of Xijing Hospital and written informed consent was obtained from all patients. Disc tissues were collected from 13 patients diagnosed with IVDD and idiopathic scoliosis disease. The patients’ age was 41.9 ± 21.5 (range 16 to 71 years). The NP samples were digested with 0.2% pronase (Gibco, Grand Island, NY, USA) for 40 min with gentle shaking, followed by incubating at 37 °C for 4 h with 0.025% Type II collagenase with gentle shaking. Then, the samples were passed through a 70 mm nylon mesh to remove any remaining tissue pieces. Subsequently, the NP cells (NPc) were incubated at 37 °C in DMEM/F12 medium supplemented with 10% foetal bovine serum (FBS). No ascorbic acid was applied to the media.

Serial centrifugation with ultracentrifugation was used to isolate dNPc-exo and nNPc-exo. Briefly, non-degenerated and degenerated NPc were inoculated with 1 × 10⁶ cells and grown in basal medium of DMEM-F12. When the cells reached 80% confluence, exosome-free FBS (Thermo Fisher Scientific, Waltham, MA, USA) was used to culture cells. After 48 h, collected the medium and centrifuged at 300×g (4 °C) for 10 min to remove dead cells. Then, obtained the supernatant and centrifuged at 2000×g (4 °C) for 20 min to remove cell debris. Next, the supernatant was centrifuged at 10,000×g (4 °C) for 30 min to remove large EVs. To remove substructures and vesicles larger than 220 nm, we filtered the supernatant through a 0.22-µm filter (Millipore, US). Then, pelleted the exosomes using ultracentrifugation at 100,000×g for 80 min (4 °C). After resuspending in sterile PBS, the exosomes were stored at -80 °C.

To determine the exosomes morphology, we dropped 10uL of exosomes suspensions onto a 400 mesh carbon-coated copper grid and allowed it to dry for 20 min. The grids were then rinsed with PBS, fixed with 1% glutamate for 10 min, and rinsed with deionized water. To further stain the grids, they were treated with uranyl oxalate for 10 min.

The exosomes microstructure was scanned via TEM (Hitachi H-7650, Tokyo, Japan). Nanoparticle tracking analysis (NTA) was used to analyze the exosomes particle concentration and size distribution using ZetaView PMX 110. The samples were diluted with PBS and administered under controlled flow. The data was analyzed via ZetaView Software 8.04.02.

Pierce BCA Protein Assay Kit (Cat#23,225, Thermo Fisher Scientific, Waltham, USA) was used following the manufacturer’s instructions to determine the exosomes protein concentration. Briefly, exosomes were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) and the proteins were collected. 96-well plate was added with 10 µL protein samples and 200 uL of working reagent. Then, after incubating the plate at 37 °C for 30 min, the absorbance was measured at 562 nm. The protein concentration of exosomes samples was determined using the standard curve method.

THP-1 human leukemia mononuclear cells were obtained from the Cell Bank of Chinese Academy of Sciences. The cells in exponential growth phase and with good growth conditions were seeded uniformly in 10 cm diameter culture dishes at a 1 × 10⁶ cells/ml density and stimulated with PMA (Sigma-Aldrich, Saint Louis, USA) (100 ng/ml) for 24 h to induce adherent M0 macrophages. The success of the induction was determined by monitoring changes in cell surface markers and morphology.

PKH26 red fluorescence cell linker mini kit (Sigma MINI26-1KT) were labeled with the exosomes following the manufacturer’s instructions. In brief, the exosomes were suspended in Diluent C solution, and PKH26 was diluted in Dilution C. The mixture was incubated at 37 °C for 5 min and then 8 mL of 15% complete medium of exosome-free was added to quench the staining reaction. Then, the exosomes were incubated overnight at 4 °C and centrifuged for 70 min at 100,000×g. Next, PBS (200 µl) were applied to resuspend the exosomes and then incubated with M0 macrophages, which were induced by THP-1 cells, for 24 h. Subsequently, the M0 macrophages were washed with PBS and fixed with 4% paraformaldehyde (PFA). The M0 macrophages were then permeabilized with Triton X-100. After blocking with 10% goat serum, the cells were incubated overnight with the primary antibody against F4/80 (1:100, ab16911, Abcam) and then stained with secondary antibodies and DAPI.

