Advanced oxidation protein products induce annulus fibrosus cell senescence through a NOX4-dependent, MAPK-mediated pathway and accelerate intervertebral disc degeneration

Ethics approval

All the animal experimental procedures had been approved by the Laboratory Animal Care and Use Committee of Nanfang Hospital, Southern Medical University (NFYY-2020-78). All the steps described in this study meet the standards of the Eighth Edition Guidelines for the Care and Use of Laboratory Animals published by the National Academy of Sciences (Washington, DC, USA).

Preparation of AOPPs-RSA

AOPPs-RSA were prepared based on the procedure described previously (Zhu et al., 2018). In brief, RSA (Rat Serum Albumin; HUIJIA Biotechnology, China) solution (20 mg/ml) was incubated with 40mM HOCl in phosphate-buffered saline (PBS, pH = 7.4) for 30 min at room temperature. Prepared samples were dialyzed for 24 h against PBS that change every 6 h to remove free HOCl. Native RSA was dissolved in PBS alone as control incubation.

Animal and experimental design

Thirty-six adult male Sprague–Dawley (SD) rats(500 ± 50 g) were randomized into six groups. Caudal discs were exposed without puncture for the sham-operated control (Group 1). Under anesthesia, two segmental caudal discs (Co6/7, Co8/9) were exposed through a posterior incision. Disc puncture was performed using a 21-gauge needle each on Co6/7, Co8/9 to cause IVDD, as mentioned previously (Group 2) (Keorochana et al., 2010; Zhang et al., 2011). To make sure that the needle did not penetrate too deeply, the length of the needle was pre-determined by approximately five mm, as reported previously (Keorochana et al., 2010; Zhang et al., 2011). Untreated Co7/8 served as the internal control. Based on our previous work demonstrates that intra-articular injections of AOPPs in rabbit OA models (Yu et al., 2015) and intraperitoneal injection in mice OA models (Liao et al., 2020) accelerate regression of cartilage, we designed Group 3 was given intra-peritoneal AOPPs (100 mg/kg/d) for 1 month and Group 4 was given intra-peritoneal AOPPs (100mg/kg/d) 3 days after disc puncture for 1 month. To evaluate the effect of AOPPs on AF cell senescence and IVDD in vivo, we prepared two solutions for intra-discal injection, including RSA (50 µg/ml) (Group 5) and AOPPs (200 µg/ml)(Group 6). 2 µl of the solution of interest was injected into each segment, and 30s were kept in the disc for each 31-gauge needle (Mao et al., 2011). The injections were conducted once every fortnight for 1 month, respectively. After surgery, each animal was allowed free, unrestricted weight-bearing and activity, fed normally, and monitored. X-ray (FX Pro, Bruker, SMU Central Laboratory, Southern Medical University) was performed 1 month after surgery for each group to measure disc height. Caudal discs were harvested for western blotting and immunohistochemical staining.

Eighteen adult male SD rats (500 ± 50 g) were randomized into three groups. In the Sham group, only the skin on the back of the lumbar vertebrae of the rats was cut and sutured. In the degeneration group, the lamina, zygapophyseal joint, and spinous process of the 1-6 lumbar vertebrae were excised, and MRI (PharmaScan70/16 US, Bruker, SMU Central Laboratory, Southern Medical University) was performed at 1 and 2 months postoperatively to examine the degeneration of the lumbar spine, and the lumbar disc was finally removed for western blotting and immunofluorescence.

Twenty-four adult male SD rats (500 ± 50 g) were randomized into four groups. In vivo blockade of NOX4 expression was achieved by using Adeno-Associated Viruses (AAV) that were purchased from the Shanghai OBiO Technology carrying the rat NOX4 shRNA-2 sequences that can block NOX4 expression and injecting 2 µl AAV into the intervertebral disc using a 31 g needle. AAV carrying the NC sequence was used as a control. RSA and AOPPs were re-injected into the intervertebral disc 3 weeks after injection, once every fortnight for 1 month, respectively. MRI was performed 1 month after surgery for each group to measure Pfirrmann grade. Caudal discs were harvested for western blotting and immunofluorescence.

