J Huazhong Univ Sci Technol［Med Sci］ 29 (3): 300-303, 2009 DOI 10.1007/s11596-009-0307-x J Huazhong Univ Sci Technol［Med Sci］ 29 (3): 2009

300

The Effects of PDTC on Interleukin-1β-induced Nitric Oxide Production in Chondrocytes Jianxiang LIU (刘建湘)1, Jingyuan DU (杜靖远)1, Shuhua YANG (杨述华)1, Xiaohua QIU (邱晓华)1, Riguang LIU (刘日光)2, Chengqing YI (易诚青)3, Xinchun LI (李新春)4 1 Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2 Department of Orthopedics, the Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China 3 Department of Orthopedics, Shanghai First Hospital, Shanghai 201620, China 4 Department of Orthopedics, Ningbo First Hospital, Ningbo 315010, China

Summary：In order to find new drugs to inhibit nitric oxide (NO) production, the effects of pyrrolidine dithiocarbamate (PDTC), a nuclear factor-kappa B (NF-κB) inhibitor, on recombinant human interleukin-1β (rhIL-1β)-induced NO production in chondrocytes were investigated. Rat chondrocytes were isolated and cultured, divided into control, P0, P1, P2, P3 and P4 groups. The chondrocytes in the P0, P1, P2, P3 and P4 groups were treated with different concentrations of PDTC (0, 3, 10, 30, and 50 μmol/L respectively) for 1 h and then incubated with 5 U/mL rhIL-1β for 24 h. NO assay kit and RT-PCR were used to detect the NO content and the iNOS mRNA expression in the chondrocytes. The expression level of iNOS mRNA in control, P0, P1, P2, P3 and P4 groups was 0.02±0.01, 1.24±0.13, 1.21±0.14, 0.61±0.11, 0.40±0.09, 0.21±0.06, and the relative content of NO was 15.8±2.7, 100±14.8, 92.6±9.3, 68.3±14.2, 27.5±9.8, 19.8±3.6, respectively. In the P0, P1, P2, P3 and P4 groups, the expression of iNOS mRNA and NO production were significantly increased as compared with those in the control group. As compared with the P0 group, the expression of iNOS mRNA and NO content in control group were lower. In the P2, P3 and P4 groups, PDTC could significantly inhibit the expression of iNOS and NO production induced by rhIL-1β in a concentration-dependent manner. It is suggested that PDTC can inhibit NO production and iNOS mRNA expression induced by IL-1β, which may provide an alternative method for the treatment of osteoarthritis. Key words：pyrrolidine dithiocarbamate; chondrocyte; nitric oxide; nuclear factor-kappa B

The destruction of articular cartilage is the most important and basic pathological change of osteoarthritis. Nitric oxide (NO) plays an important role in the development of osteoarthritis. As an important inflammatory transmitter, NO mainly causes degradation of cartilage matrix, inhibits synthesis of proteoglycan and collagen, and promotes the apoptosis of chondrocytes[1-3]. Inhibiting NO production can slow down the cartilaginous degradation, therefore treating osteoarthritis. It has been indicated in an animal experiment that NO inhibitors can cure the osteoarthritis[4]. When affected by exterior stimuli, chondrocytes can induce the iNOS expression, and produce NO thereby. The promoter site of iNOS contains the binding site of nuclear factor-kappa B (NF-κB), which is necessary for the transcription of iNOS[5]. NF-κB is widely present in the body, and is a kind of gene modulatory protein that may be increased by inducement. It can combine with the recognition sequence of the modulated gene and control the transcription[6]. PDTC is a kind of antioxidant which is mainly used for the treatment of metallic toxicosis[7]. It has been reported that PDTC can inhibit the activation of NF-κB, and therefore control the expression of detrimental genes [8]. This study investigated the effects of PDTC on Jianxiang LIU, E-mail: [email protected]

