Animal ethic statement
This study strictly adheres to internationally recognized animal welfare and ethics guidelines. All experimental protocols have been approved by Honghui Hospital, Xi’an Jiaotong University’s ethical review committee. We have taken great care to ensure the welfare of the mice involved in the experiment. All animals are fed according to appropriate standards and live in suitable environmental conditions. In conducting the experimental procedures, every possible measure has been taken to minimize the pain and discomfort of the animals. At the conclusion of the experiment, humane euthanasia was performed on the animals.
Construction of IDD model in mice
For this study, 6–8 week-old SPF-grade C57BL/6J male mice (219, Beijing Weitonglihua Experimental Animal Technology Co., Ltd., Beijing, China) weighing between 18 and 25 g were used. All mice were housed in individual cages in an SPF-grade animal facility with a controlled lighting schedule of 12 h of darkness and 12 h of light, humidity at 60% ∼ 65%, and temperature at 22 ∼ 25 ℃. Mice had ad libitum access to food and water, and after one week of adaptation, they were observed for their health before the start of the experiment.
The method for constructing the IDD animal model in mice is as follows: Mice were deeply anesthetized using a 2% isoflurane-O2 mixture (1 L/min, R510-22-10, RWD, Shenzhen, China). Under sterile conditions, the experimental mice were placed on a surgical table, and the intervertebral disc area was exposed. The intervertebral disc on one side was punctured using a 27G needle to induce IDD. To ensure the efficacy of the degeneration, the needle was rotated 360° after puncture and held for 30 s. After modeling, the success of the procedure was confirmed through radiological examination and histological analysis. For the control group mice, similar surgical procedures were performed, but IDD was not induced. After completing all surgeries, mice received appropriate postoperative care and monitoring [33, 34].
X-ray imaging examination
The mice were anesthetized with 2% isoflurane (1 L/min) prior to the examination and were positioned in a prone position for the imaging procedure. X-ray images were captured using an exposure time of 40 s, a distance of 40 cm, and a setting of 3 mA/35 kV on Philips X-ray film. The height of the intervertebral space before and after the surgery was measured and expressed as the Intervertebral Disc Height Index (DHI). Surgimap 2.3.2.1 software (Nemaris, New York) was utilized for the measurements. For detailed measurement and calculation methods, refer to Figure S4A [35].
H&E staining
Hematoxylin and eosin (H&E) staining was performed using the Hematoxylin and Eosin Staining Kit (C0105S, Beyotime, Shanghai, China) to observe the pathological changes in the intervertebral disc tissue of mice. Partial intervertebral disc tissue from mice was fixed in 4% paraformaldehyde, followed by dehydration, transparency, and embedding in paraffin. 5 μm thick sections were obtained using a microtome and then subjected to deparaffinization, hydration, staining with hematoxylin, rinsing with distilled water, immersion in 95% ethanol, and subsequent staining with eosin. Differentiation with 70% hydrochloric acid ethanol, dehydration, and transparency was performed before finally mounting the sections with neutral gum. The morphological changes in the intervertebral disc tissue of the mice were observed using an optical microscope [35].
Safranin O-Fast green staining
The Safranin O-Fast Green Staining kit (G1371, Beijing Solabio Technology Co., Ltd., Beijing, China) was used to stain and observe the distribution of acidophilic bone and basophilic cartilage in the intervertebral disc. Following the instructions provided, the specific staining procedure was performed as follows: initially, the slides were stained with Safranin O for one hour; excess dye was then removed by rinsing with distilled water, and subsequently, gradient ethanol (50 − 80%) was used for decolorization. Subsequently, the slides were stained with Fast Green for 60 s and dehydrated using absolute ethanol. Finally, the sections were mounted in a clean xylene-based mounting medium for 5 min, followed by immediate sealing with neutral resin. The staining results were evaluated using a histological scoring method described in the literature, which assessed the staining outcomes from five different perspectives. Each category’s score ranged from 1 to 3, with a cumulative score between 5 and 15, indicating a higher score corresponding to a higher degree of degeneration [36, 37].
Immunohistochemical staining
The paraffin blocks were cooled on ice or in a refrigerated fridge set at 4 °C before embedding and sectioning. The paraffin sections were left to dry overnight while the slides were placed in an oven set at 60 °C and baked for 20 min. Subsequently, the sections were soaked in xylene for 10 min, followed by another 10-minute immersion after changing the xylene. After 5 min of hydration in absolute alcohol, a second hydration was conducted by replacing the absolute alcohol with fresh absolute alcohol for another 5 min. The sections were then sequentially hydrated in 95% and 70% ethanol, with a 10-minute soak in each type of alcohol, and finally rinsed in distilled water for 5 min. The sections were immersed in citrate buffer (pH 6.0) and heated in a high-power microwave oven for 8 min, then cooled to room temperature. The slides were washed thrice in PBS (pH 7.2–7.6), each for 3 min. To inactivate endogenous peroxidase, 3% H2O2 was added and left at room temperature for 10 min. The sections were subsequently washed three times in PBS, each time for 3 min. The sections were blocked with normal goat serum blocking solution (E510009, SBS Genetech, Shanghai, China) at room temperature for 20 min.
