Osteoarthritis (OA) is the most common form of arthritis. It is also the most common cause of disability in older adults, affecting approximately 15% of the population. As an age-related disease, it mostly affects joints, shoulders, hands, feet, and spine, with knee and hip joints being commonly affected. The disease is characterized by deterioration of cartilage in joints, resulting in bones rubbing together that causes stiffness, pain, and impaired movement. Its risk factors include occupational injury, trauma, obesity, bone density, gender, lack of exercise, and genetic predisposition [1
]. As a main cause of disability, OA reduces the quality of life with socio-economic burdens due to limited social activities, mood, memory loss, suicide, economic cost, and decreased productivity. OA is not only a problem in developing countries, but also a problem in developed countries [5
]. To overcome these burdens, effective protective strategies need to be established by targeting leading causes of OA and improving the functional status of chondrocytes.
Age-related oxidative stress refers to increased intracellular levels of reactive oxygen species (ROS) known to play a critical role in the development and progression of OA [11
]. Oxidative stress can lead to OA due to cell death of chondrocytes, telomerase shorting, DNA damage, and mitochondrial dysfunction, which can stimulate signaling pathways to initiate biological processes [11
]. ROS can stimulate nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathways. Both NF-κB and Nrf2 are transcription factors implicated in gene expression of antioxidant enzymes [15
]. However, a high concentration of ROS causes apoptosis in chondrocytes [11
]. NF-κB and Nrf2 signaling pathways contribute to ROS reduction and provide protection to chondrocytes against oxidative stress [15
]. Thus, an effective protective strategy needs to be established by targeting oxidative stress through NF-κB and Nrf2 signaling pathways to maintain redox balance and chondrocyte homeostasis.
ROS are known to be cytotoxic to most species including human. However, cells can neutralize ROS through molecular mechanisms using antioxidants and antioxidant enzymes. These antioxidants and antioxidant enzymes can convert superoxide to H2
O and O2
, regulate the intracellular redox, and counteract ROS to protect chondrocytes from oxidative damage [11
]. Chondrocytes express a variety of antioxidants and antioxidants enzymes to prevent harmful effects of ROS. However, due to age-related decline in the expression of antioxidant enzymes and increased intracellular levels of ROS, homeostasis maintenance is lost and articular cartilage is degenerated, resulting in chondrocyte apoptosis and increased vulnerability to ROS-induced cell death [21
]. Therefore, there is a need to search for ways protect chondrocytes against oxidative stress and maintain homeostasis. Recently, natural compounds such as cyanidin, delphinidin, malvidin, and pelargonidin were used as therapeutics for OA and several diseases [24
]. Due to their potential as antioxidants, anthocyanins can protect chondrocytes against oxidative stress via various pathways, thus suppressing the progression of OA [25
]. Among various kinds of anthocyanins, delphinidin is a specific class of polyphenols that can protect chondrocytes and inhibit the progression of OA [28
]. However, how delphinidin protects chondrocytes and inhibits the progression of OA remains elusive.
Autophagy is a metabolic process that can sequester protein aggregates, damaged cellular organelles, and pathogen autophagosomes to be fused with lysosomes in the cytoplasm for degradation. This phenomenon is intrinsically adopted by cells to support them in a metabolically defiant microenvironment, to escape toxicity, and to reduce apoptosis induced by ROS [29
]. Autophagy has dual and context-dependent roles in progression, protection, and death promotion in OA pathogenesis as in cancer [31
]. Chondrocytes use autophagy as a very efficient housekeeping program for homeostasis maintenance and protection against various stresses [34
]. The loss or impairment of autophagy in articular cartilage under mechanical or inflammatory stress was linked to aging-related cell death and increased OA severity [38
]. However, the relationship between autophagy and cell death in chondrocytes and its mechanism are not fully understood yet. Additional studies are needed to decipher the underlying cell signaling mechanisms of autophagy.
In this study, we evaluated the potential role of delphinidin in protecting human chondrocytes against hydrogen peroxide (H2O2)-induced oxidative stress. Furthermore, we investigated which mechanism might be associated with delphinidin-induced cell protection in human chondrocytes. Cell survival is generally associated with apoptosis or autophagy. However, we do not yet know which pathway delphinidin uses to prevent the progression of OA. Thus, the purpose of this study was to investigate whether delphinidin might be cytoprotective for human chondrocytes and to further elucidate mechanisms associated with the effect of delphinidin. We hypothesized that delphinidin could prevent the progression of OA via autophagy.
