Apoptosis (2014) 19:1354–1363 DOI 10.1007/s10495-014-1012-1
ORIGINAL PAPER
Resveratrol protects rabbit articular chondrocyte against sodium nitroprusside-induced apoptosis via scavenging ROS Qian Liang • Xiao-ping Wang • Tong-sheng Chen
Published online: 8 July 2014 Ó Springer Science+Business Media New York 2014
Abstract This study aims to investigate the mechanism by which resveratrol (RV) prevents sodium nitroprusside (SNP)-induced chondrocyte apoptosis, which is a characteristic feature of osteoarthritis (OA). Rabbit articular chondrocytes were pre-incubated with 100 lM RV for 18 h before 1.5 mM SNP co-treatment for 6 h. Cell viability was evaluated by CCK-8. Annexin V/PI double staining and Hoechst 33258 staining were used to determine the fashion of SNP-induced chondrocytes death. Mitochondrial membrane potential (DWm) was measured by using flow cytometry (FCM) with TMRM and Rhodamine 123 staining. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were confirmed by FCM analysis with DCFH-DA and DAF-FM DA staining. Cytoskeleton proteins of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red were validated by confocal microscopy. SNP induced time- and dose-dependent chondrocytes apoptosis with decline of DWm, activation of caspases as well as cytoskeletal remodeling. SNP induced a significant induction of both ROS and NO. RV remarkably prevented SNP-induced ROS production and apoptosis as well as cytoskeletal remodeling, but did not prevent SNPinduced NO production. Pretreatment with NO scavengers did not significantly prevent SNP-induced apoptosis and cytoskeletal remodeling. SNP induces NO-independent ROS production which dominates rabbit articular chondrocyte Q. Liang X. Wang (&) Department of Pain Management, The First Affiliated Hospital of Jinan University, Guangzhou 510630, China e-mail: [email protected] T. Chen MOE Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510630, China
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apoptosis, and RV protects chondrocytes against SNPinduced apoptosis via scavenging ROS instead of NO. Keywords Reactive oxygen species Resveratrol Nitric oxide Chondrocyte Sodium nitroprusside Osteoarthritis
Introduction Osteoarthritis (OA) is often a progressive and disabling disease [1, 2]. Chondrocyte apoptosis has been reported to be correlated with the severity of OA [3]. Nitric oxide (NO) and reactive oxygen species (ROS) have been suggested to be key factors to mediate chondrocyte apoptosis [4–6]. Excessive ROS may lead to irreversible damage of mitochondrial DNA, resulting in mitochondrial dysfunction and ultimately cell death [7, 8]. It was reported that NO and ROS can be produced by chondrocytes in response to stimulation such as interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a), fibroblast growth factor (FGF) and transforming growth factor-b (TGF-b) [9–12]. NO can inhibit cytochrome oxidase in mitochondria, resulting in a reduction of the electron transport chain and the formation of superoxide anions (O2-) [13]. Peroxynitrite from the reaction of O2- and NO can nitrate tyrosine residues to form 3-nitrotyrosine that is often considered as the oxidative damage of cellular proteins correlating with OA in human articular cartilage [4, 14, 15]. Sodium nitroprusside (SNP) is generally considered as the donor of NO to study the mechanism of chondrocyte apoptosis induced by NO [16–19]. Many mitochondrial events occurred during SNP-induced chondrocyte apoptosis, including reduction of the enzyme mitochondrial activity of complex IV, decline of mitochondrial membrane potential (DWm) [20] and release of
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both cytochrome c (Cyt c) and AIF [21]. In addition, it was reported that SNP-induced NO upregulated endogenous ROS to mediate chondrocyte apoptosis [19, 22]. Resveratrol (RV) has therapeutic potential for OA due to its anti-inflammatory and antioxidative properties [23–25]. RV could prevent IL-1b-induced chondrocytes apoptosis via activating SIRT1 and reversing the expression of inducible nitric oxide synthase (iNOS), and the activation of caspase-3 [23, 26, 27]. IL-1b-induced p53- and ROS-dependent apoptosis can also be significantly inhibited by RV [28]. This study is designed to explore the mechanism by which RV protects rabbit articular chondrocytes against SNP-induced apoptosis. Our findings for the first time demonstrate that SNP induces NO-independent ROS to dominate chondrocytes apoptosis, and RV protects rabbit articular chondrocytes against SNP-induced apoptosis via scavenging ROS instead of NO decomposed from SNP.
Materials and methods Reagents RV, SNP, dimethyl sulphoxide (DMSO), 20 ,70 -Dichlorofluorescin diacetate (DCFH-DA), Carboxy-PTIO (PTIO), Nacetyl cysteine (NAC), Rhodamine 123 (Rho 123) and Hoechst 33258 were from Sigma (St. Louis, USA). Dulbecco’s modified Eagle medium (DMEM) was from Gibco (Carlsbad, California, USA), fetal bovine serum (FBS) was from Sijiqing (Hangzhou, China). Cell Counting Kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan). Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was from Bender Medsystems (Vienna, Austria). 3-Amino, 4-aminomethyl-20 ,70 difluorescein, diacetate (DAF-FM DA), Hemoglobin (HG), Actin-Trakcer Green and Tubulin-Trakcer Red were from Beyotime Institute Biotechnology (Jiangsu, China). Mouse monoclonal anti-Bak (Ab-2) and anti-Bax (Ab-6) were from Calbiochem (San Diego, CA). Secondary goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) antibody, Tetramethylrhodamine methyl ester (TMRM), Trypsin and type II collagenase were from Invitrogen (California, USA). Z-IETD-fmk, z-DQMD-fmk and z-LEHD-fmk were from BioVision (San Francisco, USA). Ac-LEHD-AFC and Ac-DEVD-AFC were from Alexis (Switzerl). Ac-IETD-AFC was from BD (New Jersey, USA). RV and PTIO were dissolved in DMSO, and the final concentration of DMSO was less than 1 % in all experiments. SNP was freshly dissolved in phosphate buffered solution (PBS). NAC and HG were dissolved in ultrapure water. To scavenge NO, rabbit articular chondrocytes were pretreated with 100 lM PTIO or 20 lM HG for 2 h, and 2 mM NAC pre-incubation for 2 h was used to scavenge ROS, and then co-treated with SNP.