To investigate whether NPc-exo could carry miR-27a-3p to affect macrophages, NPc was transfected with the Cy3-labelled miR-27a-3p (Tsingke Biotechnology, China). Subsequently, exosomes were purified from the transfected cells and used to co-culture with M0 macrophages. The results were detected using immunofluorescence as described previously. Briefly, F4/80 (1:100, ab16911, Abcam) and DAPI were used to stain M0 macrophages. Olympus FluoView 1200 confocal microscope was used to acquire images.

Well-conditioned NPc or THP-1 cells were seeded in cell culture plates. Once the cells reached 60–80% confluency, they were transfected with miR-27a-3p mimic (Ribo Bio, Guangzhou, China), miR-27a-3p inhibitor (Ribo Bio, Guangzhou, China), plasmid of PPARγ (Tsingke Biotechnology, China) or siRNA against PPARγ (#AM16708, Invitrogen by Thermo Fischer Scientific, MA USA), along with the corresponding Negative Control (NC), at a concentration of 100 nM using HiPerFect transfection reagent (Qiagen, Germantown, MD, USA) or Lipofectamine 3,000 (Invitrogen, MA, United States) following the recommended protocols.

To assess the target genes expression in dNPc-exo, total RNA was extracted using RNAiso for Small RNA (Takara, Shiga, Japan) following the provided protocols. Then, a NanoDrop ND-2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to confirm the purity and concentration of the total RNA. the miRNA 1st strand cDNA synthesis kit (Accurate Biology, China; Cat#AG11717) was used to perform the reverse transcription and all primers were commercially synthesized by Tsingke Biotechnology Co., Ltd. The RT-qPCR was conducted with SYBR Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, China; Cat#AG11719) using the CFX96 Real-Time System (Bio-Rad, Hercules, CA). The primer sequences can be found in Additional Table 1 of this study.

For the identification of exosomes through western blot analysis, Alix, HSP90, and TSG101 markers were used. RIPA lysis buffer (Beyotime, Shanghai, China) was used to collect the exosomes protein samples and Pierce TM BCA Protein Assay Kit (Cat#23,225, Thermo Fisher Scientific, Waltham, USA) was used to determine the concentration. The western blot analysis was performed as previously described. In brief, protein samples were separated on SDS-PAGE gels and detected using appropriate antibodies. Western blot analysis was performed using following antibodies: CD9 (1:1000; #13,174, CST, USA), CD63 (1:1000; ab134045, Abcam), TSG101 (1:1000; #72,312, CST, USA). Anti-mouse IgG or anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody (1: 2000; CST, USA) was used as a secondary antibody, and the chemiluminescent signals were visualized using ECL Western Blot detection kit (WBKLS0100, Millipore).

In the vitro experiment, the protein extracts were separated on SDS-PAGE gels and probed with the following primary antibodies: Anti-CoL2 (1:1000; 28459-1-AP, Proteintech), Anti-iNOS (1: 1000; ab283655, Abcam), Anti-PI3K (1:500; 20584-1-AP, Proteintech), Anti-AKT (1:5000; 60203-2-Ig, Proteintech), Anti-p-AKT (1:5000; 66444-1-Ig, Proteintech), Anti-PPARγ (1: 1000; ab178860, Abcam), Anti-NFκB (1:1000; 10745-1-AP, Proteintech), Anti-MMP3 (1:1000; 17873-1-AP, Proteintech), Anti-CA12 (1:4000; 15180-1-AP, Proteintech), Anti-HIF-1 alpha (1:4000; 20960-1-AP, Proteintech), Anti-TNF alpha (1: 1000; ab6671, Abcam), Anti-GAPDH (1: 1000; ab8245, Abcam). Anti-mouse IgG or anti-rabbit IgG, horseradish peroxidase (HRP)- linked antibody (1: 2000; CST, USA) was used as a secondary antibody, and the immunoreactive bands were visualized by Bio-RAD imaging system (Bio-Rad, CA) and ECL Western Blot detection kit (WBKLS0100, Millipore).

For flow cytometry analysis, single cells were collected and suspended. Cells were then incubated with 1% bovine serum albumin and incubated with CD86-FITC antibody (1:100; 305,213, Biolegend). Subsequently, the cells were determined with a flow cytometer (ACEA NovoCyte) and the data was analyzed using NovoExpress 1.5.6 flow cytometer software.