After incubation, the sections were incubated overnight at 4 °C with primary antibodies against COLII, Aggrecan, MMP-3, COX-2, and CFIm25 (ab34712, ab216965, ab52915, ab179800, and ab183660, respectively, Abcam, UK), diluted at 1:100, 1:100, 1:1000, 1:100, and 1:500, respectively. The sections were then washed three times with PBS and incubated with secondary antibodies, goat anti-rabbit IgG (ab6721, diluted at 1:5000, Abcam, Cambridge, UK), for 30 min. Afterward, the sections were incubated with SABC (Streptavidin-Biotin Complex, P0603, Beyotime, Shanghai, China) at 37 °C for 30 min. DAB chromogen (P0203, Beyotime, Shanghai, China) was added to each specimen, followed by a 6-minute incubation for color development. Counterstaining was performed with a hematoxylin solution for 30 s. The sections were subsequently dehydrated in 70%, 80%, 90%, 95% ethanol, and finally in absolute ethanol, each for 2 min. The sections were then immersed in xylene twice for 5 min before being mounted with neutral resin. Observations and statistical analysis were conducted using a BX63 upright microscope (Olympus, Japan), with five random high-power fields selected per slide. Image-Pro Plus 6.0 software was utilized to analyze the average pixel density [36]. This experiment was repeated three times.
Preparation of IVD tissue for NP
Lumbar paravertebral exposure surgery was performed on male C57BL/6J mice (n = 4, aged 6–8 weeks). In the experimental group, mice underwent unilateral IVD injury using a 27G needle, while the sham control group underwent the same lumbar paravertebral exposure surgery without injury. At 2, 4, and 8 weeks post-surgery, euthanasia was conducted, and NP tissue was isolated and pooled from the intervertebral disc, ensuring the removal of peripheral muscles, ligaments, and attachments (Figure S1). The groups included the Sham group (n = 1, sham surgery), the 2-week recovery group (n = 1, 2 weeks post-surgery), the 4-week recovery group (n = 1, 4 weeks post-surgery), and the 8-week recovery group (n = 1, 8 weeks post-surgery). The isolated NP tissue was digested in a culture medium containing 0.2% type II collagenase (17101015, Thermo Fisher, USA) and 0.3% trypsin (10165921001, Sigma-Aldrich, UK), followed by centrifugation at 1000 rpm for 10 min at 37℃ in a 5% CO2 environment to obtain cell pellets. The culture medium was removed, and cells were resuspended in PBS. The suspension was filtered through a 70 μm filter to remove unwanted cell fragments. After another centrifugation at 1000 rpm for 10 min, the resulting cell pellet was resuspended in PBS, obtaining single-cell transcriptome sequencing (scRNA-seq) samples [33].
scRNA-seq
We collected samples of mouse NP tissue at 2 weeks, 4 weeks, and 8 weeks after model simulation. The Sham group represents normal mouse NP tissue, while the IDD group represents IDD in mouse NP tissue. Each group consisted of one sample. The cell viability and integrity of the samples were assessed and counted using a microscope and flow cytometer. Qualified individual cells were captured using the C1 Single-Cell Auto Prep System (Fluidigm, Inc., South San Francisco, CA, USA) (Figure S1). After cell capture, mRNA was extracted, and cDNA was synthesized through reverse transcription. The cDNA, post-lysis, and reverse transcription were pre-amplified on a microfluidic chip for subsequent sequencing. The amplified cDNA was used to construct libraries and subjected to single-cell sequencing on the HiSeq 4000 Illumina platform, with parameters set as paired-end reads, read length of 2 × 75 bp, and approximately 20,000 reads per cell [38].
scRNA-seq data analysis
The data was analyzed using the “Seurat” package in R software. Quality control criteria were set as 200 < nFeature_RNA < 5000 and percent.mt < 20 to filter out highly variable genes. To reduce the dimensionality of the scRNA-Seq dataset, a Principal Component Analysis (PCA) was performed on the top 2000 highly variable genes. The first 20 principal components were selected for downstream analysis using the Elbowplot function in the Seurat package. The main cell subgroups were identified using the FindClusters function in Seurat with the default resolution set at res = 1. The UMAP algorithm was then applied to achieve nonlinear dimensionality reduction of the scRNA-seq sequencing data. The Seurat package was utilized to identify marker genes for various cell subgroups, and the “SingleR” package was used for cell annotation. Cell communication analysis was conducted using the “CellChat” package in R, while cell trajectory analysis was performed with the “Monocle2” package. Differential expression genes (DEGs) in the scRNA-Seq dataset were identified using the “Limma” package in R, focusing on DEGs between different samples [39, 40].
Preparation of IVD tissue for high-throughput transcriptome sequencing
Male C57BL/6J mice (n = 3), aged 6–8 weeks, underwent surgical exposure of the L4-L6 lumbar vertebrae through the peritoneum. Single-sided injury to the L4-5 and L5-6 IVDs was performed using a 27G needle. After a 4-week recovery period, the mice were euthanized, and NP tissue was isolated and pooled from either L4-5 and L5-6 discs (IDD group, representing IDD) or L2-3 and L3-4 discs (Control group, Normal control). Peripheral muscles, ligaments, and attachments were carefully removed during tissue dissection (Figure S2). The isolated NP tissue was digested in a culture medium containing 0.2% type II collagenase and 0.3% trypsin at 37 °C and 5% CO2 for 4 h. The culture medium was then centrifuged at 1000 rpm for 10 min to obtain a cell pellet. The culture medium was aspirated, and the cells were resuspended in PBS. The suspension was filtered through a 70 μm filter to remove unwanted cell debris. Subsequently, the cell pellet was resuspended in PBS after centrifugation at 1000 rpm for 10 min to obtain the high-throughput transcriptome sequencing samples.