Immunohistochemical staining and immunofluorescence

After 24–48 h of paraformaldehyde fixation, discs tissues were decalcified for at least eight weeks with Ethylene Diamine Tetraacetic Acid (EDTA). Then after dehydrated and paraffin-embedded, samples were sectioned to 4 µm. After being treated with 0.3% hydrogen peroxide for 10 min to reduce the activity of endogenous peroxidase, nonspecific staining was blocked by incubating the sections with 10% goat serum for 30 min. Then slices were incubated with primary antibodies against NOX4 (1:50, Affinity, DF6924), p16 (1:100, Abclonal, A0262), AOPPs (1:100, Department of Immunology, Southern Medical University), p21 (1:100, CST, #2946), p53 (1:100, Abclonal, A11232), IL-1β (1:100, Affinity, AF5103), TNF-α (1:100, Affinity, AF7014), p-p38 (1:100, Affinity, AF4001), p-ERK (1:100, Affinity, AF1015), p-JNK (1:100 dilution, Affinity, AF3318) at 4 °C overnight, followed by incubated with HRP-conjugated secondary antibodies or Cy3 fluorescent secondary antibody for 1 h at room temperature. DAB Horseradish Peroxidase Color Development Kit(Beyotime) was used for immunohistochemical positive cells color rendering. Nuclei were counterstained with hematoxylin or DAPI. All sections were viewed examined with a Leica DM5000B (Leica, Germany) and ZEISS AXIO Imager D2 (Germany). Fluorescent images are taken at the same exposure time for each group of target proteins, and the grey scale values are automatically measured by Imager D2.

Rat AF cell isolation and culture

As reported previously (Zhao & Tian, 2019), AF tissue was separated under a dissecting microscope and then was digested using 0.1% Type II collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 5 h with shaking at 37 °C. The isolated AF cell pellets were re-suspended in DMEM/F12 medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 1% (v/v) penicillin-streptomycin (Gibco, Waltham, MA, USA), then seeded onto a 21 cm² culture dish at 37 °C in a humidified atmosphere of 5%CO₂–95%air. Up to 80%–90% confluence, the second or third passage AF cells were used in each assay.

Western blot analysis

Total proteins of AF cells or disc AF tissues were extracted using a protein extraction reagent (FUDE Biological Technology) and quantified using a BCA kit(Thermo Scientific). Proteins mixed with 1/4 volume loading buffer (FUDE Biological Technology) were electrophoresed on SDS gels and then were transferred to PVDF membranes (Millipore). The membranes were blocked by using 5% Bovine Serum Albumin in TBST for 1 h at room temperature. Next, the membranes were incubated with primary antibodies against GAPDH (1:1000, Bioworld, BS60630), p16 (1:500, Abclonal, A0262), p21 (1:1000, CST, #2946), AOPPs (1:1000, Department of Immunology, Southern Medical University), Albumin (1:500, Affinity, DF6396), p53 (1:500, Abclonal, A11232), p38 (1:500, Affinity, AF6456), p-p38 (1:500, Affinity, AF4001), ERK1/2 (1:500, Affinity, AF0155), p-ERK1/2 (1:500, Affinity, AF1015), JNK (1:500, Affinity, AF6318), p-JNK (1:500, Affinity, AF3318), IL-1β (1:500, Affinity, AF5103), TNF-α (1:500, Affinity, AF7014) and NOX4 (1:500, Affinity, DF6924) with slowly shaking overnight at 4 °C, followed by incubation with the HRP-conjugated secondary antibodies (1:5000 dilution, Abmart, M21003) at room temperature for 1 h. Immunolabeling was detected using an ECL kit (Affinity, KF001). The total optical density (OD) of blot bands was measured by using Image J software (National Institutes of Health, Bethesda, MD, USA). The relative expression of each protein = OD of each protein / OD of each GAPDH. The relative phosphorylation level of each protein = OD of each phosphor-protein/ OD of each total protein.