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Cell isolation and culture Articular chondrocytes for primary culture were harvested from slices of shoulder, knee and hip-joint cartilage from 6-week-old New Zealand white rabbits. Chondrocytes were isolated by enzymatic digestion of 0.25 % Trypsin in PBS for 1 h and 0.2 % type II collagenase in DMEM for 4–6 h. After collection by centrifugation, chondrocytes were resuspended in DMEM supplemented with 10 % FBS and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin), and then seeded in culture flasks at 37 °C in humidified 5 % CO2 as monolayer culture. The primary cells were subcultured to generation 2 that were cultured in DMEM supplemented with 10 % FBS and antibiotics for at least 24 h before different treatment. Cell viability and apoptosis assay Cell viability was detected by CCK-8 assay using auto microplate reader (Infinite M200, Tecan, Austria). All experiments were performed in quadruple occasions. Apoptosis rates were assessed by flow cytometry (FCM) analysis with Annexin V-FITC/PI kit as previously described [29], and for each FCM analysis 10,000 events were recorded. Chondrocytes stained with Hoechst 33258 for 30 min were imaged by a fluorescent microscope (Axiovert 200 M, Zeiss). Measurement of intracellular ROS and NO Intracellular ROS or NO was quantified by FCM using DCFH-DA staining or DAF-FM DA staining which is cellpermeable [30]. Chondrocytes were stained with 20 lM DCFH-DA for 30 min or with 5 lM DAF-FM DA for 20 min at 37 °C in the dark, and re-washed with PBS three times, subsequently 500 ll of cell suspension was added in 5 ml FCM tube and analyzed quantitatively using FCM. Measurement of mitochondrial membrane potential Chondrocytes stained with 10 lM TMRM or Rho 123 for 30 min at 37 °C in the dark were harvested, and were then washed with PBS solution twice and subsequently assayed by FCM. Results were expressed as the proportion of cells with low TMRM or Rho 123 fluorescence indicating the loss of DWm. Fluorometric determination of caspase enzymatic activation Activities of caspase-8, -9 and -3 were measured using fluorogenic substrate Ac-IETD-AFC Ac-LEHD-AFC and AcDEVD-AFC, respectively, according to the manufacturer’s
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instructions as described previously [31]. The caspase activation level in control cells was normalized to 1.0. FCM analysis of the activation of Bak and Bax Chondrocytes were fixed with 3.7 % formaldehyde in PBS for 10 min at room temperature, and treated with ice-cold 100 % methanol for 15 min to permeabilize the cells. The fixed cells were blocked in PBS solution containing 1 % bovine serum for 30 min at room temperature and then were incubated with either anti-Bax (6A7) or anti-Bak (Ab-2) (1:100) at room temperature for 60 min and then incubated with FITC-conjugated goat anti mouse IgG (1:200) for 30 min in the dark. After washing, the samples were analyzed by FCM.
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Fluorescent substrate assay showed that SNP induced a significant activation of caspase-3 and a modest but significant activation of caspase-8 and -9 (Fig. 1d). Moreover, inhibition of any one of casapse-8, -9 and -3 significantly prevented SNP-induced apoptosis (Fig. 1e). In addition, FCM assay showed that compared with control cells, the mean fluorescence intensity of SNP-treated cells increased to 130.54 % for active Bak and 126.79 % for active Bax, respectively (Fig. 1f, g), indicating that SNP induced the activation of Bak and Bax. FCM analysis with TMRM or Rho 123 staining showed that SNP treatment for 1 h induced a modest but significant decrease of DWm (Fig. 1h, i). Collectively, the caspases-dependent intrinsic and extrinsic pathways were involved in SNP-induced rabbit articular chondrocyte apoptosis.
Confocal microscopic imaging of cytoskeletons Cells were fixed with 3.7 % formaldehyde in PBS for 10 min at room temperature, then permeabilized and blocked in PBS containing 0.1 % Triton X-100 and 1 % fetal calf serum for 20 min. The fixed cells were washed with PBS containing 0.1 % Triton X-100 three times and then incubated for 1 h with 2 % Actin-Trakcer Green and 0.4 % Tubulin-Trakcer Red in PBS containing 0.1 % Triton X-100 and 1 % fetal calf serum at room temperature, re-washed, and then observed using a confocal microscope (LSM510Meta, Zeiss, Jena, Germany). The excitation wavelengths for Actin-Trakcer Green and Tubulin-Trakcer Red are 488 and 543 nm, and the corresponding emission channels are BP 505–530 nm and LP 560 nm. Statistical analysis Data were presented as mean ± SD from at least three experiments. Data were analyzed by student’s t test using SPSS 13.0. P values less than 0.05 were considered to be statistically significant.
Results SNP induces apoptosis in rabbit articular chondrocytes CCK-8 assays showed that SNP induced dose- (Fig. 1a) and time- (Fig. 1b) dependent cytotoxicity. Treatment with 1.5 mM SNP for 6 h was adopted in following experiments without indication. FCM analysis with Annexin V-FITC/PI staining showed that SNP treatment for 6 h induced a remarkable chondrocytes apoptosis (Q2 (PI positive and Annexin V-FITC positive) ? Q4 (PI negative and Annexin V-FITC positive)) from 13.2 % (control) to 65.6 % (Fig. 1c), demonstrating that SNP induced chondrocyte death in apoptotic fashion.