IL-1β-induced NO production in order to find new drugs to inhibit NO production. 1 MATERIALS AND METHODS 1.1 Materials DMEM, fetal calf serum, type Ⅱ collagenase, recombinant human IL-1β (rh IL-1β), and NO kit were purchased from Yafa Biological Co. (China). PDTC was gifted by Prof. Yu Kangmin (Sigma, USA). iNOS primers were synthesized by Baoshengwu Biological Co. (China) and the sequences were as follows: upper stream, 5'-CTGGGAGAAAACCCCAGGT GCT-3' (101–122 bp), and lower stream, 5'-ATGGCCG ACCTGATGTTGCCAC-3' (721–742 bp), 642 bp in length; The sequences of GAPDH primers were as follows: upper stream, 5'-ACGGATTTGGCCTTATTGGC CGC-3' (49–72 bp), and lower stream, 5'-TGGTCCTCA GTGTAGCCCAGGAT-3' (840–863 bp), 815 bp in length[9]. 1.2 Culture of Rat Chondrocytes After male Wistar rats (weighing 180–200 g) were killed, the cartilages from femoral head, patella, condyle of femur and tibia plateau were taken under sterile condition, cut into pieces, washed with PBS twice, digested with 0.25% pancreatin, and washed with PBS twice. Type II collagenase solution (0.2%) was added at a ratio

301

J Huazhong Univ Sci Technol［Med Sci］ 29 (3): 2009

of 1 to 10 and the tube was gently shaken in the water of 37°C for 4 h until the cartilaginous pieces disappeared and the solution became turbid. The solution was centrifuged at 1500 r/min for 10 min, and the supernatant was discarded. The cells were washed with PBS, and added with DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, 10–8 mol/L dexamethasone, 50 μg/mL vitamin C. The suspension from the cellular agglomerate was made and filtered with a 200-mesh sieve. The filtrate was collected, and the cells were inoculated at a density of 6×106/mL and cultured in an incubator with 5% CO2 at 37°C.. After culture for 3 days, the medium was changed, and afterwards the medium was displaced every 2 days. Passage was performed when the culture bottles were full of the cells. The cells at the first generation were used for the experiment. 1.3 Grouping and Treatment The cells were inoculated in a 24-well culture plate at a density of 2×105/mL, and divided into 6 groups with 4 wells in each group: control (C), P0, P1, P2, P3 and P4 group. Prior to the addition of rhIL-1β, the cells in the P1, P2, P3 and P4 groups were treated with different concentrations of PDTC (3, 10, 30 and 50 μmol/L respectively) for 1 h, and then the chondrocytes in P0, P1, P2, P3 and P4 groups were incubated with 5U/mL rhIL-1β for 24 h. 1.4 Detection of NO After the condrocytes were incubated with rhIL-1β for 24 h, the supernatant in each group was harvested for detection of NO according to the manufacturer’s instructions. The experiment was repeated at least 3 times. NO was defined as relative content. The NO production in P0 group was taken as 100, and the NO content in the other groups were expressed by the ratio to the P0 group. 1.5 Detection of the iNOS mRNA Expression by RT-PCR Twenty-four h after the chondrocytes were treated with rhIL-1β, the cells were collected, and the total RNA was extracted and amplified for 23–28 cycles by RT-PCR. The amplified product was subjected to electrophoresis on a 1.5% agarose gel. The band analysis was performed on a Qianpin Imaging Analysis System (Wuhan Qianpin Imaging Limited Liability Company, China). The expression level of iNOS mRNA was defined as the value of iNOS/GAPDH. 1.6 Statistical Analysis All the data was expressed as ±s. The comparison among the groups was conducted by using t test. A P<0.01 was considered to be statistically significant. 2 RESULTS 2.1 The Morphological Changes of Chondrocytes The chondrocytes cultured were globular when seeded under an invert microscope, and 10 h later, attached growth began. The confluent primary chondrocytes presented round or oval shapes, and excrete a little amount of extracellular matrix. The culture bottles were full of cells after 1-week culture. After passage, the cells became polygonal, the extracellular matrix was increased, and the cells had the property of refraction. 2.2 NO Contents in Different Groups The chondrocytes in the control group excreted a

