Back pain is a leading cause of disability and loss of workplace productivity in otherwise healthy young and middle-aged individuals. IVD degeneration is widely recognized as a contributor to low back pain, and several factors such as age, genetic predisposition, and mechanical stress have been suggested to contribute to the initiation and progression of IVD degeneration [1
]. The degenerating IVD constitutes an inflammatory environment, with the accumulation of senescent cells. Cell senescence is a durable cell-cycle arrest that is triggered by a myriad of extracellular and intracellular stimuli [2
]. Stress-induced premature senescence can be triggered by DNA damage, oxidative stress, adverse load, and inflammation [3
]. Senescent cells, although permanently non-dividing, remain metabolically active and secrete a range of pro-inflammatory cytokines, chemokines, proteases, and growth factors, called the SASP. The SASP influences the tissue microenvironment, accelerates aging, and contributes to local and systemic dysfunction in diseases such as osteoarthritis [4
]. Furthermore, it can, in a paracrine manner, induce senescence, which further intensifies tissue deterioration [6
]. Several markers have been used to identify senescent cells. Activation of the lysosomal enzyme senescence-associated β-galactosidase (SA-β-gal) was the first reliable indicator of senescent cells. More specific molecular biomarkers, such as the cyclin-dependent kinase inhibitor 2A (also known as p16INK4a
), are now also used [7
Drugs are emerging that either kill (senolytic) or reverse cellular senescence (senomorphic). Clinically approved senolytic drugs, including glucocorticoids, metformin, and rapamycin inhibitors, can restore tissue homeostasis [2
], and recent data suggest that they can prevent or even treat cartilage-related diseases like sarcopenia, osteoarthritis, kyphosis, and herniated discs [4
]. Examples of senomorphic compounds, include free radical scavengers and inhibitors of IkB kinase (IKK), nuclear factor (NFkB) [8
] and the Janus kinase (JAK) pathways [9
]. These drugs suppress markers of senescence or their secretory phenotype without inducing apoptosis. Some compounds can be senolytic and/or senomorphic, depending on the cell type. For example, fisetin is a natural compound that has senomorphic effects on senescent human lung fibroblasts (IMR90) or pre-adipocytes, and selectively kills IR-induced senescent but non-proliferating human umbilical vein endothelial cells [10
One of the compounds of interest in this study, curcumin (diferuloylmethane), has therapeutic benefits via its antioxidant and anti-inflammatory properties [12
]. It has been shown to exert cytotoxic effects on cancer cells without being toxic to non-malignant cells [13
]. However, curcumin has poor bioavailability, low water solubility, and chemical instability. An increase in interest has therefore been raised with regard to the biological properties of curcumin’s metabolites [14
]. o-Vanillin (2-hydroxy-3-methoxybenzaldehyde) is the main metabolite, and like curcumin, it has documented anti-inflammatory and antioxidative properties [15
] and is present in high concentrations in circulation after curcumin consumption [16
]. Both compounds interfere with multiple cellular processes, including cell proliferation, cell survival and apoptosis [17
]. To the best of our knowledge, no study has yet evaluated the potential senolytic and/or senomorphic effects of curcumin and o-Vanillin on human IVD cells.
2. Experimental Section
2.1. Study Approval and Tissue Collection
Collection of human disc tissue was approved by the ethical review board at McGill University (IRB#s A04-M53-08B and A10-M113-13B). The tissue demographics are presented in Table 1
. Nucleus pulposus (NP) and annulus fibrosis (AF) cells were isolated separately, as previously described [19
]. Briefly, NP and AF tissue were separated microscopically according to their different macroscopic morphologies. Next, the samples were washed in phosphate-buffered saline solution (PBS, Sigma-Aldrich, Oakville, ON, Canada) and Hank’s-buffered saline solution (HBSS, Sigma-Aldrich, Oakville, ON, Canada) supplemented with PrimocinTM
(InvivoGen, San Diego, CA, USA) and Fungiozone (Sigma-Aldrich, Oakville, ON, Canada). Then, the matrix was minced and digested in 0.15% collagenase type II (Gibco) for 16 h at 37 °C. Cells were passed through a 70-μm filter and re-suspended in DMEM (Sigma-Aldrich, Oakville, ON, Canada) supplemented with 10% fetal bovine serum (FBS (Gibco)), PrimocinTM
(100 mM), Glutamax (1X) (Sigma-Aldrich, Oakville, ON, Canada), and maintained in a 5% CO2
incubator at 37 °C.