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Prevention of SNP-induced apoptosis in rabbit articular chondrocytes by RV CCK-8 assay showed that treatment with 100 lM RV for 24 h did not induce significant cytotoxicity (Fig. 2a, b), and that pretreatment with 100 lM RV for 12, 18, and 24 h respectively presented a potent prevention on the cytotoxicity of SNP (Fig. 2c). We also assessed the prevention of different dosage of RV (50, 75 and 100 lM) on SNPinduced apoptosis (data not shown), and found that 100 lM of RV showed the best protection effects. Pretreatment with 100 lM RV for 18 h was used in all following experiments without indication. Microscopic images showed that RV significantly prevented SNPinduced chondrocytes death (Fig. 2d). FCM analysis with Annexin V-FITC/PI staining also showed that RV potently prevented SNP-induced apoptosis (Fig. 2e), which was further verified by the microscopic images of chondrocytes stained with Hoechst 33258 (Fig. 2f). RV prevents SNP-induced apoptosis via scavenging ROS instead of NO We first used FCM assay to assess the SNP-induced ROS and NO production by using DCFH-DA and DAF FM-DA, respectively, and found that SNP treatment for 6 h induced a significant generation of both ROS (Fig. 3a) and NO (Fig. 3b). Pretreatment with RV markedly prevented SNPinduced ROS production, but did not prevented SNPinduced NO production (Fig. 3a, b). We also used FCM to assess the effects of NAC, a scavenger of ROS, and PTIO or HG, the scavengers of NO, on the SNP-induced ROS and NO production. As shown in Fig. 3c, d, NAC pretreatment largely prevented the SNP-induced ROS and NO, RV pretreatment markedly prevented SNP-induced ROS but modestly enhanced SNP-induced NO production, and pretreatment with PTIO or HG significantly prevented
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1358 b Fig. 1 SNP induces apoptosis. a SNP induced dose-dependent
cytotoxicity. **P \ 0.01, compared with control. b SNP induced time-dependent cytotoxicity. **P \ 0.01, compared with control. c SNP induced time-dependent apoptosis by FCM analysis with Annexin V-FITC/PI staining. d SNP induced activation of caspase-3, -8 and -9. *P \ 0.05 and **P \ 0.01, compared with control. e Inhibition of caspase-3, -8, and -9 by casapse inhibitors significantly prevented SNP-induced cytotoxicity. After pretreatment with each caspase inhibitor (10 lM) for 2 h or 100 lM RV for 18 h or RV combined with each caspase inhibitor, the cells were then co-treated with SNP or not. *P \ 0.05 and **P \ 0.01, compared with control; ## P \ 0.01, compared with SNP alone; $$P \ 0.01, compared with the combined treatment of RV and SNP. f, g SNP induced activation of both Bax (g) and Bak (f) by FCM analysis. Compared with Ctrl, the mean flourescence intensity of SNP-treated cells increased to 126.79 % for active Bax and 130.54 % for active Bak respectively. h, i SNP induced a rapid loss of DWm by FCM analysis with TMRM (h) and Rho 123 (i) staining
SNP-induced NO production but did not prevented SNPinduced ROS production. These data demonstrated that SNP induced ROS production independent of NO and RV pretreatment remarkably prevented SNP-induced ROS instead of NO. We next assessed the dynamics of ROS and NO generation after SNP treatment, and found that SNP treatment induced a rapid ROS generation that reached to saturation within 0.5 h and a slow NO generation that had significant increase at 2 h after SNP treatment (Fig. 3e), further demonstrating the notion that SNP induced ROS production independent of NO decomposed from SNP. CCK-8 assay showed that pretreatment with RV and/or NAC remarkably prevented SNP-induced apoptosis, but pretreatment with PTIO did not show any prevention on SNP-induced apoptosis (Fig. 3f). In addition, FCM analysis about DWm showed that pretreatment with RV or NAC induced a modest but significant prevention on SNPinduced loss of DWm (Fig. 3g). Collectively, SNP induced NO-independent ROS production that played a key role in SNP-induced chondrocytes apoptosis, and RV protected rabbit articular chondrocytes from SNP-induced apoptosis via scavenging ROS instead of NO. Prevention of SNP-induced cytoskeletal remodeling by RV Confocal microscopy was used to image the cytoskeleton of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red (Fig. 4). In untreated chondrocytes, the long-form and regular F-actin filament as well as uniformly microtubule cytoskeleton were observed. SNP not only shortened F-actin filaments but also induced disruption of microtubule structure and cell shrinkage, which could be significantly prevented by pretreatment with RV and/or NAC but not PTIO (Fig. 4a), and the statistical results on the percentages of remodeling chondrocytes
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under different conditions from three independent experiments were shown in Fig. 4b.