RNA purity and integrity were verified using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher) to measure the RNA sample concentration by OD260/280, ensuring the absence of protein and organic contamination. The RNA concentration was determined using the Qubit RNA Assay Kit (Q33221, Thermo Fisher, USA). Total RNA samples meeting the following criteria were used for subsequent experiments: RNA integrity number (RIN) ≥ 7.0 and 28 S:18 S ratio ≥ 1.5 [33, 41].
High-throughput transcriptome sequencing
The sequencing libraries were generated and sequenced by CapitalBio Technology (Beijing, China). A total of 5 µg of RNA was used for each sample. Briefly, we employed the Ribo-Zero Magnetic Kit (MRZG12324, Epicentre, USA) to remove ribosomal RNA (rRNA) from the total RNA. Subsequently, the Illumina NEB Next Ultra RNA Library Prep Kit (E7760S, NEB, USA) was utilized to construct the sequencing libraries. The RNA was fragmented using NEB Next First Strand Synthesis Reaction Buffer (5x), resulting in fragment lengths of approximately 300 base pairs (bp). Next, the first-strand cDNA was synthesized using reverse transcription primers and random primers, followed by the preparation of the second-strand cDNA in the second-strand synthesis reaction buffer containing dUTP Mix (10x). The cDNA fragments were subjected to end repair, including the addition of polyA tails and ligating sequencing adapters. After the ligation of Illumina sequencing adapters, the second strand of the cDNA was digested using the USER enzyme (M5508, NEB, USA) to construct a strand-specific library. Subsequently, the library DNA was amplified and purified, followed by PCR enrichment. Finally, library identification was conducted using the Agilent 2100 system, and quantification was performed using the KAPA Library Quantification Kit (KK3605, Merck, USA). Ultimately, paired-end sequencing was carried out on the Illumina NextSeq CN500 platform [42, 43].
Transcriptome sequencing data analysis
The raw sequencing data was examined for the quality of paired-end reads using FastQC software v0.11.8. Preprocessing of the raw data involved removing Illumina sequencing adapters and poly (A) tails sequences using Cutadapt software 1.18. A perl script was employed to eliminate reads with an N content exceeding 5%. Furthermore, the FASTX Toolkit software 0.0.13 was used to extract reads with a base quality of at least 20, accounting for 70% of the bases. The BBMap software was utilized to correct the paired-end sequences. Eventually, the filtered high-quality read fragments were aligned to the mouse reference genome using hisat2 software (0.7.12).
Differential analysis was performed on mRNA read counts using the “Limma” package in the R programming language. The criteria for selecting differentially expressed genes were set as |log2FC| > 1 and P.value < 0.05. The “heatmap” package in R generated a heat map displaying the intersecting genes. The protein-protein interaction (PPI) relationships of key factors were analyzed using the STRING database (https://string-db.org/), with a minimum required interaction score set to 0.700. The Cytoscape 3.5.1 software was used to visualize the network of interaction relationships, and the built-in CytoHubba tool was employed to identify core genes.
For the aforementioned intersecting genes, functional enrichment analysis of Gene Ontology (GO) was conducted using the “ClusterProfiler” package in R, including analysis of biological processes (BP), molecular functions (MF), and cellular components (CC). The results of GO enrichment were visualized through bubble plots and circle plots, with a screening criterion of P < 0.05. Additionally, based on the p-values, the “ClusterProfiler” package in R was used to conduct the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for the candidate targets. The results of the KEGG enrichment analysis were illustrated through bubble plots and circle plots [41].
Isolation and handling of mouse NP cells
After euthanizing the mice, the skin, muscles, and other soft tissues from the lumbar and tail regions were aseptically removed. The intervertebral disc tissue was extracted and washed several times with PBS to eliminate blood and impurities. The NP tissue was then dissected and placed in DMEM/F12 medium (11320033, Thermo Fisher, USA). It was transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min to remove the supernatant, retaining the pellet. PBS was added for three additional washes. The tissue was minced and placed in a digestion solution containing 0.25% trypsin (25200056, Thermo Fisher, USA) and EDTA and digested at 37℃ for 30 min. During digestion, pipetting was required to separate cells. After digestion, the supernatant was removed by centrifugation at 1000 rpm for 5 min, followed by three washes with PBS. Subsequently, the tissue was exposed to DMEM/F12 medium containing 0.1% type II collagenase at 37℃ for 4 h, followed by three washes with PBS. Finally, the cells were seeded in appropriate culture plates using DMEM/F12 complete culture medium containing 10% FBS (10100147 C, Thermo Fisher, USA) and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, 15140163, Thermo Fisher, USA), and cultured at 37℃ with 5% CO2. The culture medium needed to be regularly replaced, and cell growth was monitored [36, 44].