2.2. In Vitro Disc Cell Cultures
Experiments were performed with NP and AF separately. Cells were used directly following digestion for pellet cultures and within passages 0–1 for monolayer cultures.
Monolayer culture: 20,000 or 250,000 cells per well were seeded in 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) and 6-well plates (Sarstedt, TC plate 6-well, Cell+, F) respectively. Cells were serum-starved in DMEM with ITS (1X) (Thermo Fisher, Waltham, MA, USA) for 2 h prior to treatment with 5 μM curcumin (Sigma-Aldrich, Oakville, ON, Canada), 100 μM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada), 50 μM peroxide (Sigma-Aldrich, Oakville, ON, Canada), or vehicle (DMSO (0.01%, (Sigma-Aldrich, Oakville, ON, Canada) for 1 and 6 h.
Pellet culture: 300,000 NP cells/tube were collected by centrifugation at 500× g for 5 min. Pellets were incubated in 1 mL DMEM (2.25 g/L glucose, 5% FBS, ascorbic acid (5 μM) (Sigma-Aldrich, Oakville, ON, Canada)) at 37 °C and 5% CO2. Culture media was changed every 3 days. Cell pellets were washed in PBS, cryoprotected in 10–30% sucrose, then embedded in Optimum Cutting Temperature compound (OTC, Thermo Fisher, Waltham, MA, USA), and finally flash-frozen and kept at −80 °C. Sections 5-µm-thick were cut with a cryostat (Leica Microsystems, Richmond Hill, ON, Canada) and placed on slides for immunostaining.
2.3. SA-β-Gal Staining
SA-β-gal staining was carried out on cells that were seeded in 8-well chamber slides, according to the manufacturer’s protocol (Sigma-Aldrich, Oakville, ON, Canada). Following treatment, the culture media was removed, and the cells were washed twice with PBS, then fixed with the provided fixation buffer for 10 min at room temperature. After rinsing three times with PBS, the staining mixture was added, and the plate was sealed with Parafilm and incubated overnight at 37 °C. Coverslips were mounted using Aqua Polymount (Polysciences, Warrington, PA, USA), and slides were visualized using the Zeiss Axioskop40 microscope and an AxioCam MR camera. Images were processed using AxioVision LE64 software (Zeiss, Oberkochen, Germany). Ten fields, randomly distributed across the well, were analyzed, and the number of positive (blue stained) and total cells were counted, and the percentage of senescent cells was calculated.
Monolayer cultures were washed with PBS, fixed with 4% paraformaldehyde (Thermo Fisher, Waltham, MA, USA), and blocked in PBS with 1% BSA (Sigma-Aldrich, Oakville, ON, Canada), 1% serum, and 0.1% Triton X-100 (Sigma-Aldrich, Oakville, ON, Canada) for 1 h. Slides were then incubated with the p16INK4a CINtec PLUS Kit (Roche, Ventana laboratories, Mississauga, ON, Canada) according to the manufacturer’s instructions. Slides were also exposed to primary antibodies specific to Ki-67 (Novus, Oakville, ON, Canada), caspase 3 (Sigma-Aldrich, Oakville, ON, Canada), Nrf2, or p65 (Cell signalling) overnight at 4 °C. After washing, slides were incubated with the appropriate Alexa Fluor® 488-conjugated secondary antibody (Thermo Fisher, Waltham, MA, USA) for 1 h at room temperature, and then counterstained with DAPI for p65 and Nrf2 immunofluorescence. A mouse- and rabbit-specific HRP/DAB (ABC) Detection IHC Kit (ab64264, Abcam, Cambridge, Ma, USA) was used for caspase-3 and Ki-67 staining, and counterstaining was performed by Mayer’s hematoxylin (Sigma-Aldrich, Oakville, ON, Canada). Apoptosis was detected using a commercial kit (ab176749, Abcam, Cambridge, Ma, USA) according to the manufacturer’s instructions. Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera (Olympus, Tokyo, Japan). Bright-field images were acquired as described for SA-β-gal staining.