little amount of NO. There was no significant difference in NO production between groups P1 and P2, indicating 3 μmol/L PDTC couldn’t significantly inhibit the NO production in the chondrocytes treated with IL-1β. But significant differences were noted in NO content between P2, P3, P4 and P0 groups, suggesting when the concentration of PDTC reached or surpassed 10μmol/L, PDTC could significantly inhibit NO production and the inhibitory effect was in a concentration-dependent manner (table 1). 2.3 iNOS mRNA Expression by RT-PCR Table 1 and fig.1 showed that the expression of iNOS mRNA in the control group was weak. The iNOS mRNA in the condrocytes of P0 group was highly expressed. The expression levels of iNOS mRNA in the P2, P3, and P4 groups were significantly lower than in P0 groups, suggesting PDTC at a concentration of 10 μmol/L or above could significantly inhibit the rhIL1β-induced iNOS mRNA expression in the condrocytes. Table 1 The effect of PDTC on NO production and iNOS mRNA expression NO production iNOS mRNA expression (% of P0) (iNOS/GAPDH) Control 15.8±2.7* 0.02±0.01* P0 100±14.8 1.24±0.13 P1 92.6±9.3 1.21±0.14 P2 68.3±14.2 0.61±0.11* * P3 27.5±9.8 0.40±0.09* * P4 19.8±3.6 0.21±0.06* * P<0.01 as compared with P0 group.

Groups

Fig. 1 RT-PCR for iNOS mRNA 1, 2, 3, 4, 5, 6 : Control, P0, P1, P2, P3, P4 groups respectively; M: Marker

3 DISCUSSION Osteoarthritis is a common disease that severely affects the quality of life of the senile. The incidence of osteoarthritis is increasing with the aging society. At present, there are a variety of treatment options for the disease, such as physical therapy, drugs therapy, surgical therapy, gene technique and so on. However, the ideal strategy that can prevent the destruction and degeneration of the cartilages in joints is not yet available. Osteoarthritis is characterized by the degradation and breakage of the cartilage matrix. Therefore, how to restrain the degradation and breakage of the cartilage matrix becomes the key of the treatment of osteoarthritis. Previous Studies have already shown that in osteoarthritis, chondrocytes could synthesize many inflammatory mediaters such as IL-1, TNF-α, NO, MMPs and so on, which would destroy the cartilage matrix[10-13].