Discussion These results of this report support the notion that SNP induces ROS-dependent and NO-independent apoptosis in rabbit articular chondrocytes. Furthermore, this report for the first time demonstrates that RV remarkably prevents SNP-induced apoptosis by scavenging the SNP-triggered ROS independent of NO decomposed from SNP. In contrast to the acknowledged notion that SNP-induced NO mediates ROS generation to regulate cell apoptosis, our observations demonstrate that SNP induces ROS generation independent of NO to mainly mediate SNP-induced chondrocytes apoptosis. SNP is generally used as NO donor to study the role of NO in chondrocytes apoptosis [16, 18, 21, 32–34]. From the inhibitory effect of NAC on SNP-induced apoptosis in rabbit chondrocytes, Nakagawa and co-workers speculated that ROS acted as the downstream factor of NO to mediate SNP-induced apoptosis [22]. This inference is based on the hypothesis that NO is the key upstream mediator to mediate SNP-induced chondrocyte apoptosis [16–18]. Similarly, from the increase of intracellular ROS after exposure of human articular chondrocyte to SNP, Wu and co-workers inferred that intracellular ROS was produced by NO decomposed from SNP [19]. These studies did not assess the rigorous relationship between NO and ROS after SNP treatment, we herein focused on the roles of ROS and NO in SNP-induced chondrocytes apoptosis. In our system, the results that SNP induces a very rapid ROS generation within 0.5 h and slow NO generation that has a significant increase at 2 h after SNP treatment (Fig. 3e) and pretreatment with PTIO or HG significantly prevent SNP-induced NO production but does not prevent SNP-induced ROS production (Fig. 3c, d) demonstrate that SNP induces ROS generation independent of NO. It is generally considered that NO decomposed from SNP dominates SNP-induced chondrocyte apoptosis via an intrinsic apoptosis pathway [19, 35, 36]. Our data support this notion that the intrinsic apoptotic pathway participated in SNP-induced rabbit articular chondrocyte apoptosis (Fig. 1). However, HG or PTIO potently decreased SNPinduced NO production (Fig. 3d), but did not prevent SNPinduced cytotoxicity (Figs. 3f, 4). In strong contrast, RV remarkably prevented SNP-induced cytotoxicity (Fig. 3f) as well as almost completely prevented SNP-induced ROS production (Fig. 3c), but not NO (Fig. 3d). In addition, SNP treatment for 2 h only induced a very slight increase in intracellular NO level (Fig. 3e), but led to 40 % decrease of cell viability (Fig. 1b). Therefore, we concluded that ROS instead of NO dominated SNP-induced rabbit
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Fig. 2 Prevention of SNP-induced apoptosis by RV. a, b Cytotoxicity of RV on chondrocytes. **P \ 0.01, compared with control. c RV markedly prevented SNP-induced cytotoxicity. **P \ 0.01, compared with control; #P \ 0.05 and ##P \ 0.01, compared with SNP alone. d Microscopic images of chondrocytes after different
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treatments. Original magnification: 9200. e RV prevented SNPinduced apoptosis analyzed by FCM with Annexin V/PI double staining. f RV prevented SNP-induced nuclear compaction of chondrocytes stained with Hoechst 33258. Original magnification: 9400
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Fig. 3 RV protects chondrocytes from SNP by scavenging ROS. a, b FCM analysis of SNP-induced ROS (a) and NO (b) generation. c, d Prevention of SNP-induced intracellular ROS (c) and NO (d) by RV, NAC, PTIO and HG, respectively. **P \ 0.01, compared with control; ##P \ 0.01, compared with SNP alone. e Dynamic increase
of intracellular ROS and NO level after SNP treatment. f Prevention of SNP-induced cytotoxicity by RV, NAC and PTIO respectively. *P \ 0.05 and **P \ 0.01, compared with control; ##P \ 0.01, compared with SNP alone. g RV or NAC showed a modest but significant prevention on SNP-induced loss of DWm
articular chondrocyte apoptosis, and RV protected chondrocytes against SNP via scavenging ROS instead of NO. It was reported that SNP could lead to the reduction of the enzyme mitochondrial activity of complex IV [20]. Complex IV is formed by Cyt c oxidase, cytochrome oxidase and Cyt c-O2 oxidoreductase [37]. Reduction of the enzyme mitochondrial activity of complex IV may increase the concentration of reduced Cyt c and obstruct electron transfer. Cyt c can mediate electron transfer between itself and p66Shc protein to produce ROS, triggering mitochondria-dependent apoptosis [38]. Decrease of DWm could lead to depolarization of the mitochondrial membrane,
resulting in the release of ROS from mitochondria to the cytoplasm and subsequent cell death [39]. In our system, SNP induced a rapid ROS generation (Fig. 3e) and rapid loss of DWm within 0.5 h (Fig. 1h, i). In combination with the observation that SNP induced a rapid and significant decrease of cell viability within 2 h (Fig. 1b), it seems to be reasonable to surmise that SNP triggers mitochondria directly to produce ROS to mediate apoptosis. However, the exact mechanism is unclear. Interestingly, compared with pretreatment with RV or NAC alone, co-pretreatment with RV and NAC showed better protection against SNPinduced apoptosis (Fig. 3f). NAC did significantly
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Fig. 4 a Confocal images of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red. Scale Bar 10 lm. b Statistical results of the percentages of remodeling chondrocytes under different conditions (Color figure online)
mechanism by which RV protects against SNP-induced ROS-dependent apoptosis is unclear. According to our data, we summarize the signaling pathways related to SNP-induced chondrocytes apoptosis and the RV-mediated protection in Fig. 5. SNP induces ROS and NO generation separately, and ROS but not NO plays a key role in SNP-induced apoptosis in which the intrinsic and extrinsic pathways were involved. RV potently prevents SNP-induced chondrocytes apoptosis via scavenging ROS instead of NO.
Fig. 5 Schematic diagram showing apoptotic signaling pathway induced by SNP in rabbit articular chondrocytes
scavenge SNP-induced ROS and NO (Fig. 3c, d), while RV almost completely scavenged SNP-induced ROS (Fig. 3c), but did not scavenged SNP-induced NO (Fig. 3d), suggesting that NAC and RV have different scavenging mechanisms for ROS and NO. The better prevention of SNP-induced apoptosis by co-pretreatment with both NAC and RV implies that NAC and RV may have complementary mechanisms contributed to the protection against SNP-induced apoptosis in our system. However, the exact
Acknowledgments This work was supported by National Natural Science Foundation of China (81071491 and 61178078), Key Project of the Department of Education and Finance of Guangdong Province (cxzd115) and the Opening Project of MOE Key laboratory of Laser Life Science, South China Normal University. Conflict of interest
The authors declare no conflicts of interest.