Pellet sections were heated at 60 °C for 30 min, then washed in PBS, and fixed in 4% paraformaldehyde. Cells were permeabilized with 0.3% Triton X-100 in PBS, saturated with 1% BSA, 1% serum, and 0.1% Triton X-100 for 1 h, and then incubated overnight at 4 °C for p16INK4a
and 1 h at room temperature for Ki-67 and caspase-3 primary antibodies. The HRP/DAB Detection IHC Kit was used for detection. Samples were also stained with Safranin-O (Sigma-Aldrich, Oakville, ON, Canada) and antibodies to collagen type II (ab34712, Abcam, Cambridge, Ma, USA). Regarding p16INK4a
detection, only nuclear and nuclear with cytoplasmic immunostaining were considered positive [20
]. Coverslips were mounted using Aqua Polymount, and images were captured as described for SA-β-gal and analyzed with ImageJ and MatLab script [22
2.5. Metabolic Activity
Metabolic activity was assessed by the Alamar Blue assay [23
]. Briefly, NP and AF cells (1 × 104
) were seeded in 96-well tissue culture plates for 12 h prior to exposure to 0, 5, 10, 20, 30, 40, 50, 75, 100, or 200 μM curcumin and o-Vanillin, for 6 h. After exposure, 10% Alamar Blue reagent (Thermo Fisher, Waltham, MA, USA) was added to each well and incubated for 4 h at 37 °C. Fluorescence (Ex560/Em590) was measured by using a spectrophotometer (Tecan Infinite T200, Männedorf, Switzerland) equipped with Magellan software (Tecan, Männedorf, Switzerland). Results are presented as a percentage of metabolic activity compared to the control. Experiments were performed (3–6) times in triplicate wells for each compound and concentration.
2.6. Caspase 3/7 Activity Assay
After treatment, the caspase 3/7 activity of degenerate and non-mildly-degenerate NP cells was measured using the Amplite Fluorimetric Caspase 3/7 Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) as per the manufacturer’s protocol. Briefly, cells were incubated with the caspase 3/7 assay solution, which contained caspase substrate (Z-DEVD-R110), at room temperature for 1 h in the dark. Fluorescence intensity was then measured at 490 nm excitation and 525 nm emission. The results are expressed as a percentage of the mean of the control group (set at 100%). Each experiment was performed in triplicate and carried out three times from each round of cell isolation.
Following treatment, RNA was extracted using the TRIzol chloroform extraction method, as previously described [24
]. Briefly, 500 ng of RNA was reverse-transcribed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA) with an Applied Biosystems Verti Thermocycler (Thermo Fisher, Waltham, MA, USA). RT-qPCR was performed using an Applied Biosystems StepOnePlus machine (Thermo Fisher, Waltham, MA, USA) with PerfecCTa SYBR Green Fast Mix (Quanta Biosciences, Beverly, MA, USA). Previously published primers for senescence, inflammatory markers, and housekeeping genes are described in Table 2
. All reactions were conducted in triplicate, and fold-changes in gene expression were calculated by using the 2−ΔΔCt
], after normalizing to the housekeeping gene and vehicle-treated cells.
SASP factors (IL6, IL8, MMP3 and MMP13) in 100 μL pooled cell pellet culture media from day 0 through to day 21 were determined using ELISA kits, according to the manufacturer’s instructions (RayBiotech, Norcoss, GA, USA). Colorimetric absorbance was measured with a Tecan Infinite M200 PRO (Tecan, Männedorf, Switzerland) spectrophotometer and analyzed with i-control 1.9 Magellan software (Tecan, Männedorf, Switzerland). Protein levels of the treated conditions and the vehicle control were then compared.
DMMB assays to quantify sulfated glycosaminoglycans (sGAG) in the media of degenerate NP pellets with or without curcumin or o-Vanillin treatment were performed as previously described [27
]. Chondroitin sulfate was used to generate the standard curve. Conditioned media samples were pooled from days 4 to 21. All samples were diluted to fall within the middle of the linear portion of the standard curve, then placed in triplicate into clear 96-well plates (Costar, Corning, NY, USA); later, DMMB dye was added to the wells. Samples were analyzed for absorbance at room temperature immediately after adding DMMB dye, using a spectrophotometer (Tecan Infinite T200, Männedorf, Switzerland).
2.10. Western Blot Analysis
Following treatment, cells were washed once with ice-cold PBS (pH 7.4) and then lysed in hot Laemmli sample buffer. Twenty micrograms of protein/sample of the homogenate was resolved with 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with 5% skim milk, and incubated overnight with antibodies recognizing total or phosphorylated AKT, ERK1/2, JNK, Nrf2, p38, p65, and β-actin (Cell Signalling, Beverly, MA, USA). HRP-coupled secondary antibodies (Abcam, Cambridge, Ma, USA) were used, and detection was performed using Western Lightning Plus (NEL103E001EA, PerkinElmer, Woodbridge, ON, Canada). The intensity of each band was normalized to that of β-actin, and the data are presented as relative intensity.