The isolated NP cells were seeded in a 12-well plate. Once the cells adhered to the surface, they were treated with IL-1β (10 ng/mL, HY-P7073, MedChemExpress, USA) or TNF-α (50 ng/mL, HY-P7090, MedChemExpress, USA) for 48 h to induce NP cell degeneration. After treatment, fresh culture medium was replaced, or cells and culture medium were collected after 24 h for subsequent analysis and experiments, such as cell viability assays and gene and protein expression analysis, to evaluate the effectiveness of the degeneration model [45].
Immunofluorescence staining
In addition to the culture medium, cells were rinsed three times with PBS for 2 min each time. They were then fixed with − 20℃ ice-cold methanol for 30 min, and excess methanol was removed. After that, they were washed three times with PBS for 5 min each time. The cells were treated with 0.1% Triton X-100 at room temperature for 15 min, followed by three washes with PBS for 5 min each time. Blocking was performed with BSA for 30 min, and then rabbit anti-COLII or Aggrecan antibodies (catalog numbers PA5-99159 and PA1-1746, respectively, diluted at 1:500, Thermo Fisher, USA) were added and incubated at 37℃ for 60 min. The cells were washed with PBS for 5 min three times. Goat anti-rabbit secondary antibodies labeled with FITC (A-11008 or A-11012, diluted at 1:500, Abcam, UK) were added and incubated at 37℃ in the dark for 60 min, followed by three washes with PBS for 3 min each time. The cells were stained with DAPI for 10 min, and then excessive DAPI was removed by washing with PBS. Finally, a 20 µL mounting medium was added to let it dry before observing and capturing images with a fluorescence microscope. For quantitative analysis, the fluorescence coverage area in six randomly selected fields of view was measured using a 40× objective lens, and the average was calculated [35].
Alcian blue staining
NP cells were uniformly seeded in a 6-well plate, with each well containing a 2 mL complete culture medium to provide optimal growth conditions. Alcian Blue staining of cells was performed using the alcian blue staining kit (G1563, Beijing Solabel Science & Technology Co., Ltd., Beijing, China), following the detailed instructions provided by the manufacturer. The fluorescence area was measured, and the density of each image was integrated using Image-Pro Plus 6.0 software to analyze the relative staining level of each image while ensuring consistency in parameters [36].
Enzyme-linked immunosorbent assay (ELISA)
The serum or supernatant collected from each group of mice or NP cells was subjected to ELISA using the ELISA assay kit (Elabscience, Wuhan, China) according to the manufacturer’s instructions to detect the expression of IL-6, iNOS, TNF-α, and IL-1β. The kit numbers used were E-EL-M0044, E-EL-M0696, E-EL-M3063, and E-EL-M0037. In brief, standard samples and specimens were transferred to individual wells and incubated to allow protein binding with immobilized antibodies. After washing, biotinylated specific antibodies were added. After a certain incubation period, unbound biotinylated antibodies were washed away, followed by the addition of HRP conjugate and subsequent washing. 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate was added, turning the solution blue, which further turned yellow upon the addition of the stop solution. The optical density (OD) values were measured at 450 nm after subtracting the blank well values. The expression of each inflammatory factor was calculated based on the protein concentration curve of standard samples [45].
Cell viability assay using CCK-8
The NP cells to be tested were digested and resuspended, and the cell concentration was adjusted to 1 × 105 cells/mL. The cells were seeded in a 96-well plate with 100 µL per well and incubated overnight. Cell viability was determined using the CCK-8 assay kit (C0041, Beyotime, Shanghai, China) according to the manufacturer’s instructions at 12, 24, 36, and 48 h after incubation. For each measurement, 10 µL of CCK-8 detection reagent was added and incubated at 37℃ with 5% CO2 for 1 h. The absorbance at 450 nm was then measured using an enzyme-linked immunosorbent assay reader to calculate cell viability [46].
Edu staining
The NP cells to be tested were seeded in a 24-well plate with a density of 1 × 105 cells per well, and each cell group was replicated in 3 different wells. A solution of 5-Ethynyl-2’-deoxyuridine (Edu) at 10 µmol/L was added to the culture medium from ST067 (Beyotime, Shanghai, China). The plate was then incubated in a cell culture incubator for 2 h. The culture medium was removed, and the cells were fixed with a PBS solution containing 4% paraformaldehyde for 15 min at room temperature. Subsequently, the cells were washed twice with PBS containing 3% BSA. A PBS solution containing 0.5% Triton-100 was added, and the cells were incubated at room temperature for 20 min. After two washes with PBS containing 3% BSA, 100 µL of the staining solution was added to each well, and the plate was incubated at room temperature in the dark for 30 min. DAPI was then added to stain the cell nuclei for 5 minutes. Following that, 6–10 random fields of view were observed under a fluorescence microscope (FM-600, Shanghai Putian Optical Instrument Co., Ltd.), and the number of positive cells in each field was recorded. The Edu labeling rate (%) was calculated as the number of positive cells divided by the sum of positive and negative cells multiplied by 100% [46]. Each experiment was performed in triplicate.
Transwell migration experiment
After various treatments, the NP cells were subjected to a Transwell migration assay. First, the NP cells were diluted in basal culture medium without FBS to a 2.5 × 104 cells/mL. Then, 100 µL of the cell suspension was added to the upper chamber of each Transwell, while 500 µL of medium containing 10% FBS was added to the lower chamber. After 24 h, the upper chamber was removed, and cells in the upper chamber were gently removed using a cotton swab. Next, the cells were fixed with a solution containing 4% paraformaldehyde at room temperature for 30 min. Subsequently, the cells were stained with 0.1% crystal violet (C0121, Beyotime, Shanghai, China) for 30 min. Five random areas were selected, and the cells were photographed and counted under an inverted microscope (IXplore Pro, Olympus, Japan) [47]. Each experiment was performed in triplicate.