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ORIGINAL PAPER
Resveratrol protects rabbit articular chondrocyte against sodium nitroprusside-induced apoptosis via scavenging ROS Qian Liang • Xiao-ping Wang • Tong-sheng Chen
Published online: 8 July 2014 Ó Springer Science+Business Media New York 2014
Abstract This study aims to investigate the mechanism by which resveratrol (RV) prevents sodium nitroprusside (SNP)-induced chondrocyte apoptosis, which is a characteristic feature of osteoarthritis (OA). Rabbit articular chondrocytes were pre-incubated with 100 lM RV for 18 h before 1.5 mM SNP co-treatment for 6 h. Cell viability was evaluated by CCK-8. Annexin V/PI double staining and Hoechst 33258 staining were used to determine the fashion of SNP-induced chondrocytes death. Mitochondrial membrane potential (DWm) was measured by using flow cytometry (FCM) with TMRM and Rhodamine 123 staining. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were confirmed by FCM analysis with DCFH-DA and DAF-FM DA staining. Cytoskeleton proteins of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red were validated by confocal microscopy. SNP induced time- and dose-dependent chondrocytes apoptosis with decline of DWm, activation of caspases as well as cytoskeletal remodeling. SNP induced a significant induction of both ROS and NO. RV remarkably prevented SNP-induced ROS production and apoptosis as well as cytoskeletal remodeling, but did not prevent SNPinduced NO production. Pretreatment with NO scavengers did not significantly prevent SNP-induced apoptosis and cytoskeletal remodeling. SNP induces NO-independent ROS production which dominates rabbit articular chondrocyte Q. Liang X. Wang (&) Department of Pain Management, The First Affiliated Hospital of Jinan University, Guangzhou 510630, China e-mail: [email protected] T. Chen MOE Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510630, China
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apoptosis, and RV protects chondrocytes against SNPinduced apoptosis via scavenging ROS instead of NO. Keywords Reactive oxygen species Resveratrol Nitric oxide Chondrocyte Sodium nitroprusside Osteoarthritis
Introduction Osteoarthritis (OA) is often a progressive and disabling disease [1, 2]. Chondrocyte apoptosis has been reported to be correlated with the severity of OA [3]. Nitric oxide (NO) and reactive oxygen species (ROS) have been suggested to be key factors to mediate chondrocyte apoptosis [4–6]. Excessive ROS may lead to irreversible damage of mitochondrial DNA, resulting in mitochondrial dysfunction and ultimately cell death [7, 8]. It was reported that NO and ROS can be produced by chondrocytes in response to stimulation such as interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a), fibroblast growth factor (FGF) and transforming growth factor-b (TGF-b) [9–12]. NO can inhibit cytochrome oxidase in mitochondria, resulting in a reduction of the electron transport chain and the formation of superoxide anions (O2-) [13]. Peroxynitrite from the reaction of O2- and NO can nitrate tyrosine residues to form 3-nitrotyrosine that is often considered as the oxidative damage of cellular proteins correlating with OA in human articular cartilage [4, 14, 15]. Sodium nitroprusside (SNP) is generally considered as the donor of NO to study the mechanism of chondrocyte apoptosis induced by NO [16–19]. Many mitochondrial events occurred during SNP-induced chondrocyte apoptosis, including reduction of the enzyme mitochondrial activity of complex IV, decline of mitochondrial membrane potential (DWm) [20] and release of
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both cytochrome c (Cyt c) and AIF [21]. In addition, it was reported that SNP-induced NO upregulated endogenous ROS to mediate chondrocyte apoptosis [19, 22]. Resveratrol (RV) has therapeutic potential for OA due to its anti-inflammatory and antioxidative properties [23–25]. RV could prevent IL-1b-induced chondrocytes apoptosis via activating SIRT1 and reversing the expression of inducible nitric oxide synthase (iNOS), and the activation of caspase-3 [23, 26, 27]. IL-1b-induced p53- and ROS-dependent apoptosis can also be significantly inhibited by RV [28]. This study is designed to explore the mechanism by which RV protects rabbit articular chondrocytes against SNP-induced apoptosis. Our findings for the first time demonstrate that SNP induces NO-independent ROS to dominate chondrocytes apoptosis, and RV protects rabbit articular chondrocytes against SNP-induced apoptosis via scavenging ROS instead of NO decomposed from SNP.
Materials and methods Reagents RV, SNP, dimethyl sulphoxide (DMSO), 20 ,70 -Dichlorofluorescin diacetate (DCFH-DA), Carboxy-PTIO (PTIO), Nacetyl cysteine (NAC), Rhodamine 123 (Rho 123) and Hoechst 33258 were from Sigma (St. Louis, USA). Dulbecco’s modified Eagle medium (DMEM) was from Gibco (Carlsbad, California, USA), fetal bovine serum (FBS) was from Sijiqing (Hangzhou, China). Cell Counting Kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan). Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was from Bender Medsystems (Vienna, Austria). 3-Amino, 4-aminomethyl-20 ,70 difluorescein, diacetate (DAF-FM DA), Hemoglobin (HG), Actin-Trakcer Green and Tubulin-Trakcer Red were from Beyotime Institute Biotechnology (Jiangsu, China). Mouse monoclonal anti-Bak (Ab-2) and anti-Bax (Ab-6) were from Calbiochem (San Diego, CA). Secondary goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) antibody, Tetramethylrhodamine methyl ester (TMRM), Trypsin and type II collagenase were from Invitrogen (California, USA). Z-IETD-fmk, z-DQMD-fmk and z-LEHD-fmk were from BioVision (San Francisco, USA). Ac-LEHD-AFC and Ac-DEVD-AFC were from Alexis (Switzerl). Ac-IETD-AFC was from BD (New Jersey, USA). RV and PTIO were dissolved in DMSO, and the final concentration of DMSO was less than 1 % in all experiments. SNP was freshly dissolved in phosphate buffered solution (PBS). NAC and HG were dissolved in ultrapure water. To scavenge NO, rabbit articular chondrocytes were pretreated with 100 lM PTIO or 20 lM HG for 2 h, and 2 mM NAC pre-incubation for 2 h was used to scavenge ROS, and then co-treated with SNP.