2.11. Statistical Analysis
The data was analyzed using Graph Prism 6 (Graph Pad, La Jolla, CA, USA). A paired t-test was used to analyze the two groups, and a multiple pairwise comparison (Analysis of Variance (ANOVA) was used to evaluate the variance with Bonferroni’s post hoc test. The significance was set at p < 0.05.
Senescent cells were long thought to be passive bystanders, but recent data suggest that they actively participate in age-related diseases such as osteoarthritis, heart disease and cancer. Here, we show that curcumin and its metabolite o-Vanillin have senolytic and senomorphic effects on senescent cells of degenerating human IVDs. Reducing the number of senescent cells promoted the proliferation of the remaining cells and generated a more favorable and less inflammatory environment with a reduced SASP. Glucose concentration in the culture media can affect cellular senescence, and standard cell culture media containing 4.5 g/L glucose is often used to culture IVD cells, but it has previously been shown to induce senescence in rat IVD and notochordal cells [32
]. Here, we found a higher number of senescent cells following culture in 4.5 g/L than in 2.25 g/L glucose. Thus, 2.25 g/L glucose was used in all subsequent experiments to minimize culture-induced senescence.
While cell cycle arrest is a hallmark of senescence, not all non-dividing cells are senescent, and different markers could give different results. We determined how p16INK4a
and SA-β-gal correlate in the detection of positive cells and found significant differences in the exact number of cells labeled as senescent with the two markers. However, both methods indicated a similar fold change (~40%), with higher levels of senescent cells from degenerate compared to non-mildly-degenerate IVDs. SA-β-gal staining indicated a higher level of positive cells but it has been suggested to also identify quiescent cells in addition to truly senescent cells [33
]. This marker may therefore overestimate the number of senescent cells. p16INK4a
staining, on the other hand, could identify a sub-population of senescent cells, thereby underestimating the total number. However, independent of the exact number of cells detected as senescent, our results corroborate with other studies relating cellular senescence to IVD degeneration [34
]. In addition, the higher number of senescent cells from degenerating tissue was associated with an increase in SASP factor production.
Extrinsic factors like age, sex and lifestyle are known to affect cell senescence, confounding a direct link to IVD degeneration. Thus, we quantified the number of senescent cells and SASP factor expression from degenerate and non-mildly-degenerate discs of the same individual. Radiographic [29
] and macroscopic [30
] grading systems were used to define the overall degree of degeneration prior to cell isolation. Interestingly, both degenerate NP and AF tissues had significantly more senescent cells and a higher expression of key genes encoding the senescence marker p16INK4a
, ECM-degrading enzymes (MMP3 and MMP13), and pro-inflammatory cytokines (IL6 and IL8). These results directly correlate with IVD degeneration, with the accumulation of senescent cells and SASP factors in human IVD. To our knowledge, this is the first study to evaluate the basal level of senescent cells in IVDs, with different degrees of degeneration from the same individual.
We found a higher percentage of p16INK4a
-positive cells in non-mildly-degenerate tissue from donors aged 62 ± 14.68 years, compared to younger (40.42 ± 17.23 years) donors. However, the accumulation was clearly higher in degenerate tissue, independent of age. Our findings were in accordance with previous reports suggesting that ageing promotes cell senescence [34
Curcumin and o-Vanillin are well-known for their anti-oxidative and anti-inflammatory properties. We used 5 μM curcumin and 100 μM for o-Vanillin; the concentrations were selected based on their effects on metabolic activities and on concentrations used in previous publications. In our experiments, curcumin concentrations above 50 µM were toxic. Interestingly, similar and higher concentrations of curcumin were not cytotoxic in mixed population of IVD cells [35
]. Furthermore, o-Vanillin (200 μM) showed no cytotoxicity in our cultures, corroborating with previous studies [36
]. Treatment with both compounds reduced the number of senescent cells and SASP factors, and it was accompanied with an increase in cell proliferation. Curcumin has been shown to activate caspase-3, and to induce time- and dose-dependent apoptosis in cancer cells [37
]. To evaluate if curcumin and o-Vanillin are senolytic to senescent IVD cells, we next investigated whether the two compounds increased proliferation (Figure 3
D) and apoptosis (Figure 3
E), by selectively killing off the non-proliferating senescent cells, or by increasing the proliferation and apoptosis of non-senescent cells. Cells from degenerate (with about 30% of the cells were p16INK4a
-positive cells) and non-mildly-degenerate (6% were p16INK4a
-positive cells) IVDs were evaluated. Our results demonstrate that both compounds selectively induce apoptosis in senescent (p16INK4a
positive) but not proliferating (Ki-67 positive) cells (Figure 4
A). Moreover, curcumin and o-Vanillin increased metabolic activity (Figure 4
B), caspase 3/7 activity (Figure 4
C), and apoptosis (Figure 4
D) in cells from degenerate IVDs, but not in cells from non-mildly-degenerate IVDs. These results suggest senolytic potential, where curcumin and o-Vanillin are killing off senescent cells isolated from human IVDs.