Scratch experiment
On the bottom surface of a 6-well plate, evenly draw lines at intervals of 0.5–1 cm using a ruler and a marker. Each hole should be intersected by at least 5 lines. NP cells were seeded into the wells at a density of 5 × 105 cells per well and allowed to grow until reaching 100% confluence. A scratch was made using a 200 µL pipette tip perpendicular to the marked lines, followed by a medium change to serum-free culture medium. The distance between the wound edges was measured and recorded under an optical microscope (model: DM500, Leica) at 0 h and 24 h. Images of the cell groups were captured using an inverted microscope to observe their migration ability. Image-Pro Plus 6.0 was used to analyze the distance between the scratches, and the wound healing rate was calculated according to the following formula [48].
$$Wound\;healing\;rate\; = \;\frac{{dis\tan c{e_0}{\;_h}\; – \;dis\tan c{e_{24\;}}_h}}{{dis\tan c{e_0}{\;_h}}}$$
In this study, the variables distance0h and distance24h represent the distances between scratched cells at 0 h and 24 h after the scratch, respectively.
Knockout of CFIm25 using CRISPR-Cas9 technology
CFIm25-KO cells were generated using the CRISPR/Cas9 technology. The sgRNA targeting the Nudt21 gene was designed using the CRISPOR online tool. The sgRNA sequences used were as follows: Nudt21-sgRNA: Forward: 5’-TCTCGACTGTTAATGGCGGG-3’ (PAM: TGG), Reverse: 5’-AAGAGGCGCGGAACACACGC-3’ (PAM: CGG).
The sgRNA was inserted into the Lenti-CRISPR v2 vector (HanBio, Shanghai, China) containing the Streptococcus pyogenes Cas9 nuclease gene. Cell transduction was performed using the lentiviral Lenti-CRISPR v2 vector, followed by the generation of CFIm25-KO cells using the CRISPR/Cas9 editing system.
Transfected cells, including the sgRNA plasmid and donor sequence, were selected with 4 µg/mL puromycin (HY-K1057, MedChemExpress, USA). Surviving cells were then subjected to limited dilution cloning, and CFIm25-KO cells were selected through RT-qPCR and Western blot analysis [45, 49].
Overexpression of CFIm25 using lentivirus
The plasmid vector pCMV6-AC-GFP (LM-2069, LMAI Bio, Shanghai, China) was used by LMAI Bio (Shanghai, China) to construct the CFIm25 plasmid. To generate CFIm25-UP lentivirus (CFIm25-UP-LTEP-s, hereafter referred to as CFIm25-UP) and control lentivirus (NC-LTEP-s, hereafter referred to as Mock), HEK293T cells (Bio-72947, Beijing Biodex Biotechnology Co., Ltd.) were employed. Plasmids and lentivirus packaging services were provided by LMAI Bioengineering. The constructed luciferase reporter gene plasmids (Mock-luc, CFIm25-UP-luc), along with the helper plasmid, were co-transfected into HEK293T cells using Lipofectamine 2000 reagent (11668030, Thermo Fisher, USA). The transfected cells were assessed, amplified, and purified to obtain packaged lentivirus.
For lentiviral-mediated cell transduction, 5 × 105 cells were seeded in a 6-well plate. Once the NP cell confluency reached 60–70%, the medium containing an appropriate amount of packaged lentivirus (MOI = 10, with a working titer of approximately 5 × 106 TU/mL) and 5 µg/mL polybrene (TR-1003, Sigma-Aldrich, UK) was added for transduction. After 4 h of transduction, an equal medium volume was added to dilute polybrene. Following a 24-hour transduction period, fresh medium was replaced. After 48 h of culturing, the transduction efficiency was assessed using the luciferase reporter gene, and the cells were further maintained in a medium supplemented with 2 µg/mL puromycin (E607054, LMAI Bioengineering, Shanghai, China). During cell passaging, the puromycin concentration was gradually increased in a gradient of 2, 4, 6, 8, and 10 µg/mL to select resistant cell lines. Once the cells no longer died in a medium containing puromycin, they were collected, and the knockout efficiency was validated using RT-qPCR [50].
The cell groups were as follows: CFIm25-WT NP cells (wild-type cells), CFIm25-KO NP cells (CFIm25 knockout cells), CFIm25-UP NP cells (CFIm25 overexpression cells), and Mock NP cells (cells transfected with empty lentivirus).