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Cell isolation and culture Articular chondrocytes for primary culture were harvested from slices of shoulder, knee and hip-joint cartilage from 6-week-old New Zealand white rabbits. Chondrocytes were isolated by enzymatic digestion of 0.25 % Trypsin in PBS for 1 h and 0.2 % type II collagenase in DMEM for 4–6 h. After collection by centrifugation, chondrocytes were resuspended in DMEM supplemented with 10 % FBS and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin), and then seeded in culture flasks at 37 °C in humidified 5 % CO2 as monolayer culture. The primary cells were subcultured to generation 2 that were cultured in DMEM supplemented with 10 % FBS and antibiotics for at least 24 h before different treatment. Cell viability and apoptosis assay Cell viability was detected by CCK-8 assay using auto microplate reader (Infinite M200, Tecan, Austria). All experiments were performed in quadruple occasions. Apoptosis rates were assessed by flow cytometry (FCM) analysis with Annexin V-FITC/PI kit as previously described [29], and for each FCM analysis 10,000 events were recorded. Chondrocytes stained with Hoechst 33258 for 30 min were imaged by a fluorescent microscope (Axiovert 200 M, Zeiss). Measurement of intracellular ROS and NO Intracellular ROS or NO was quantified by FCM using DCFH-DA staining or DAF-FM DA staining which is cellpermeable [30]. Chondrocytes were stained with 20 lM DCFH-DA for 30 min or with 5 lM DAF-FM DA for 20 min at 37 °C in the dark, and re-washed with PBS three times, subsequently 500 ll of cell suspension was added in 5 ml FCM tube and analyzed quantitatively using FCM. Measurement of mitochondrial membrane potential Chondrocytes stained with 10 lM TMRM or Rho 123 for 30 min at 37 °C in the dark were harvested, and were then washed with PBS solution twice and subsequently assayed by FCM. Results were expressed as the proportion of cells with low TMRM or Rho 123 fluorescence indicating the loss of DWm. Fluorometric determination of caspase enzymatic activation Activities of caspase-8, -9 and -3 were measured using fluorogenic substrate Ac-IETD-AFC Ac-LEHD-AFC and AcDEVD-AFC, respectively, according to the manufacturer’s
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instructions as described previously [31]. The caspase activation level in control cells was normalized to 1.0. FCM analysis of the activation of Bak and Bax Chondrocytes were fixed with 3.7 % formaldehyde in PBS for 10 min at room temperature, and treated with ice-cold 100 % methanol for 15 min to permeabilize the cells. The fixed cells were blocked in PBS solution containing 1 % bovine serum for 30 min at room temperature and then were incubated with either anti-Bax (6A7) or anti-Bak (Ab-2) (1:100) at room temperature for 60 min and then incubated with FITC-conjugated goat anti mouse IgG (1:200) for 30 min in the dark. After washing, the samples were analyzed by FCM.
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Fluorescent substrate assay showed that SNP induced a significant activation of caspase-3 and a modest but significant activation of caspase-8 and -9 (Fig. 1d). Moreover, inhibition of any one of casapse-8, -9 and -3 significantly prevented SNP-induced apoptosis (Fig. 1e). In addition, FCM assay showed that compared with control cells, the mean fluorescence intensity of SNP-treated cells increased to 130.54 % for active Bak and 126.79 % for active Bax, respectively (Fig. 1f, g), indicating that SNP induced the activation of Bak and Bax. FCM analysis with TMRM or Rho 123 staining showed that SNP treatment for 1 h induced a modest but significant decrease of DWm (Fig. 1h, i). Collectively, the caspases-dependent intrinsic and extrinsic pathways were involved in SNP-induced rabbit articular chondrocyte apoptosis.
Confocal microscopic imaging of cytoskeletons Cells were fixed with 3.7 % formaldehyde in PBS for 10 min at room temperature, then permeabilized and blocked in PBS containing 0.1 % Triton X-100 and 1 % fetal calf serum for 20 min. The fixed cells were washed with PBS containing 0.1 % Triton X-100 three times and then incubated for 1 h with 2 % Actin-Trakcer Green and 0.4 % Tubulin-Trakcer Red in PBS containing 0.1 % Triton X-100 and 1 % fetal calf serum at room temperature, re-washed, and then observed using a confocal microscope (LSM510Meta, Zeiss, Jena, Germany). The excitation wavelengths for Actin-Trakcer Green and Tubulin-Trakcer Red are 488 and 543 nm, and the corresponding emission channels are BP 505–530 nm and LP 560 nm. Statistical analysis Data were presented as mean ± SD from at least three experiments. Data were analyzed by student’s t test using SPSS 13.0. P values less than 0.05 were considered to be statistically significant.
Results SNP induces apoptosis in rabbit articular chondrocytes CCK-8 assays showed that SNP induced dose- (Fig. 1a) and time- (Fig. 1b) dependent cytotoxicity. Treatment with 1.5 mM SNP for 6 h was adopted in following experiments without indication. FCM analysis with Annexin V-FITC/PI staining showed that SNP treatment for 6 h induced a remarkable chondrocytes apoptosis (Q2 (PI positive and Annexin V-FITC positive) ? Q4 (PI negative and Annexin V-FITC positive)) from 13.2 % (control) to 65.6 % (Fig. 1c), demonstrating that SNP induced chondrocyte death in apoptotic fashion.