SA-β-gal staining

The SA-β-gal staining of AF cells was investigated using the SA-β-gal staining kit (Solarbio). According to instruction, fixative AF cells seeded in 6-well culture plates were incubated with the staining solution at 37 °C overnight. Nine random fields per well were imaged using a phase-contrast microscope (Olympus) to calculate the mean percentage of SA-β-gal positive cells.

Cell cycle analysis

To determine cell cycle distribution, NP cells were re-suspended in ice-cold 70% ethanol and incubated at 4 °C overnight. The next day, the cells were washed with PBS again and stained with propidium iodide (PI) using the cell cycle and apoptosis analysis kit (Beyotime, C1052) for 30 min. Cells were analyzed using flow cytometry (BD FACSCalubur™, USA) and cell proportions (in%) were measured using FlowJo7.6.1 software. Gate strategy: gate 1, horizontal coordinate (FSC), vertical coordinate (SSC), circles out the main population of cells based on their forward and lateral angles. Then put this group of cells in Gate 2, horizontal coordinate (PI-W), vertical coordinate (PI-A), and circle out the target cells according to the width and peak of PI. Finally, put this cluster of cells in (PI-A) and divide it into three phases according to the amount of PI staining.

Transfections of adenovirus

Adenovirus with shNOX4 and adenovirus with NC sequences were purchased from the Shanghai OBiO Technology. The sequence of rat NOX4 shRNA-1 is GCTTCTACCTATGCAATAA. The sequence of rat NOX4 shRNA-2 is GCAACAAACCTGTCACCAT. The sequence of rat NC shRNA is CCTAAGGTTAAGTCGCCCTCG. Transfections of adenovirus into AF cells were according to the manufacturer’s instructions. Approximately 72 h after transfection, virus delivery was determined by observing green fluorescent protein (GFP) under the fluorescence microscope (Olympus). The ratio of GFP-positive cells to total cells was used to define transfection efficiency. Cells with a transfection efficiency of more than 90% were used in each assay. Cell lysates were collected, and Western blotting was used to examine their efficiency.

Dimethylmethylene blue

For GAGs level determination, total protein extract from disc tissue was measured using a 1,9-dimethyl methylene blue (DMMB) method with Bovine chondroitin 4-sulfate(Sigma-Aldrich) as a standard. 200 µl DMMB solution was added to the wells on a 96-well plate containing 40 µl of a sample. The DMMB solution was made by mixing 16 mg DMB, 3.04 g glycine, 1.6 g NaCl and 95 ml of 0.1 M Acetic Acid in 1 L of distilled water. The absorbance was measured at λ = 525nm on a SpectraMax M5 system (Molecular Devices, San Jose, CA, USA).

Statistical analysis

All experiments were repeated independently at least three times and the data are presented as the mean ± SD. All statistical analyses were performed by using Statistical Packages for Social Sciences v20.0 software (SPSS, Chicago, IL). Results for continuous variables such as grey values of western blot and immunofluorescence, SA-β-gal Staining, Cell cycle analysis, and Dimethyl methylene blue were analyzed using one-way ANOVA. Nonparametric data like disc Pfirrmann grade were analyzed with the Kruskal-Wallis H test. Post hoc two-by-two comparison using adjusted α value. P ≤ 0.05 was considered significant.

AOPPs accumulate in IVDD, up-regulate the expression of NOX4, senescence markers, and senescence-associated inflammatory proteins, and accelerate IVDD

To verify whether AOPPs are involved in the course of natural IVDD, we constructed a model of persistent lumbar disc degeneration in rats. A model of lumbar instability was constructed by removing the posterior bony structures of the lumbar 1-6 vertebrae, i.e., the lamina, zygapophyseal joint, and spinous process, while preserving muscle tissue to avoid excessive damage to the lumbar mobility of the rat, thereby accelerating IVDD without directly damaging the disc.