The cells were fixed with 4% PFA for 30 min. After that, the cells were permeabilized with 0.5% Triton X-100, blocked with goat serum and incubated with primary antibodies against F4/80 (1:50; ab16911, Abcam), iNOS (1:50; ab283655, Abcam). Next, secondary antibodies (1:100; Proteintech) were utilized followed by DAPI staining. The images were required with confocal laser scanning microscope and analyzed via ImageJ software. To facilitate quantitative comparison of immunofluorescence signals, all images within the same experiment were captured using the identical exposure time.

RNA extraction was carried out on dNPc-exo and nNPc-exo samples using RNAiso for Small RNA kit (Takara, Shiga, Japan). RNA quality was assessed for degradation and contamination, and concentration and purity were determined. Small RNAs were reverse transcribed into cDNA to create a cDNA library after being cut into 18-30nt. Illumina HiSeqTM 2500 by Gene Denovo Biotechnology Co (Guangzhou, China) was used to sequence the cDNA. Raw reads were filtered, aligned, and microRNAs were identified. Further analysis included miRNA expression profiles, Principal Component Analysis of miRNA, clustering of miRNA expression patterns, identification of differentially expressed miRNAs (DE miRNA), target gene predictions, and functional enrichment of target genes. DE miRNA was identified based on a fold change greater than 1.5 and a Q-value less than 0.001. Thresholds were established for up-regulated and down-regulated miRNAs.

For the change of Col2A1 and Aggrecan content with the IVDD severity, NP tissues were fixed in 4% PFA and 20% Sucrose solution. Next, the tissue samples were sectioned to a thickness of 7 μm and blocked with 5% goat serum. Immunohistochemical staining was performed using Anti-aggrecan (1:400; 13880-1-AP, Proteintech) and Anti-CoL2 (1:400; 28459-1-AP, Proteintech) primary antibodies. The sections were then developed with DAB solution (GeneTech, China) and counterstained with hematoxylin. Microscopy was used to capture images and analyzed using ImageJ software.

To verify the miR-27a-3p combination capacity with PPARγ, the pmirGLO-luciferase vector was used, into which the wild-type PPARG 3′-UTR and mutant 3′-UTR were inserted. HEK293T cells were co-transfected with 100 ng of reporter plasmid and 30 nM of miR-27a-3p mimic or control using Lipofectamine 3000 transfection reagent and Opti-MEM medium. After 48 h, the dual luciferase reporter assay was performed using the Dual Luciferase Reporter Assay System, and the firefly luciferase activity was normalized using Renilla luciferase activity. The corresponding primers are listed in Supplementary Table.

Animal procedures were followed the guidelines of the Institutional Ethics Review Board of Xijing Hospital. The rat IVDD models were constructed by needle puncturing, as previously described [12].

To investigate the impact of miR-27a-3p on macrophages M1 polarization and IVDD progression in vivo, we extracted NPc from Sprague-Dawley (SD) rats and regulated the expression of miR-27a-3p using mimic or inhibitor. The exosomes of the manipulated NPc were then collected. Then, SD rats (200–250 g) were used to construct IVDD model. In brief, Hypnorm and Dormicum were used to anesthetize the SD rats. The caudal spine was identified by palpation. A longitudinal incision was made in the rat tail skin, and the muscle tissue and subcutaneous connective tissue were separated. Next, an 18-gauge sterile needle was inserted into the disc center to a 5 mm depth, rotated 360 degrees and held for 30 s. Then, a total of 5µL rat-miR-27a-3p-mimic or rat-miR-27a-3p-inhibitor was injected into the rat model via a 27G needle. We monitored the rats daily after surgery to ensure their health condition. After 4 weeks, the rats were put to death and IVDs were obtained from different groups for further experiments. The degenerative control (DC) group consisted of rats treated with PBS after puncture surgery, while the normal control (NC) group consisted of rats that underwent no surgery. A total of 30 SD rats were used for the animal experiment. The primer sequences of miR-27-3p mimic or inhibitor can be found in Additional Table 1.

The disc height of IVD was assessed using X-ray (GE XR650, USA) Digital X-ray fluoroscopy and micro-computed tomography scanning (PerkinElmer, Waltham, USA). Radiographs of the caudal vertebrae at the experimental level (Co7/8) were taken from rats both before and four weeks after surgery. The disc height index (DHI) was calculated using Image J software and a published method [12], and the DHI% (postoperative DHI/preoperative DHI) was used to express changes in DHI. 3D and 2D reconstructions were generated via CTvox software.