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Prevention of SNP-induced apoptosis in rabbit articular chondrocytes by RV CCK-8 assay showed that treatment with 100 lM RV for 24 h did not induce significant cytotoxicity (Fig. 2a, b), and that pretreatment with 100 lM RV for 12, 18, and 24 h respectively presented a potent prevention on the cytotoxicity of SNP (Fig. 2c). We also assessed the prevention of different dosage of RV (50, 75 and 100 lM) on SNPinduced apoptosis (data not shown), and found that 100 lM of RV showed the best protection effects. Pretreatment with 100 lM RV for 18 h was used in all following experiments without indication. Microscopic images showed that RV significantly prevented SNPinduced chondrocytes death (Fig. 2d). FCM analysis with Annexin V-FITC/PI staining also showed that RV potently prevented SNP-induced apoptosis (Fig. 2e), which was further verified by the microscopic images of chondrocytes stained with Hoechst 33258 (Fig. 2f). RV prevents SNP-induced apoptosis via scavenging ROS instead of NO We first used FCM assay to assess the SNP-induced ROS and NO production by using DCFH-DA and DAF FM-DA, respectively, and found that SNP treatment for 6 h induced a significant generation of both ROS (Fig. 3a) and NO (Fig. 3b). Pretreatment with RV markedly prevented SNPinduced ROS production, but did not prevented SNPinduced NO production (Fig. 3a, b). We also used FCM to assess the effects of NAC, a scavenger of ROS, and PTIO or HG, the scavengers of NO, on the SNP-induced ROS and NO production. As shown in Fig. 3c, d, NAC pretreatment largely prevented the SNP-induced ROS and NO, RV pretreatment markedly prevented SNP-induced ROS but modestly enhanced SNP-induced NO production, and pretreatment with PTIO or HG significantly prevented
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1358 b Fig. 1 SNP induces apoptosis. a SNP induced dose-dependent
cytotoxicity. **P \ 0.01, compared with control. b SNP induced time-dependent cytotoxicity. **P \ 0.01, compared with control. c SNP induced time-dependent apoptosis by FCM analysis with Annexin V-FITC/PI staining. d SNP induced activation of caspase-3, -8 and -9. *P \ 0.05 and **P \ 0.01, compared with control. e Inhibition of caspase-3, -8, and -9 by casapse inhibitors significantly prevented SNP-induced cytotoxicity. After pretreatment with each caspase inhibitor (10 lM) for 2 h or 100 lM RV for 18 h or RV combined with each caspase inhibitor, the cells were then co-treated with SNP or not. *P \ 0.05 and **P \ 0.01, compared with control; ## P \ 0.01, compared with SNP alone; $$P \ 0.01, compared with the combined treatment of RV and SNP. f, g SNP induced activation of both Bax (g) and Bak (f) by FCM analysis. Compared with Ctrl, the mean flourescence intensity of SNP-treated cells increased to 126.79 % for active Bax and 130.54 % for active Bak respectively. h, i SNP induced a rapid loss of DWm by FCM analysis with TMRM (h) and Rho 123 (i) staining
SNP-induced NO production but did not prevented SNPinduced ROS production. These data demonstrated that SNP induced ROS production independent of NO and RV pretreatment remarkably prevented SNP-induced ROS instead of NO. We next assessed the dynamics of ROS and NO generation after SNP treatment, and found that SNP treatment induced a rapid ROS generation that reached to saturation within 0.5 h and a slow NO generation that had significant increase at 2 h after SNP treatment (Fig. 3e), further demonstrating the notion that SNP induced ROS production independent of NO decomposed from SNP. CCK-8 assay showed that pretreatment with RV and/or NAC remarkably prevented SNP-induced apoptosis, but pretreatment with PTIO did not show any prevention on SNP-induced apoptosis (Fig. 3f). In addition, FCM analysis about DWm showed that pretreatment with RV or NAC induced a modest but significant prevention on SNPinduced loss of DWm (Fig. 3g). Collectively, SNP induced NO-independent ROS production that played a key role in SNP-induced chondrocytes apoptosis, and RV protected rabbit articular chondrocytes from SNP-induced apoptosis via scavenging ROS instead of NO. Prevention of SNP-induced cytoskeletal remodeling by RV Confocal microscopy was used to image the cytoskeleton of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red (Fig. 4). In untreated chondrocytes, the long-form and regular F-actin filament as well as uniformly microtubule cytoskeleton were observed. SNP not only shortened F-actin filaments but also induced disruption of microtubule structure and cell shrinkage, which could be significantly prevented by pretreatment with RV and/or NAC but not PTIO (Fig. 4a), and the statistical results on the percentages of remodeling chondrocytes
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under different conditions from three independent experiments were shown in Fig. 4b.