As shown in Fig. 1A, after resection of the posterior lumbar structures, we found significant degeneration of the lumbar 1/2,2/3 and 3/4 discs, while the lumbar 4/5 and 5/6 discs showed protrusion into the spinal canal at two months after surgery. In Fig. 2C, the results of the DMMB suggested that the GAG content of the lumbar intervertebral discs decreased significantly from one month postoperatively and dropped to a minimum in two months postoperatively, suggesting the lumbar IVDD in rats. Immunofluorescence experiments in Fig. 2B showed a significant accumulation of AOPPs in the degenerated AF tissues at two months after surgery. In subsequent WB analysis, we found that albumin, which could be oxidized to AOPPs, also accumulated in the degenerated lumbar discs. At the same time, AOPPs, NOX4, senescence markers: p53, p21, and p16, as well as senescence-associated inflammatory proteins: IL-1β and TNF-α were highly expressed in the degenerated discs (Fig. 1D).

From the mechanism of AOPPs formation and the above experimental results, we can speculate that AOPPs may not be the initiating factor in disc degeneration and that their pathological effects may be more focused on accelerating disc degeneration. To test this conjecture, we constructed a rat caudal disc puncture model. Interestingly, after being punctured, the albumin was accumulated in the degenerative discs. As a result, AOPPs were also accumulated, accompanied with high expression of NOX4, p53, p21, p16, IL-1β, and TNF-α, especially higher in the intra-discal injection of AOPPs group (Fig. 1E). In Fig. 1F, the radiographs showed that the disc height index (DHI) of the punctured discs were all low, suggesting disc space collapse and significant disc degeneration, with the lowest DHI in the intra-discal injection AOPPs group. The dimethylmethylene blue assay (DMMB) of the disc tissue showed (Fig. 1J) that the GAG were lower in all groups of discs after the puncture, with the lowest levels in the intra-discal injection AOPPs group. Based on the results of DHI and DMMB, it’s obvious that degeneration was observed in all puncture groups, and the intra-discal AOPPs injection caused the most serious damage, while the intra-peritoneal(ip) AOPPs injection could not cause more damage no matter puncture or not. Finally, in the AF tissue immunohistochemistry staining, we detected the expression of p16 (Figs. 1G and 1H) and NOX4 (Figs. 1G and 1I) that was positive in puncture discs and much higher after exogenous AOPPs stimulation.

AOPPs up-regulate NOX4 expression, induce senescence in AF cells, and increase the secretion of senescence-associated inflammatory proteins

Based on the above findings, we used rat AF primary cells as an in vitro model to investigate whether the accumulation of AOPPs directly triggers the senescence of AF cells. We first studied the cell cycle distribution of AF cells by quantitative fluorescence-activated cell sorting (FACS) analysis. AOPPs were used to treat cells at varying doses (50–400 µg/mL, 24 h) and time (200 µg/mL, 3-48 h), and it was found that the proportion of AF cells arrested in G1 phase arrest was increased (Figs. 2A and 3A). We further used SA- β-gal staining to confirm senescence. As shown in Figs. 2B and 3B, AOPPs increased the proportion of senescent AF cells at different concentrations and times. Similarly, expression of NOX4, p53, p21, p16, IL-1β, and TNF-α was up-regulated by AOPPs treatment at different doses and times (Figs. 2C and 3C).

The AOPPs-induced senescence of AF cells is mediated by NADPH oxidase

Previous studies in our group suggest that NADPH oxidase was the main effector molecule of AOPPs (Liao et al., 2020; Zhu et al., 2018). To confirm the role of NADPH oxidase in AOPPs-induced AF cell senescence, we pre-incubated cells with two NADPH oxidase inhibitors: Setanaxib (GKT137831, Selleck) (GKT) and Apocynin (NSC2146, Selleck) (APO), before AOPPs exposure, respectively. Blocking down NADPH oxidase restored the AOPPs-induced G1 phase arrest (Fig. 4A), as well as the positive cells of SA-β-gal staining (Fig. 4B). Similarly, High expression of NOX4, p53, p21, p16, IL-1β and TNF-α induced by AOPPs was down-regulated in response to inhibitor treatment (Fig. 4C).