2. Material and Methods
C28/I2 human chondrocyte cells (SCC043) were purchased from Merck (Darmstadt, Germany). Fetal bovine serum (FBS; 16000-044), Dulbecco’s modified Eagle’s medium (DMEM, 11995-065), Roswell, and Hank’s buffered salt solution (HBSS, 14025-092) were purchased from Gibco and Life Technologies (Pittsburgh, PA, USA). Hydrogen peroxide solution (H2O2) (#MKBV3835V), N-acetylcysteine (NAC), DCFDA (2′,7′-dichloroflourescin diacetate), acridine orange hydrochloride hydrate (#318337), and monodansylcadaverine (MDC) (#30432) were bought from Sigma (St. Louis, MO, USA). Delphinidin chloride (cat no. ALX-385-028-M010, Lot no. L29971) was obtained from Enzo life sciences (Farmingdale, NY, USA). Chloroquine (C6628) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (R-5000) was purchased from LC Laboratories (Woburn, England). CCK-8 (Cell Counting Kit-8) was purchased from Dojindo (Tokyo, Japan). The DeadEnd™ Fluorometric terminal uridine nick-end labeling (TUNEL) system (Ref#G3250, Lot #0000007545) was obtained from Promega (Madison, WI, USA.). The β-actin antibody (A5441) was from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against LC3A/B (12741), caspase-3 (#9662), cleaved caspase-3 (#9661), NF-κB p65 (#8242), phospho-NF-κB p65 Ser536 (#3033), and cleaved poly(ADP-ribose) polymerase N-acetylcysteine (PARP) (#9541) were bought from Cell Signaling Technology (Beverly, MA, USA). Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (#p36966) was purchased from Invitrogen (Calsbad, CA, USA). Antibodies against MAP1LC3 (Microtubule-associated proteins 1A/1B light chain 3B)(SC-376404) and Nrf2 (SC-722) were purchased from Santa Cruz biotechnology (Dallas, TX, USA). Secondary antibodies against rabbit immunoglobulin G (IgG) (STAR208P) and mouse IgG (STAR117P) were purchased from Bio-Rad (Hercules, CA, USA). Secondary antibodies for immunocytochemistry (fluorescein isothiocyanate, FITC) were obtained from Santa Cruz (Dallas, TX, USA).
2.2. Cell Viability
C28/I2 human chondrocyte cells (SCC043) were obtained from Merck and cultured at 37 °C in a 5% CO2 humidified atmosphere in DMEM medium supplemented with 5% (v/v) FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cell viability of C28/I2 human chondrocytes was determined using the CCK-8 kit. Cells (~5 × 104 cells/well) in 96-well plates were treated with hydrogen peroxide solution (H2O2) in combination with other compounds such as NAC, delphinidin chloride, chloroquine, or rapamycin. Each well containing 100 µL of media and CCK-8 reagent (10 µL) was added into each well, and cells were further incubated at 37 °C for 4 h in a 5% CO2 humidified atmosphere. Cell viability was determined by measuring absorbance at 485 nm using a microplate reader (Hidex 1 FN/Chameleon; Turku, Finland).
2.3. Determination of Intracellular ROS
C28/I2 human chondrocyte (~5 × 104 cells/well) were seeded in 96-well plates in DMEM medium (200 µL) supplemented with 5% FBS for 24 h. After incubation, cells were then treated with 500 µM H2O2 in the presence or absence of 40 µM of delphinidin and 5 mM NAC, incubated at 37 °C for 2 h. and 4 h. Intracellular ROS levels were determined with a DCFDA cellular ROS detection assay kit (cat. no. ab113851; Abcam, Burlingame, CA, USA) After adding 30 µM DCFDA dissolved in DMSO/phosphate-buffered saline (PBS) to each well, plates were incubated at 37 °C for 30 min in the dark. Plates were then read with a GloMax® detection system (Model #E 8032; Promega, Sunnyvale, CA, USA) at 485/535 nm.
2.4. TUNEL Assay
TUNEL (terminal uridine nick-end labeling) assays were performed to measure the apoptosis by using the Promega DeadEnd™ Fluorometric TUNEL assay system. Cells were cultured on coverslips for 24 h and then treated with hydrogen peroxide solution (H2O2) in the presence or absence of NAC (N-acetylcysteine), delphinidin chloride, chloroquine, and rapamycin. Cells were fixed with 4% (w/v) paraformaldehyde for 30 min and permeabilized with PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cells were blocked with 5% horse serum in PBS for 1 h, and then TUNEL assays were performed to measure the apoptosis accordance with the manufacturer’s instruction of the Promega DeadEnd™ Fluorometric TUNEL assay system for 1 h. After washing with PBS, cells the glass coverslips were mounted onto glass slides using a mounting medium containing DAPI. Slides were analyzed with a florescence microscope (BX51-DSU; Olympus, Tokyo, Japan). More than 1500 nuclei were counted per field. The experiment was repeated three times.