For the analysis of rat tail data, a MRI scanner (Philips Eclipse, Aachen, Germany) was utilized. The NP structure was evaluated using a T2-weighted sequence. The parameters for the MRI scanner were set as follows: a spin-echo repetition time of 3000ms, an echo time of 90ms, and no phase wrap. Two researchers separated evaluated the signal intensity of rat intervertebral discs on T2-weighted sagittal images and used the Pfirrmann classification to assess the rat IVDD grades. The data were analyzed with Image J.

The paraffin-embedded tissue specimens were cut into 8 mm sections. H & E staining and immunofluorescence were then performed on the specimens. The histological score of IVD degeneration were evaluated via a grading scale as previously described [13]. Two independent observers assessed the degree of IVDD blindly. A normal intervertebral disc has a histological score of 5 points, while a moderately degenerated intervertebral disc has a score of 6–11 points. A severely degenerated intervertebral disc, on the other hand, has a score of 12–14 points.

To investigate the impact of M1 macrophages on the progression of IVDD, we extracted bone marrow-derived macrophages (BMMs) from 8-week old Sprague-Dawley (SD) male rats. BMMs were extracted following a previously described method [14]. Briefly, the SD rats were euthanized, and the femurs were cut at both ends. MEM-α medium (Gibco, Grand Island, NY, USA) was then flushed through the femurs using a syringe to collect the bone marrow. Tissue debris was removed using a 70 μm cell strainer, and the BMMs were cultured in MEM-α medium supplemented with 10% FBS. To induce differentiation of M0 macrophages into M1 macrophages, we stimulated them with 10 ng/mL LPS (Sigma, L2630). Subsequently, the rat-NPc was seeded in 6-well plates (Corning), while the rat M1 macrophages were cultured in transwell inserts (0.4 μm pores; Corning) placed in the same well as rat-NPc. A 1:1 mixture of DF12 and MEM-α medium was added to the 6-well plates.

Statistical analysis was conducted with GraphPad Prism 8.3.0 software. Student’s t-test was used to compare two groups. For group > 2, one-way or two-way ANOVA test was used. Statistical significance was set at P < 0.05. All data were presented as mean ± standard error of mean (SEM). Graphic abstract, Figs. 1G and 6J were created by Figdraw (www.figdraw.com↗).

In this study, NP cells were acquired from IVDD or idiopathic scoliosis patients (Fig. 1A). The results of immunohistochemistry confirmed the degeneration of IVD by showing a reduced expression of Aggrecan and Col2A1 in degenerated IVD compared to that of the idiopathic scoliosis group (Fig. 1D and E). The number of macrophages, especially M1 macrophages, significantly increased in degenerated IVD tissue, as shown by immunofluorescence staining and flow cytometry analysis (Fig. 1B, C and F, Fig S1). Additionally, western blot analysis was used to detect the NP markers CA12 and HIF-1α (Fig. S3 A, B). Differential centrifugation was used to isolate and collect dNPc-exo and nNPc-exo from NPc culture medium (Fig. 1G). TEM analysis revealed that NPc-exo exhibited a classic “cup-shape” morphology, and the average diameter was around 100 nm (Fig. 1H). The nanoparticle tracking analysis (NTA) found that most exosomes were below 200 nm in size and had a peak diameter of 120 nm (Fig. 1I). Western blot analysis showed high expression of CD9, CD63 and TSG101 in dNPc-exo (Fig. 1J). To confirm if dNPc-exo could be internalized by M0 macrophages, PKH26 was used to label dNPc-exo and added to M0 macrophages cultures. Punctated red fluorescence was observed after incubation, indicating internalization of dNPc-exo by macrophages. F4/80 was used to show the macrophages cellular outline as background staining (Fig. 1K).

To investigate the effect of dNPc-exo on macrophages polarization, dNPc-exo and nNPc-exo were co-cultured with M0 macrophages for 48 h, respectively. Following analysis showed that dNPc-exo could induce M1 polarization of macrophages compared with nNPc-exo (immunofluorescence staining, Fig. 2A, B. RT-qPCR, Fig. 2C. western blot, Fig. 2D, E. flow cytometry. Figure 2 F, G).