AOPPs induce AF cell senescence by activating phosphorylation of MAPK

Previous studies in our group suggested MAPK were major effector molecules in the pathology mediated by AOPPs (Liao et al., 2020; Zhu et al., 2018). To determine whether the MAPK pathway is involved in AOPPs-induced senescence, we evaluated MAPK phosphorylation in AF cell cultures treated with AOPPs. Our findings revealed that phosphorylation of p38 MAPK appeared after 60 min of AOPPs stimulation, while ERK phosphorylation appeared 5 min after AOPPs stimulation, and JNK phosphorylation similarly began to rise 5 min after AOPPs stimulation, while AF cells stimulated with RSA for 60 min showed a slight rise in JNK phosphorylation (Figs. 5A, 5B and 5C). To further evaluate the role of the MAPK pathway in senescence, AF cell cultures were incubated with a p38 MAPK inhibitor (SB202190; Beyotime, Shanghai, China), an ERK1/2 inhibitor (FR180204; Beyotime, Shanghai, China), a JNK inhibitor (SP600125; Beyotime, Shanghai, China) respectively, before AOPPs treatment. Our findings revealed that the AOPPs-induced senescence in AF cells was ameliorated by all three inhibitors (Figs. 5D, 5E and 5F).

To better confirm that NADPH oxidase was activated earlier than MAPK, AF cells were incubated with GKT and APO before AOPPs treatment. Our findings showed activation of AOPPs-induced MAPK phosphorylation was ameliorated by GKT and APO (Figs. 5G, 5H and 5I).

AOPPs induce AF cell senescence via the NOX4-MAPK pathway

Based on the findings that both GKT and APO can ameliorate the AOPPs-induced senescence, and as NOX1 is mainly presented in colon epithelium (Szanto et al., 2005), we decided to further confirm the role of NOX4 in AOPPs-induced senescence in AF cells. We pre-incubated cells with two NOX4-specific shRNAs packaged by adenoviruses before AOPPs exposure. NC shRNA packaged by adenoviruses was used as a control. As shown in Fig. 6A, NOX4-specific shRNAs significantly inhibited AOPPs-induced G1 phase arrest. The positive cells of SA-β-gal staining were reduced (Fig. 6B). AF cells transfected with ADV packed with NOX4-specific blocking shRNA sequences did not show cell cycle arrest and increased positive staining for senescence, suggesting that the transfected AF cells were in good condition. As shown in Fig. 6C, NOX4 shRNA-1 only blocked p53 and p21, but not p16, IL-1β and TNF-α expression (data not shown), whereas as shown in Fig. 6D, NOX4 shRNA-2 was well blocked by both. AOPPs-induced up-regulation of NOX4, p53, p21, p16, IL-1β, TNF-α, and hyper-phosphorylation levels of p38, ERK, and JNK were reduced after transfection with NOX4-specific blockade of shRNA-2 sequences (Figs. 6C and 6D).

Blocking NOX4 expression in vivo by AAV reduces the expression of senescence markers and senescence-related proteins and delays IVDD

As suggested by the MRI in Fig. 7A, in vivo blocking NOX4 expression by AAV reduced the Pfirrmann grade of disc degeneration caused by RSA and AOPPs and delayed IVDD. HE staining and staining with saffron O/fast green showed that in discs with blocked NOX4 expression, disc damage caused by injections of AOPPs and RSA was less severe, the growth plate and part of the cartilage endplate were preserved, the morphology of the AF tissue was improved, the number of surviving AF cells was higher and, overall, the corresponding disc damage scores were lower (Figs. 7B and 7C). Furthermore, blocking NOX4 expression before stimulation with RSA and AOPPs reduced the expression of p53, p21, p16, p-p38, p-ERK, p-JNK, IL-1β, and TNF-α in the disc (Figs. 7D, 7E and 7F).