302 NO, an important inflammatory mediater, plays a critical role in osteoarthritis. NO is generated by NO synthesizing enzyme, which has three types: construction NOS (cNOS), neuron NOS (nNOS) and inducing NOS (iNOS). Chondrocytes of humans and animals predominantly express iNOS. NO could inhibit the growth of chondrocytes, facilitate the apoptosis and restrain the synthesis of extracellular matrix[1-3]. There is a binding site for NF-κB at the promoter site of iNOS gene[5]. The translocation of NF-κB from cytoplasm to nucleus is essential for the IL-1- or LPS-induced transcription of iNOS[14]. NF-κB is a sort of protein belonging to NF-κB/Rel family. The NF-κB/Rel family consists of five members: Rel A (P65), Rel B, C-Rel, NF-κB1 (P50), and NF-κB2 (P52). They share the same N-terminal known as Rel domain (RHD) including nearly 300 amino acids. The RHD contains the DNA-binding and dimerization domains and the nuclear localization signal of the Rel proteins. Only P65 has transcription-activated regions and can act directly on the transcription-binding site and invoke the transpcription. NF-κB combined with activated DNA is a dimer. Usually, the dimer of P65/P50 is referred to as NF-κB. I-kappa B (IκB) is the inhibitor of NF-κB[15-17]. NF-κB in the resting form is combined with IκB. IκB combined with NF-κB could prevent the translocation of NF-κB from cytoplasm to nucleus. And NF-κB could not bind to the specific amino acid sequence in the promoter region of the aimed gene, and the transcription of the gene is inhibited. Extracellular stimulation may activate protein kinases by different signaling pathways, therefore phosphorylating IκB. NF-κB is separated from IκB, which is followed by the degradation of IκB. With the loss of IκB, NF-κB in the cytoplasm becomes activated, transfers into nucleus, combines with NF-κB binding sites in the promoter region of modulating genes on DNA, and consequently promotes gene transcription. Diethyldithiocarbamate (DDTC), an antioxidant, is used for the treatment of metal toxicosis by metal chelation. Recently, this substance was found to inhibit the activation of NF-κB in vitro [7]. Pyrrolidine dithiocarbamate (PDTC) is a derivative of DDTC and can maintain stable for a long time under physical condition. It has a great potential for clinical application for its anti-oxidation effect and inhibition of the activation of NF-κB[8]. Our study demonstrated that PDTC could inhibit NO production through NF-κB signaling pathway in chondrocytes. NO is produced by iNOS, and iNOS gene has NF-κB binding sites. iNOS expression in chondrocytes can be induced by a single stimuli such as IL-1 or tumor necrosis factor-alspha (TNF-α)[18, 19]. Moreover, NO also promotes IL-1 generation, which plays an important role in cartilage destruction. In theory, inhibiting NO production can treat osteoarthritis. And It was also observed in animal osteoarthritis model that articular cartilage score was improved and cartilage destructive factors were reduced by inhibiting NO generation[4, 20, 21]. The expression of iNOS mRNA begins when NF-κB is activated, which is followed by the generation of NO. It is acknowledged that NF-κB is induced by some up-regulated pro-inflammatory factors such as IL-1β and TNF-α, indicating NF-κB plays a key role in NO production in chondrocytes when stimulated by pro-inflammatory factors. Therefore, inhibiting NF-κB

J Huazhong Univ Sci Technol［Med Sci］ 29 (3): 2009

will block the key network in cartilage destruction. In this study, we used IL-1β to stimulate chondrocytes as IL-1β is a very important cartilage destructive factor for osteoarthritis. The pathological changes by administration of chondrocytes with IL-1β mimick the inflammation-mediated cartilage degeneration that was found in osteoarthritis. We found that the chondrocytes in the control group produced little amount of NO. NO content was significantly increased in the chondrocytes induced by IL-1β. And PDTC could inhibit the production of NO when the concentration reached 10 μmol/L (P2 group). The production of NO was decreased with PDTC concentration increasing (P2, P3, and P4 groups). In addition, we detected iNOS mRNA expression by RT-PCR. The expression of iNOS mRNA in the chondrocytes was low in the absence of IL-1β treatment. And it was significantly enhanced in the chondrocytes treated with IL-1β. The iNOS mRNA expression was decreased by the use of PDTC (10 μmol/L or above), and the decrease was in a concentration-dependant manner. Our findings were consist with those by Cuzzorea[22]. In our study, it was found that NO production and iNOS mRNA expression dropped rapidly when PDTC concentration was increased from 3 to 30 μmol/L, while the trend slowed down when PDTC concentration was increased from 30 to 50 μmol/L. The phenomenon may be ascribed to that most NF-κB activation would be suppressed when PDTC reached a certain concentration, e.g., 30 μmol/L in this study. This study proved that PDTC could inhibit the production of NO in chondrocytes, which may provide an alternative way for the treatment of osteoarthritis. However, the experiment was conducted in vitro, and the application of PDTC in vivo to treat osteoarthritis awaits further investigation. REFERENCES 1 Castro RR, Cunha FQ, Silva FS Jr, et al. A quantitative approach to measure joint pain in experimental osteoarthritis--evidence of a role for nitric oxide. Osteoarthritis Cartilage, 2006,14(8):769-776 2 Min BH, Kim HJ, Lim H, et al. Effects of ageing and arthritic disease on nitric oxide production by human articular chondrocytes. Exp Mol Med, 2001,33(4):299-302 3 Notoya K, Jovanovic DV, Reboul P, et al. The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2. J Immunol, 2000,165(6): 3402-3410 4 Pelletier JP, Jovanovic D, Fernandes JC, et al. Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum, 1998,41(7):1275-1286 5 Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem, 1994,269(7):4705-4708 6 Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol, 1997,17(1):3-9 7 Schreck R, Meier B, Mannel DN, et al. Dithiocarbamates as potent inhibitors of nuclear factor kapaB activation in intact cells. J Exp Med, 1992,175(5):1181-1194 8 Nurmi A, Vartiainen N, Pihlaja R, et al. Pyrrolidine