Discussion These results of this report support the notion that SNP induces ROS-dependent and NO-independent apoptosis in rabbit articular chondrocytes. Furthermore, this report for the first time demonstrates that RV remarkably prevents SNP-induced apoptosis by scavenging the SNP-triggered ROS independent of NO decomposed from SNP. In contrast to the acknowledged notion that SNP-induced NO mediates ROS generation to regulate cell apoptosis, our observations demonstrate that SNP induces ROS generation independent of NO to mainly mediate SNP-induced chondrocytes apoptosis. SNP is generally used as NO donor to study the role of NO in chondrocytes apoptosis [16, 18, 21, 32–34]. From the inhibitory effect of NAC on SNP-induced apoptosis in rabbit chondrocytes, Nakagawa and co-workers speculated that ROS acted as the downstream factor of NO to mediate SNP-induced apoptosis [22]. This inference is based on the hypothesis that NO is the key upstream mediator to mediate SNP-induced chondrocyte apoptosis [16–18]. Similarly, from the increase of intracellular ROS after exposure of human articular chondrocyte to SNP, Wu and co-workers inferred that intracellular ROS was produced by NO decomposed from SNP [19]. These studies did not assess the rigorous relationship between NO and ROS after SNP treatment, we herein focused on the roles of ROS and NO in SNP-induced chondrocytes apoptosis. In our system, the results that SNP induces a very rapid ROS generation within 0.5 h and slow NO generation that has a significant increase at 2 h after SNP treatment (Fig. 3e) and pretreatment with PTIO or HG significantly prevent SNP-induced NO production but does not prevent SNP-induced ROS production (Fig. 3c, d) demonstrate that SNP induces ROS generation independent of NO. It is generally considered that NO decomposed from SNP dominates SNP-induced chondrocyte apoptosis via an intrinsic apoptosis pathway [19, 35, 36]. Our data support this notion that the intrinsic apoptotic pathway participated in SNP-induced rabbit articular chondrocyte apoptosis (Fig. 1). However, HG or PTIO potently decreased SNPinduced NO production (Fig. 3d), but did not prevent SNPinduced cytotoxicity (Figs. 3f, 4). In strong contrast, RV remarkably prevented SNP-induced cytotoxicity (Fig. 3f) as well as almost completely prevented SNP-induced ROS production (Fig. 3c), but not NO (Fig. 3d). In addition, SNP treatment for 2 h only induced a very slight increase in intracellular NO level (Fig. 3e), but led to 40 % decrease of cell viability (Fig. 1b). Therefore, we concluded that ROS instead of NO dominated SNP-induced rabbit
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Fig. 2 Prevention of SNP-induced apoptosis by RV. a, b Cytotoxicity of RV on chondrocytes. **P \ 0.01, compared with control. c RV markedly prevented SNP-induced cytotoxicity. **P \ 0.01, compared with control; #P \ 0.05 and ##P \ 0.01, compared with SNP alone. d Microscopic images of chondrocytes after different
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treatments. Original magnification: 9200. e RV prevented SNPinduced apoptosis analyzed by FCM with Annexin V/PI double staining. f RV prevented SNP-induced nuclear compaction of chondrocytes stained with Hoechst 33258. Original magnification: 9400
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Fig. 3 RV protects chondrocytes from SNP by scavenging ROS. a, b FCM analysis of SNP-induced ROS (a) and NO (b) generation. c, d Prevention of SNP-induced intracellular ROS (c) and NO (d) by RV, NAC, PTIO and HG, respectively. **P \ 0.01, compared with control; ##P \ 0.01, compared with SNP alone. e Dynamic increase
of intracellular ROS and NO level after SNP treatment. f Prevention of SNP-induced cytotoxicity by RV, NAC and PTIO respectively. *P \ 0.05 and **P \ 0.01, compared with control; ##P \ 0.01, compared with SNP alone. g RV or NAC showed a modest but significant prevention on SNP-induced loss of DWm
articular chondrocyte apoptosis, and RV protected chondrocytes against SNP via scavenging ROS instead of NO. It was reported that SNP could lead to the reduction of the enzyme mitochondrial activity of complex IV [20]. Complex IV is formed by Cyt c oxidase, cytochrome oxidase and Cyt c-O2 oxidoreductase [37]. Reduction of the enzyme mitochondrial activity of complex IV may increase the concentration of reduced Cyt c and obstruct electron transfer. Cyt c can mediate electron transfer between itself and p66Shc protein to produce ROS, triggering mitochondria-dependent apoptosis [38]. Decrease of DWm could lead to depolarization of the mitochondrial membrane,
resulting in the release of ROS from mitochondria to the cytoplasm and subsequent cell death [39]. In our system, SNP induced a rapid ROS generation (Fig. 3e) and rapid loss of DWm within 0.5 h (Fig. 1h, i). In combination with the observation that SNP induced a rapid and significant decrease of cell viability within 2 h (Fig. 1b), it seems to be reasonable to surmise that SNP triggers mitochondria directly to produce ROS to mediate apoptosis. However, the exact mechanism is unclear. Interestingly, compared with pretreatment with RV or NAC alone, co-pretreatment with RV and NAC showed better protection against SNPinduced apoptosis (Fig. 3f). NAC did significantly
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Fig. 4 a Confocal images of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red. Scale Bar 10 lm. b Statistical results of the percentages of remodeling chondrocytes under different conditions (Color figure online)
mechanism by which RV protects against SNP-induced ROS-dependent apoptosis is unclear. According to our data, we summarize the signaling pathways related to SNP-induced chondrocytes apoptosis and the RV-mediated protection in Fig. 5. SNP induces ROS and NO generation separately, and ROS but not NO plays a key role in SNP-induced apoptosis in which the intrinsic and extrinsic pathways were involved. RV potently prevents SNP-induced chondrocytes apoptosis via scavenging ROS instead of NO.
Fig. 5 Schematic diagram showing apoptotic signaling pathway induced by SNP in rabbit articular chondrocytes
scavenge SNP-induced ROS and NO (Fig. 3c, d), while RV almost completely scavenged SNP-induced ROS (Fig. 3c), but did not scavenged SNP-induced NO (Fig. 3d), suggesting that NAC and RV have different scavenging mechanisms for ROS and NO. The better prevention of SNP-induced apoptosis by co-pretreatment with both NAC and RV implies that NAC and RV may have complementary mechanisms contributed to the protection against SNP-induced apoptosis in our system. However, the exact
Acknowledgments This work was supported by National Natural Science Foundation of China (81071491 and 61178078), Key Project of the Department of Education and Finance of Guangdong Province (cxzd115) and the Opening Project of MOE Key laboratory of Laser Life Science, South China Normal University. Conflict of interest
The authors declare no conflicts of interest.
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