To investigate the mechanism of dNPc-exo induced macrophages M1 polarization, RNA-seq was used to analyze the gene expression of nNPc-exo and dNPc-exo. miRNAs that expressed differently between the two groups were detected and further analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Among these microRNAs, seven microRNAs (hsa-miR-136-3p, miR-27-y, hsa-miR-485-5p, miR-7070-x, novel-m0003-5p, hsa-miR-27a-3p, novel-m0001-5p) were significantly upregulated in dNPc-exo. As shown in Fig. 2H and I, the heatmap indicates the two-way hierarchical clustering results of miRNAs. The distribution of miRNA in the two groups were displayed using a volcano plot (Fig. 2J). And then, the KEGG Pathway analysis results showed the relative signaling pathways (Fig. 2K). Next, the expression of seven microRNAs (hsa-miR-136-3p, miR-27-y, hsa-miR-485-5p, miR-7070-x, novel-m0003-5p, hsa-miR-27a-3p, novel-m0001-5p) in dNPc-exo were confirmed (Fig. 2L). Subsequently, miRNA mimics were used to upregulate the seven miRNAs expression in M0 macrophages, and iNOS gene expression was validated by RT-qPCR. Among these miRNAs, miR-27a-3p showed significant effect of inducing M1 polarization of macrophages (Fig. 2M).

To confirm whether dNPc-exo could transport miR-27a-3p into macrophages, Cy3-labelled miR-27a-3p mimic were incubated with dNPc. The dNPc-exo were isolated and co-cultured with M0 macrophages. Immunofluorescence staining results showed a red fluorescent signal in macrophages co-cultured with the Cy3-labelled dNPc-exo, indicating that miR-27a-3p was transferred from dNPc to macrophages via dNPc-exo (Fig. 3A). After co-culturing, miR-27a-3p expression in M0 macrophages was upregulated in macrophages of the dNPc-exo co-culture group as compared with nNPc-exo and control group (Fig. 3B and C). To confirm the effect of dNPc-exo on macrophages miRNA expression, dNPc-exo was incubated with M0 macrophages. As shown in Fig. 3D, compared with nNPc-exo and control group, miR-27a-3p expression was upregulated in macrophages of the dNPc-exo group. Subsequently, dNPc-exo, miR-27a-3p mimic or miR-27a-3p inhibitor was incubated with M0 macrophages. We found that iNOS expression were up-regulated in macrophages in the dNPc-exo and miR-27a-3p mimic group, indicating that both dNPc-exo and miR-27a-3p could promote M1 polarization of macrophages (Fig. 3E and F). Collectively, these results revealed that dNPc-exo-carried miR-27a-3p could effectively promote M1 polarization of macrophages.

To determine the pathway targets of miR-27a-3p on macrophages, the potential target genes were predicted via Targetscan (http://www.targetscan.org/↗). We discovered that PPARγ has a binding sequence with miR-27a-3p (Fig. 4A). This was further supported by the luciferase reporter assay (Fig. 4B). Then, we found that the dNPc-exo-mimic group, where dNPc was co-cultured with miR-27a-3p mimic, showed a significant increase in the expression level of miR-27a-3p compared to the dNPc-exo group. This suggests that the incubation with miR-27a-3p mimic enhanced the loading efficiency of miR-27a-3p into exosomes (Fig. 4C). Studies have shown the essential role of PPARγ/NFκB/PI3K/AKT signaling pathway in the regulation process of macrophages polarization [15, 16]. Therefore, plasmid or siRNA was used to regulate PPARγ gene expression in M0 macrophages. The analysis (Fig. 4D, F and G) revealed that PPARγ upregulation led to a significant increase in PI3K and AKT expression, while PPARγ downregulation resulted in a reduction of these proteins. Additionally, PPARγ upregulation led to a decrease in NFκB expression, while PPARγ downregulation increased its expression. The M1 polarization marker iNOS and pro-inflammatory marker TNF-α was found to be downregulated in the PPARγ upregulation group, whereas iNOS and TNF-α was upregulated in PPARγ downregulation group. And then, miR-27a-3p mimic or inhibitor was used to regulate miR-27a-3p expression in M0 macrophages. As shown in Fig. 4E, H and I, miR-27a-3p levels were significantly increased in miR-27a-3p mimic-treated M0 macrophages, while decreased in miR-27a-3p inhibitor-treated M0 macrophages. The results of western blot and RT-qPCR analysis showed that miR-27a-3p mimic downregulated the expression of PPARγ, PI3K and AKT but enhanced the expression of NFκB in M0 macrophages. Conversely, miR-27a-3p inhibitor enhanced the expression of PPARγ, PI3K, and AKT while downregulated the expression of NFκB in M0 macrophages. Additionally, the expression of M1 polarization marker iNOS and TNF-α was increased in the miR-27a-3p mimic group while decreased in the miR-27a-3p inhibitor group. Collectively, the results revealed that miR-27a-3p carried by dNPc-exo can regulate M1 polarization of macrophages by targeting the PPARγ/NFκB/PI3K/AKT signaling pathway.