Detection of gene expression by RT-qPCR
Total RNA from tissues and cells was extracted using the Trizol Reagent kit (A33254, Thermo Fisher, USA). Reverse transcription was performed using the reverse transcription kit (RR047A, Takara, Japan) to obtain the corresponding cDNA. The reaction system was prepared using the SYBR® Premix Ex TaqTM II kit (DRR081, Takara, Japan) and the RT-qPCR reaction was carried out in a real-time fluorescence quantitative PCR instrument (ABI7500, Thermo Fisher, USA). The PCR program was designed as follows: an initial denaturation at 95 °C for 30 s, followed by a cycling stage consisting of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and 40 cycles. Subsequently, the extension was performed at 95 °C for 15 s and 60 °C for 60 s, followed by a final extension at 90 °C for 15 s to generate amplification curves. The housekeeping gene, GAPDH, was used as an internal reference. Each RT-qPCR was set up in triplicates, and the experiment was repeated three times. The fold change in gene expression between the experimental and control groups was calculated using the 2−ΔΔCt method, where ΔΔCT = ΔCt experimental group – ΔCt control group, and ΔCt = Ct target gene – Ct reference gene. Ct represents the number of amplification cycles required for the real-time fluorescence intensity to reach the set threshold, at this point, the amplification enters the exponential phase [45]. Primer design details can be found in Table S1.
Western blot analysis
To extract total protein from tissue and cell lysates, RIPA buffer (P0013B, Beyotime, Shanghai, China) containing 1% PMSF (phenylmethanesulfonyl fluoride) was used following the manufacturer’s instructions. The protein concentration of each sample was determined using the BCA assay kit (P0011, Beyotime, Shanghai, China). The protein concentration was then adjusted to 1 µg/µL, and each sample was set at a volume of 100 µL. Samples were boiled at 100 °C for 10 min to denature the proteins and stored at -80 °C until further use.
An 8-12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was prepared based on the target protein band size. An equal amount of 50 µg protein sample was loaded onto each lane using a microsyringe. Electrophoresis separation was performed at a constant voltage of 80 V for 2 h, followed by 120 V for an additional 90 min at a constant current of 250 mA. The proteins from the gel were then transferred to a PVDF membrane (1620177, Bio-Rad, USA).
The membrane was blocked at room temperature for 1 h with 1× TBST (Tris-buffered saline with Tween 20) containing 5% skim milk. After discarding the blocking solution, the membrane was washed with 1× TBST for 10 min. The primary antibody (refer to Table S2 for antibody information) was incubated overnight at 4 °C, followed by three washes with 1× TBST for 10 min each. Subsequently, the membrane was washed three times with 1× TBST at room temperature for 5 min each. HRP-conjugated goat anti-rabbit IgG (ab6721, dilution: 1:5000, Abcam, Cambridge, UK) or goat anti-mouse IgG (ab205719, dilution: 1:5000, Abcam, Cambridge, UK) secondary antibody was added and incubated at room temperature for 1 h. After three washes with 1× TBST at room temperature for 5 min each, the membrane was immersed in an ECL reaction solution (1705062, Bio-Rad, USA) and incubated at room temperature for 1 min. The liquid was removed, and the membrane was covered with plastic wrap for band exposure using the Image Quant LAS 4000 C gel imaging system (GE, USA). The relative protein expression levels were quantified by comparing the grayscale value ratio of the target band to the reference band, with GAPDH serving as an internal control for total cellular protein. Protein expression levels were analyzed for each protein [45]. Each experiment was repeated three times.
Preparation of GelMA-HAMA composite hydrogel
Synthesis of Methacrylate Gelatin (GelMA): Initially, a 10% (w/v) phosphate-buffered solution with a pH of 7.4 was prepared using type A gelatin (G2500, Sigma-Aldrich, UK) derived from pig skin. Subsequently, a solution of 20% methacrylic anhydride (MA, 276685, Sigma-Aldrich, UK) was added dropwise to the prepared gelatin solution at 50 °C and stirred for 1 h. The resultant mixture was then diluted 5-fold with a 40 °C heated phosphate-buffered solution, filtered, and dialyzed using distilled water for 1 week before freeze-drying (Figure S7A) [32].
Synthesis of Methacrylate Hyaluronic Acid (HAMA): Initially, a distilled water solution of hyaluronic acid (924474, Sigma-Aldrich, UK) was prepared at a concentration of 0.5% (w/v). Dimethylformamide (DMF) was added to the solution at a ratio of H2O: DMF = 3:2. While stirring, a 1% solution of MA (276685, Sigma-Aldrich, UK) was added dropwise. The pH was then adjusted to 8–9 using 0.5 M sodium hydroxide (S5881, Sigma-Aldrich, UK), and the solution was incubated overnight with continuous stirring at 4 °C. Following this, the solution was dialyzed using distilled water for 3 days and subsequently freeze-dried (Figure S7B) [32].
Preparation of GelMA-HAMA Microspheres: HAMA and GelMA were mixed in a 3:1 ratio, followed by the addition of 0.3% (w/v) photoinitiator (L157759, Aladdin, Shanghai, China), mineral oil (ST275, Beyotime, Shanghai, China), and 10% (w/w) Span80 (1338-43-8, Macklin, Shanghai, China) as a surfactant. Crosslinking of the hydrogel was achieved through UV irradiation (365 nm, 6.9 mW·cm− 2) in a dark setting. The flow rate was adjusted using a microfluidic device to prepare GelMA-HAMA microspheres. The collected microspheres were washed repeatedly with isopropanol and 75% ethanol to remove surfactants and mineral oil. The purified microspheres were then soaked in PBS for 4 h, repeated 6 times, to eliminate other additives. To obtain porous microspheres, the purified spheres were frozen at -20 °C for 8 h and subsequently freeze-dried for 48 h [35, 51].