IVD cells maintain their native phenotype better when cultured in a three-dimensional culture system, and it allows for matrix synthesis to be evaluated [38
]. Collecting all isolated cells in pellets also reduces the risk of selection bias, based on their adhesion to plastic culture dishes. In pellet cultures, we found a more profound reduction in the senescent cells following treatment, which could be explained by a higher number of cells being captured using this method. The different times of treatment for the monolayer and pellet cultures could also play a role. The short course of treatment (6 h) in monolayer cultures resulted in a rapid reduction in senescent cells, reinforcing the hypothesis of senolytic activity. In contrast, the longer treatment (4 days) and the following culture (21 days) gave both compounds more time to suppress senescence markers, and/or prevent secondary senescence, adding to the senolytic and senomorphic effects consistent with their anti-inflammatory properties [11
The loss of proteoglycan and decreased collagen type II production are regarded as typical pathological changes in IVD degeneration. Interestingly, curcumin and o-Vanillin significantly increased the proteoglycan contents of both NP cell pellets and the surrounding culture media. Both compounds also increased collagen type-II production and reduced the secretion of SASP factors. These results suggest a comparable effect of the two compounds and highlight the beneficial effects of curcumin and o-Vanillin on IVD health, and they are potential therapeutics for IVD degeneration.
Curcumin has been reported to act on multiple cellular targets and signaling pathways in cells of different origins [39
]. NFkB and Nrf2 signaling pathways are both important in the defense mechanism against oxidative stress and cellular senescence [40
]. Our findings demonstrate higher expression and nuclear translocation of p65 and Nrf2 in cells from degenerate IVDs. Both decreased following treatment with curcumin and o-Vanillin. The lower level of NFkB nuclear translocation indicates a downregulation of inflammatory gene transcription. Also, the decrease in Nrf2 expression highlights the homeostatic condition where Nrf2 is maintained at low levels by the inhibitory factor Keap1 [41
]. These results corroborate with reported data that curcumin modulates the Nrf2-Keap1 complex, leading to Nrf2 binding to the antioxidant responsive element [42
] and the translocation inhibition of NFkB into the nucleus [43
The dysregulation of multiple signaling pathways has been shown to be implicated in IVD degeneration. The transcription factor Nrf2 is activated by upstream kinases, including PI3K/AKT, JNK/SAPK, ERK1/2, p38, and NFkB [44
]. Therefore, major MAPK pathways were also investigated following treatments with curcumin and o-Vanillin. JNK activation triggers senescence [45
], and a link between JNK/SAPK and p16INK4a
has been reported [46
]. Interestingly, our results demonstrate a decrease of JNK activation in the treated cells, suggesting that this pathway is a mediator of the senolytic effect.
PI3K/AKT plays a critical role in IVD degeneration [47
], and silencing of AKT induces the apoptosis of human-degenerated NP cells [48
]. We found a decrease in the phosphorylation levels of AKT in the treated groups, suggesting that curcumin and o-Vanillin mediate apoptosis through AKT inhibition. Curcumin modulation of the AKT pathway has been reported in cancer cells, and a crosstalk between the AKT and NFkB signaling pathways [49
] has been described. Both the ERK and p38 pathways were activated in NP cells when they were exposed to curcumin and o-Vanillin. ERK1/2 phosphorylation has been described to stimulate IVD cell proliferation [50
]. Although p38-MAPK is reported to mediate senescence in fibroblasts [51
], an increase in p38 in human NP has also been linked to IVD cell proliferation. Indeed, p38, which is upstream of Nrf2, can both stimulate and inhibit Nrf2 nuclear translocation.
To date, few natural compounds have been shown to display senolytic activity. The data presented here suggest that curcumin and o-Vanillin may be used as senolytic and anti-inflammatory drugs for senescent IVD cells. The presented observations prompt the need for further investigations of the two compounds and their therapeutic contributions in reducing cellular senescence and retarding IVD degeneration in vivo.