2.5. Western Blot Analysis
C28/I2 human chondrocytes, after treatment with hydrogen peroxide solution (H2O2) in the presence or absence NAC (N-acetylcysteine), delphinidin chloride, chloroquine, and rapamycin, were collected and washed twice with ice-cold 1× PBS. Total proteins were extracted with a cell lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (HaltTM Protease), and the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA) method was used for determination of protein concentration. After concentration, equal amounts (30 µg) of total protein were separated by 12% or 10% SDS-PAGE. Target proteins were specifically detected by Western blotting using indicated antibodies. Proteins were visualized with enhanced chemiluminescence detection reagent (Thermo Scientific, Waltham, MA, USA). All protein levels were normalized to β-actin levels.
2.6. Immunofluorescence Staining
C28/I2 human chondrocytes were cultured on polylysine-coated coverslips for 24 h. After incubation, cells were treated with 500 µM H2O2 in the presence or absence of 40 µM delphinidin chloride for 2 h. After treatment, cells were washed twice with 1× PBS and then fixed with 4% (w/v) paraformaldehyde for 30 min. After incubation, cells were washed with 1× PBS and permeabilized with PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cells were blocked with 5% horse serum in PBS for 1 h and then incubated with primary antibodies overnight at 4 °C. After washing with PBS, cells were incubated with FITC-conjugated secondary antibodies (1:50 in PBS) at room temperature for 90 min. Slides were washed twice with PBS for 5 min each, and images were captured with a confocal microscope (FV-1000; Olympus, Tokyo).
2.7. Acridine Orange Dye Staining
C28/I2 cells were cultured on coverslips for 24 h and then treated with 500 µM H2O2 in the presence or absence of delphinidin for 2 h. After treatment, cells were washed three times with PBS, fixed with 4% formaldehyde, and permeabilized with 100% methanol. For determination of autophagy, an acridine orange (AO) assay were performed according to the given instructions. Acridine orange (AO) dye was added to cells at a final concentration of 1 μg/mL and incubated at room temperature for 15 min, protected from direct light. Red (acidic vacuoles) and green (cytoplasm) fluorescent images were captured with a confocal microscope (FV-1000; Olympus, Tokyo) using color filters for TRITC (Tetramethylrhodamine) and FITC (Fluorescein isothiocyanate), respectively. Overlapped images were then processed and quantified using NIH Image J software.
2.8. Monodansylcadaverine (MDC) Staining for Autophagic Vacuoles
C28/I2 cells were seeded into six-well plates with sterile cover slips for 24 h and then treated with 500 µM H2O2 in the presence or absence of delphinidin chloride, rapamycin, or chloroquine for 2 h. Following treatment, cells were washed with 1× PBS three times. After washing, autophagic vacuoles were labeled with MDC by incubating cells grown on coverslips with 0.05 mM MDC in PBS at 37 °C for 15 min in the dark (protected from direct exposure to light). After incubation, cells were washed with PBS four times and immediately analyzed with an inverted fluorescent microscope (Leica).
2.9. Statistical Analyses
Each experiment was done at least three times. Representative data are reported as means ± standard deviation (± SD). Differences between two groups were assessed using a two-tailed Student’s t-test. One-way analysis of variance (ANOVA) was used to compare means of three groups or more followed by a Tukey’s multiple comparison tests. Values of p < 0.05 and p < 0.01 were considered significant. All statistical analyses were performed using SPSS 18.0 for Windows (SPSS, Chicago, IL, USA).
The most important finding of this study was that delphinidin could protect human chondrocytes against oxidative stress by regulating Nrf2 and NF-κB and activating autophagy. Hydrogen peroxide (H2
) has a cytotoxic effect on human chondrocytes via ROS activation, which can be inhibited by delphinidin through its antioxidant potential to protect cells from apoptosis. This protective mechanism of delphinidin was due to activation of antioxidant response pathways such as Nrf2 and NF-κB pathways and protective autophagy in chondrocytes (Figure 6
). We hypothesized that delphinidin might have protective effects on human chondrocytes and help prevent the progression of OA by inducing autophagy. Results of this study support our hypothesis, indicating that delphinidin-induced protective effects for cells via autophagy might provide theoretical evidence and novel insight for treating OA.