Preparation of GelMA-HAMA microspheres
GelMA-HAMA microspheres conjugated with APET×2 polypeptide (GelMA-HAMA-APET×2, GA): Disperse 100 mg of GelMA-HAMA microspheres in 1 mL of MES buffer (pH = 6, HY-D0858, MedChemExpress, Shanghai, China). Then sequentially add 8 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 22980, Thermo Fisher, USA) and 12 mg of NHS (N-hydroxysuccinimide, 24500, Thermo Fisher, USA) and react at 37 ℃ for 15 min. Subsequently, add 5 µg of APET×2 (ab141849, Abcam, UK) and incubate at 37 ℃ for 6 h. Rapid centrifugation yields GA, which is washed three times and then freeze-dried for 48 h to obtain GA hydrogel [35].
GCA hydrogel loaded with Cas9-CFIm25-sgRNA recombinant protein (GelMA-HAMA-APET×2@ Cas9-CFIm25-sgRNA, GCA): Dissolve Cas9-CFIm25-sgRNA recombinant protein at a concentration of 2 mg/mL in PBS and mix it with 10% (wt) GA hydrogel. Incubate at 37 ℃ for 30 min, then wash three times, centrifuge, and dry to obtain GCA [25, 52].
Characterization of composite hydrogels
The composite hydrogel was characterized and detected using Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Briefly, SEM analysis was conducted using a Hitachi S-4800 SEM with an acceleration voltage of 3 kV. The hydrogel sample was placed on conductive tape and coated with gold using Cressington Scientific Instruments’ coating equipment in Watford, UK, for 60 s. Subsequently, the sample was observed using the SEM.
For the FTIR analysis, the hydrogel was frozen in a -20℃ freezer and freeze-dried for 48 h. The freeze-dried sample (1 mg) was mixed with 80 mg of dried KBr (221864) from Sigma, ground into a fine powder, and pressed into a pellet. The pellet was then scanned using the Nicolet 6700 infrared spectrometer from Thermo Fisher, with specific parameters for single-beam absorption mode (resolution: 4 cm− 1, wavelength range: 4000 –400 cm− 1, scan speed: 0.15 cm/s, number of scans: 128). During the scanning process, the sample was purged with pure helium at a flow rate of 5 mL/min to remove air.
In the in vitro release experiment, the GCA hydrogel solution was dissolved in 1 mL of PBS and incubated at 37℃ with 5% CO2. The supernatant was collected every 2 days and replaced with fresh PBS. The released protein quantity in the culture medium was determined using the Bradford protein assay kit from Shanghai Biotehnai Co., Ltd., following the instructions provided in the kit. [32, 35].
In vivo animal experiment
A total of 24 male C57BL/6J mice were involved in the experiment and were randomly divided into two groups. One group consisted of 6 mice, while the other group consisted of 18 mice. The IDD model was constructed in 18 mice using the method described above, and they were randomly divided into 3 groups, with 6 mice in each group. The remaining 6 mice were exposed to the intervertebral disc area but were not induced by IDD. These mice were divided into 4 groups and received different treatments: the Sham group, where intervertebral disc exposure was performed without injury, and 10 µL of PBS was injected into the intervertebral disc as treatment; the model control group (PBS group), where mice were injected with 10 µL of PBS after inducing IDD; the treatment group (GA group), where mice were injected with 10 µL of GA solution into the intervertebral disc; and the treatment group (GCA group), where mice were injected with 10 µL of GCA solution into the intervertebral disc. Drug treatment was initiated one week after constructing the intervertebral disc model, with injections administered every 2 days for a duration of 4 weeks. After completing the treatment, the mice were subjected to radiographic examination and euthanized, and their intervertebral disc tissues were extracted for subsequent biochemical experiments [35].
Proteomic analysis
Six mice were randomly selected and divided into two groups, the PBS group (n = 3) and the GCA group (n = 3), for injection therapy. The protein from the intervertebral disc NP tissue of mice was extracted using RIPA buffer supplemented with a protease inhibitor (Figure S3). During the extraction process, samples were treated with an ultrasound probe for 30 s every 5 min, three times in total, to ensure cell rupture and protein release. The protein concentration of the extracted samples was determined using the BCA protein quantification kit, and the concentration was ensured to be within an acceptable range. After adjusting the pH to 8.0, trypsin (15090046, Thermo Fisher, USA) was added at a ratio of 1:50 (enzyme to protein), and the mixture was incubated at 37 °C for 16 h for enzymatic digestion. The digested samples were then subjected to clean-up using ZipTip C18, loaded onto a high-performance liquid chromatography system, and connected to a mass spectrometer for MS/MS analysis. The data were processed using the MaxQuant software, which included protein identification and quantification.
iTRAQ labeling was performed using desalted peptides containing 0.1% formic acid (1002641000, Sigma, USA) for mass spectrometry analysis. Each sample was analyzed three times using the QSTAR Elite Hybrid MS (Applied Biosystems/MDS-SCIEX) and an online HPLC system (Shimadzu, Japan). In each analysis, 30 µL of peptide solution was injected and separated on a homemade nano-column with a nano-electrospray emitter (75 μm ID × 15 cm, 5 μm particle size) (New Objectives, Woburn, MA). A 90-minute high-performance liquid chromatography gradient was established using mobile phase A (5% (v/v) acetonitrile, 0.2% (v/v) formic acid) and mobile phase B (90% (v/v) acetonitrile, 0.2% (v/v) formic acid), with an effective flow rate of 300 nL/min. The separations were performed using a constant flow rate of 30 µL/min. The mass spectrometer was set to acquire data in positive ion mode, with a mass range of 300–2000 m/z and precursor fragmentation for charges + 2 to + 4. The three most abundant peptide ions with a count threshold greater than 5 were selected in each MS/MS spectrum. The dynamic exclusion time for selected precursor ions was set to 30 s, with a mass tolerance of 30 mDa.