To investigate the polarization effects of dNPc-exo-carried miR-27a-3p, we collected the dNPc-exo and co-cultured it with M0 macrophages. The western blot and RT-qPCR analysis revealed that the expression of NFκB, M1 polarization marker iNOS and pro-inflammatory marker TNF-α was increased, while the expression of PPARγ, PI3K and AKT was decreased after incubation (Fig. 5A, C and D). Additionally, miR-27a-3p mimic or inhibitor was incubated with dNPc, and the exosomes were collected and co-cultured with M0 macrophages. The results showed that miR-27a-3p could increase the expression of NFκB, M1 polarization marker iNOS, while decrease the expression of PPARγ, PI3K and AKT (Fig. 5B). These findings indicate that dNPc-exo is sufficient to induce M1 polarization of macrophages by transferring miR-27a-3p and targeting PPARγ/NFκB/PI3K/AKT signaling pathway.

To examine the impact of miR-27a-3p on the inducing of macrophages M1 polarization, a rat IVDD model was created using needle puncture. Besides, the rat NPc was isolated and cultured. Subsequently, the rat NPc was co-cultured with miR-27a-3p-mimic, miR-27a-3p-mimic NC, miR-27a-3p-inhibitor, or miR-27a-3p-inhibitor NC. The RT-qPCR analysis revealed that the expression level of miR-27a-3p was upregulated in the miR-27a-3p-mimic group, while it was downregulated in the miR-27a-3p inhibitor group after incubation (Fig. S2). Then, the rat model was intradiscally injected with PBS, exo-miR-27a-3p-mimic, or exo-miR-27a-3p-inhibitor using a 27G needle after surgery. After 4 weeks, the degree of disc degeneration was evaluated using disc height index (DHI) and Pffirmann grade through X-ray, MRI, and Micro-CT examinations. X-ray observations showed minimal changes in DHI of the exo-miR-27a-3p-inhibitor group, which was similar to the NC group (Fig. 6A and B). In the DC group, severe collapse of the IVD space was observed. However, the group treated with exo-miR-27a-3p-mimic showed a more severe collapse compared to the DC group. This indicates that miR-27a-3p could worsen the degradation of IVD.

The results from the MRI images and the Pffirmann grade (Fig. 6C and D) indicate that the IVD of the exo-miR-27a-3p-mimic group was significantly more degenerated than that of the NC and DC group. However, the exo-miR-27a-3p-inhibitor group showed significantly healthier results than the exo-miR-27a-3p-mimic group. Similar trends were observed in the Micro-CT images (Fig. 6E).

To confirm the histology analysis results, H & E staining was performed. The results of the staining (Fig. 6F and G) showed that exo-miR-27a-3p-inhibitor group had a relatively small loss of NP tissue. However, in the DC and exo-miR-27a-3p-mimic group, the NP tissue was gradually replaced by AF. Subsequently, immunofluorescence staining was used to demonstrate the impact of miR-27a-3p on macrophages M1 polarization. The outcome elucidated that the exo-miR-27a-3p-mimic groups had a higher iNOS expression compared to the DC group (Fig. 6H and I). These results collectively suggest that miR-27a-3p can induce M1 polarization of macrophages and contribute to IVDD in vivo.

Then, we isolated the BMMs of rat and used LPS to stimulate it into M1 macrophages. Subsequently, rat-nNPc was co-cultured with M1 macrophages (Fig. 6J), and the western blot analysis revealed a decrease in Col2A1 expression in the nNPc-M1 macrophages group. Conversely, the expression of MMP3 and TNF-α was found to be increased (Fig. 6K and L). These results suggest that M1 macrophages have the ability to enhance the expression of pro-inflammatory cytokines and matrix metalloproteinase in rat-nNPc, thus aggravating the degradation of IVD.

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Supplementary Material 4