This study demonstrated the molecular mechanism involved in the protection for chondrocytes elicited by delphinidin against H2
-induced oxidative stress. Responses of cells to cytokines and growth factors depend on the cell’s redox status, which is the result of a subtle equilibrium between ROS production and intracellular antioxidant level. Previous studies reported that OA progression is significantly associated with oxidative stress and ROS [44
]. In fact, ROS are produced at low levels in articular chondrocytes normally. Under normal circumstances, physiological levels of ROS can reversibly modify biomacromolecules which play crucial roles in regulating cell function. Furthermore, they are integral actors of intracellular signaling mechanisms contributing to the maintenance of cartilage homeostasis because they can modulate chondrocyte apoptosis, gene expression, extracellular matrix (ECM) synthesis and breakdown, and cytokine production [46
]. However, under oxidative stress, excess ROS can affect chondrocytes in multiple ways, including telomerase shorting, DNA damage, mitochondrial dysfunction, and stimulation of cell death signaling pathways [11
]. In the same vein, ROS levels due to oxidative stress were found to be elevated in patients with OA. Over-produced ROS can act as second messengers to promote the expression of matrix metalloproteinases (MMPs), which can lead to cartilage degeneration [50
]. Furthermore, ROS can directly degrade matrix components and induce cell death [53
]. Based on this theoretical evidence, the viability of cartilage cells was observed to be decreased when oxidative stress was applied to the cartilage in the present study. Furthermore, when using NAC and delphinidin as powerful antioxidants, cell viability was increased, confirming that H2
-induced oxidative stress could directly affect the redox status of cartilage cells and become harmful to cells.
Most existing pharmacologic therapies for OA such as oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) and glucosamine are limited to pain management rather than prevention and cure. Surgery is typically the last resort for treating knee OA [54
]. Indeed, efficient licensed disease-modifying drugs are currently unavailable. To overcome these burdens, effective protective strategies need to be established by targeting leading causes of OA and improving the functional status of chondrocytes. Recently, natural compounds such as anthocyanins were used as therapeutics for several diseases [24
]. Among various anthocyanins, delphinidin is a specific class of polyphenols with beneficial effects on OA progression via various pathways. Wongwichai et al. [26
] reported that anthocyanins can inhibit IL (interleukin)-1β induced expression of MMPs in human articular chondrocytes via NF-κB and the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway. Haseeb et al. [28
] reported that delphinidin can inhibit IL-1β-induced production of a cartilage-degrading molecule prostaglandin E2 (PGE2) via inhibition of cyclooxygenase-2 (COX-2) expression. Their results also verified that delphinidin could inhibit IL-1 receptor-associated kinase-1 (IRAK1Ser376
) phosphorylation by IL-1β-induced activation of NF-κB in human chondrocytes. Although biomarkers such as IL-1β, IL-6, tumor necrosis factor-α (TNF-α), PGE2, and COX-2 related to anti-inflammation were not analyzed in the present study, results of this study confirmed that the mechanism via which delphinidin protected chondrocytes from oxidative stress was through underlying cell-death mechanisms.
In general, cell death is most commonly associated with apoptosis, although it can also occur by means of another mechanism such as autophagy. Apoptosis is a highly regulated, active process of cell death involved in the development, homeostasis, and aging [55
]. Dysregulation of apoptosis can lead to pathological states such as various degenerative diseases. For instance, with increased oxidative stress, disequilibrium between DNA damage and repair can occur, resulting in cumulative un-repaired DNA damage. This accumulation could lead to altered gene transcription and the formation of altered proteins or the induction of apoptosis [56
]. To prevent the development of OA, preventing the degradation of chondrocytes is essential. Thus, it is important to regulate the induction of excessive apoptosis. Unlike apoptosis, autophagy is a homeostatic mechanism and an important process in all cells to remove damaged mitochondria and misfolded proteins known to cause production of ROS that can cause DNA damage and lead to genomic instability [60
]. However, excess autophagy induction can trigger autophagy-related apoptosis [61
]. Thus, accurate understanding of the role of apoptosis and autophagy is critical for OA treatment.
Chondrocytes are pre-mitotic cartilage cells that lack the proliferative activity in adult articular cartilage, suggesting that there must be some strategies to protect chondrocytes cells under various stresses to resist apoptosis [14
]. In this study, we found that delphinidin treatment increased levels of antiapoptotic protein (Bcl-XL
, Nrf2, and NF-κB) but decreased levels of proapoptotic proteins (caspase-3, c-PARP) in chondrocytes. Furthermore, apoptosis was much decreased after treatment with delphinidin as compared to that in the group treated with H2
only. These results confirmed our hypothesis that delphinidin could be a potential therapeutic drug to prevent oxidative stress-induced cell death and protect chondrocytes.