303

J Huazhong Univ Sci Technol［Med Sci］ 29 (3): 2009

9

10

11

12

13

14

15

dithiocarbamate inhibits translocation of nuclear factor kappa-B in neurons and protects against brain ischaemia with a wide therapeutic time window. J Neurochem, 2004,91(3):755-765 Nedelec E, Abid A, Cipolletta C, et al. Stimulation of cyclooxygenase-2-activity by nitric oxide-derived species in rat chondrocyte: lack of contribution to loss of cartilage anabolism. Biochem Pharmacol, 2001,61(8): 965-978 Shikhman AR, Kuhn K, Alaaeddine N, et al. N-Acetylglucosamine prevents IL-1beta-mediated activation of human chondrocytes. J Immunol, 2001,166(8): 5155-5160 Badger AM, Cook MN, Swift BA, et al. Inhibition of interleukin-1-induced proteoglycan degradation and nitric oxide production in bovine articular cartilage/chondrocyte cultures by the natural product, hymenialdisine. J Pharmacol Exp Ther, 1999,290(2):587-593 Saklatvala J. Tumor necrosis factor alpha stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature, 1986,322(6079):547-549 Goldring MB. The role of cytokine as inflammatory mediators in osteoarthritis: lessons from animal modes. Connect Tissue Res, 1999,40(1):1-11 Di Marco S, Mazroui R, Dallaire P, et al. NF-kappa B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release. Mol Cell Biol, 2005,25(15): 6533-6545 Yamamoto Y, Gaynor RB. IkB kinases: key regulators of

16

17 18

19

20

21

22

the NF-kappaB pathway. Trends Biochem Sci, 2004,29(2):72-79 Bouwmeester T, Bauch A, Ruffner H, et al. A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat Cell Biol, 2004,6(2): 97-105 Tak PP, Firestein GS. NF-kB: a key role in inflammatory disease. J Clin Invest, 2001,107(1):7-11 Clancy RM, Amin AR, Abramson SB. The role of nitric oxide in inflammation and immunity. Arthritis Rheum, 1998,41(7):1141-1151 Studer R, Jaffurs D, Stefanovic-Racic M, et al. Nitric oxide in osteoarthritis. Osteoarthritis Cartilage, 1999,7(4): 377-379 Pelletier JP, Lascau-Coman V, Jovanovic D, et al. Selective inhibition of inducible nitric oxide synthase in experimental osteoarthritis is associated with reduction in tissue levels of catabolic factors. J Rheumatol, 1999,26(9): 2002-2014 Pelltier JP, Jovanovic DV, Lascau-Coman V, et al. Selective inhibitor of inducible nitric oxide synthase reduces progression of experimental osteoarthritis in vivo: possible link with the reduction in chondrocyte apoptosis and caspase 3 level. Arthritis Rheum, 2000,43(6):1290-1299 Cuzzorea S, Chatterjee PK, Mazzon E, et al. Pyrrolidine dithiocarbamate attenuates the developmentof acute and chronic inflammation. Br J Pharmacol, 2002,135(2): 496-510 (Received July 11, 2008)