LC-MS/MS injections were performed utilizing automated collision energy and automatic mass spectrometry/mass spectrometry accumulation smart information dependency. The fragmentation intensity multiplier was set to 20, with a maximum accumulation time of 2 s. Three LC-MS/MS injections (technical replicates = 3) were carried out to achieve better coverage of the target proteome and improve statistical consistency. WB analysis was used for protein verification after proteomics analysis of the same sample. The parameters were as follows: (1) MS: scan range (m/z) = 350–1500; resolution = 120,000; AGC target = 4e5; maximum injection time = 50 ms; (2) HCD-MS/MS: resolution = 30,000; AGC target = 1e5; collision energy = 33; (3) DIA (data-independent acquisition). Each window overlapped by 1 m/z, and the window number was 47. The iRT kit (Ki3002, Biognosys AG, Switzerland) was added for calibration of peptide peak retention times. The DIA dataset was generated by Spectronaut V 13 (Biognosys AG, Switzerland), including data normalization and relative protein quantification. Differential expression proteins were filtered using Welch’s ANOVA test with a filtering condition of p-value < 0.05 and logFC > 1.0 [53].
TUNEL detection of cellular apoptosis
Tissue cells were stained using the TUNEL staining kit (C1088, Beyotime, Shanghai, China). Briefly, mouse intervertebral disc tissue was fixed with 4% paraformaldehyde for 30 min, followed by three washes with PBS. Then, a permeabilization solution containing 0.3% Triton X-100 in PBS was added and incubated for 3 min at room temperature, followed by 5 min at room temperature. This was followed by two additional washes with PBS. Subsequently, 50 µL of TUNEL detection solution was added, and the samples were incubated in the dark at 37 °C for 60 min. After incubation, three washes with PBS were performed, followed by a 10-minute restaining with DAPI (10 µg/mL), and finally, the slides were sealed using an anti-fluorescence quenching mounting solution. Cellular apoptosis was observed using a fluorescence microscope, with Cy3 exhibiting an excitation wavelength of 550 nm and an emission wavelength of 570 nm (red fluorescence). The apoptotic cell proportion in each group was calculated using Image-Pro Plus 6.0 software [36].
Flow cytometry analysis
Initially, cells were isolated from mouse intervertebral disc tissue samples and treated with collagenase and DNAse for approximately 1–2 h to obtain a single-cell suspension using density gradient centrifugation. Subsequently, the cells were adjusted to approximately 1 × 106 cells/mL and incubated for 5 h in a stimulant solution containing PMA (Phorbol 12-myristate 13-acetate, HY-18739, MedChemExpress, Shanghai, China), ionomycin (HY-13434, MedChemExpress, Shanghai, China), and GolgiPlug™ (Brefeldin A, 555029, BDbiosciences, USA) at 37 °C and 5% CO2. After incubation with CD16/CD32 antibodies (0.5 mg/mL, 14-0161-82, Thermo Fisher, USA) for 10 min to block nonspecific binding, surface labeling was performed as follows: APC-conjugated CD19 antibody (0.2 mg/mL, 17-0193-82, Thermo Fisher, USA) for B cells, PE-conjugated CD3 antibody (0.2 mg/mL, 12-0031-82, Thermo Fisher, USA) for T cells, FITC-conjugated CD4 antibody (0.5 mg/mL, 11-0041-82, Thermo Fisher, USA) for CD4+ T cells, PE-Cy7-conjugated CD8 antibody (0.2 mg/mL, A15385, Thermo Fisher, USA) for CD8+ T cells, APC-conjugated antibody for IFN-γ (0.2 mg/mL, 17-7311-82, Thermo Fisher, USA) for Th1 cells, and PE-conjugated antibody for IL-4 (0.2 mg/mL, 12-7041-82, Thermo Fisher, USA) for Th2 cells. Cell analysis was carried out using a flow cytometer, ensuring differentiation of cell types by setting appropriate gates and employing unstained cells and singly stained controls to verify staining specificity and accuracy. All procedures were conducted under sterile conditions to prevent cell contamination [54].
Statistical analysis
Data were obtained from at least three independent experiments and presented as the mean ± standard deviation (Mean ± SD). For comparisons between the two groups, an independent-sample t-test was used. For comparisons among three or more groups, a one-way analysis of variance (ANOVA) was employed. If the ANOVA results indicated significant differences, Tukey’s HSD post hoc test was further performed to compare the differences between each group. For non-normally distributed or heterogeneous variance data, the Mann-Whitney U test or Kruskal-Wallis H test was used. All statistical analyses were conducted using GraphPad Prism 9.5.0 (GraphPad Software, Inc.) and R version 4.2.1 (R Foundation for Statistical Computing). The significance level for all tests was set at 0.05, with a two-sided p-value less than 0.05 considered statistically significant, while a p-value greater than 0.05 was deemed non-significant.
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