Nrf2 and NF-κB signaling pathways contribute to ROS reduction and provide protection to chondrocytes under oxidative stress [15
]. Thus, an effective protective strategy could be established by targeting oxidative stress and Nrf2 and NF-κB signaling pathways to maintain redox balance and chondrocyte homeostasis. In the present study, we found that delphinidin could activate Nrf2 and NF-κB signaling pathways, suggesting the importance of Nrf2 and NF-κB signaling pathways in the protection of chondrocytes by delphinidin. A knockout mouse study showed that NF-κB has anti-apoptotic activity in non-immune cells. Many subsequent cell or tissue experiments showed that one of the most important functions of NF-κB in non-immune cells is its anti-apoptotic activity [62
]. Nrf2 inductions may also play a key role in OA pathophysiology. Abnormal ROS signaling in OA by stress is associated with increased Nrf2 activity, which plays a major chondroprotective role in the progression of OA. It is also associated with suppressed levels of IL-1β-induced MMP-1, MMP-3, MMP-13, PGE2, and nitric oxide (NO) production [64
]. Similar reports showed that other compounds with antioxidant properties such as hyaluronic acid (HA), 7,8-dihydroxyflavone (7,8-DHF), resveratrol, licochalcone A, diallyl disulfide, pterostilbene, wogonin, protandim, and 6-gingerol can exert anti-inflammatory and chondroprotective properties in joint tissues through activation of Nrf2 and NF-κB pathways [17
]. These lines of evidence demonstrate the importance of Nrf2 and NF-κB signaling pathways in maintaining redox balance and chondrocyte homeostasis during aging and in OA. However, the precise signaling pathways that elicit these effects remain incompletely understood.
Autophagy has a dual and context-dependent role in progression, protection, and death promotion in OA pathogenesis as in cancer [31
]. Chondrocytes use autophagy as a very efficient housekeeping program for homeostasis maintenance and protection under various stresses [34
]. The loss or impairment of autophagy in articular cartilage under mechanical or inflammatory stress was linked to aging-related cell death and increased OA severity [38
]. All these findings show that the relationship between autophagy and cell death in chondrocytes is not fully understood yet. Additional studies are needed to decipher the underlying cell signaling mechanisms of autophagy. Here, we found that cytoprotective mechanism of delphinidin was due to the activation of protective autophagy, since inhibition of autophagy increased apoptosis while its activation significantly decreased apoptosis. The finding that delphinidin has cytoprotective roles in oxidative stress of chondrocytes is valuable information.
Although the present study has its merits, it also has several limitations. Firstly, it was found that redox signaling can control chondrocyte function by regulating signal transduction pathways such as those involving MAPK, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), mammalian target of rapamycin (mTOR), and cyclic guanosine monophosphate (cGMP), as well as transcription factors such as hypoxia-inducible factor (HIF)-1a, activator protein-1 (AP-1), and NK-κB/p65 [71
]. Although we used delphinidin as an antioxidant for redox control, due to limitations in using various antibodies, detailed aforementioned pathways were not proven. These detailed pathways need to be identified through further studies. Secondly, based on a previous study, OA cartilage is more sensitive to oxidative stress with significantly more ROS-induced DNA damage than normal cartilage [72
]. However, this study only evaluated when delphinidin acted on oxidative stress in normal cartilage cells. Thirdly, for clinical application of delphinidin as a chemotherapeutic agent, although the present studies documented the cytoprotective effects on chondrocytes in vitro by delphinidin, concentrations of bioactive compounds sufficient enough to exert such protective effects in vivo need to be determined. For instance, the bioeffective compounds might be formed or removed in vivo due to intestinal bacterial and/or hepatic metabolism [73
]. For these reasons, to obtain the concentrations required to induce direct cell-protective activity is difficult in vivo. This is thought to be closely related to the method of application (topical, oral, or injection) of bioactive compounds and should be proven through animal models and pre-clinical trials on the basis of our findings. Our long-term clinical goal is to reduce the prevalence of osteoarthritis by demonstrating how delphinidin can prevent OA progression. Therefore, the precise mechanism and application via which delphinidin operates in OA cartilage also need to be determined in the future. Despite these limitations, this study is the first to show that delphinidin can protect cells against oxidative stress by inhibiting apoptosis and causing autophagy.