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W F1 mice
European Journal of Pharmacology 607 (2009) 102–106
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Cystamine attenuates the expressions of NOS- and TLR-associated molecules in the brain of NZB/W F1 mice Hon-Pin Wang a,1, Tsai-Ching Hsu b,1, Gwo-Jong Hsu c, Sin-Lun Li d, Bor-Show Tzang d,⁎ a
Division of Allergy, Rheumatology and Immunology, Department of Internal Medicine, Chia-Yi Christian Hospital, Chia-Yi, Taiwan Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan Division of Infectious disease and Department of Internal Medicine, Chia-Yi Christian Hospital, Chia-Yi, Taiwan d Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan b c
a r t i c l e
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Article history: Received 7 January 2009 Received in revised form 12 February 2009 Accepted 12 February 2009 Available online 4 March 2009 Keywords: SLE (systemic lupus erythematosus) NOS (Nitric Oxide Synthase) Neuropsychiatric TLR (toll-like receptor)
a b s t r a c t Evidence have indicated the impairment of central nervous system (CNS) and neuropsychiatric disorder in patients with systemic lupus erythematosus (SLE). However, little is known to improve the brain abnormality in SLE. To investigate the effect of cystamine on brain abnormality in SLE, NZB/W F1 mice were used as the animal model. Notably, significantly reduced neural Nitric Oxide Synthase (nNOS), inducible Nitric Oxide Synthase (iNOS), p53, p21WAF1/CIP1, and heat shock protein (HSP)-90 proteins were detected in the brain of NZB/W F1 mice that were treated with cystamine. In contrast, no variation was observed between the brain samples of BALB/c mice that were treated with PBS or cystamine. Moreover, significantly reduced Toll-like receptors- (TLR-) 4, 5 and 7 were detected in the brain samples of NZB/W F1 mice that were treated with cystamine whereas no variation of TLR-4, TLR-5, TLR-7, and TLR-9 was observed in BALB/c mice that were treated with PBS or cystamine. These findings demonstrated the beneficial effects of cystamine on brain abnormality in NZB/W F1 mice and probably suggested the potential of cystamine on treating patients with neuropsychiatric SLE. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Many clinical manifestations of systemic lupus erythematosus (SLE), including CNS involvement, lead to neuropsychiatric disease and mortality (Kaell et al., 1986; Jonsson et al., 1989). Indeed, various studies have indicated the increased involvement of central nervous system (CNS) or neuropsychosis in SLE patients (Feinglass et al., 1976; McCune and Golbus, 1988; Futrell et al., 1992; Sibley et al., 1992; Hanly and Liang, 1997), which is recognized as a major cause of longstanding functional impairment and mortality (Jonsson et al., 1989). However, little is known about the mechanism and treatment for brain abnormality in SLE. A number of studies have associated the nitric oxide (NO)associated tissue injury with different autoimmune diseases, including SLE (Belmont et al., 1997; Abramson et al., 2001). NO is produced by a group of enzymes denominated Nitric Oxide Synthases (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS), inducible NOS (iNOS) and mitochondrial NOS (mtNOS), which play important roles in both physiological and pathological processes (Elfering et al., 2002;
⁎ Corresponding author. Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung Taiwan, NO. 110, Jianguo N. Rd. Taichung 402, Taiwan. Tel.: +886 4 23248168; fax: +886 4 23248195. E-mail address: [email protected] (B.-S. Tzang). 1 H.P. Wang and T.C. Hsu contributed equally to this paper. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.02.039
Guix et al., 2005). In an iNOS-knockout MRL-lpr/lpr animal model, iNOS (−/−) and iNOS(+/−) mice had significantly less vasculitis and lower IgG rheumatoid factor levels as compared to iNOS(+/+) mice (Gilkeson et al., 1997). Additionally, increased NOS is related to the disease activity in SLE patients (Belmont et al., 1997; Wigand et al., 1997). Toll-like receptors (TLRs) play crucial roles in innate immunity and pathogenesis of autoimmune diseases. Sterile inflammation or autoimmunity can be induced by inappropriate activation of TLRs (Abdollahi-Roodsaz et al., 2007; Barrat and Coffman, 2008). Evidence have indicated the genetic variation in TLR-5 and TLR-9 that relates to the disease activity of SLE (Hawn et al., 2005; De Jager et al., 2006). Recently, various studies have indicated that HSP-90 could bind CpG oligonucleotides directly and act as a ligand transfer molecule in TLR-9 signaling cascade (Bandholtz et al., 2003). Similar result was reported in another study that HSP90 (heat shock protein 90)-Cdc37 chaperone module directly influences the magnitude of TLR-mediated macrophage activation by binding interleukin-1 receptor associated kinase (IRAK-1), a key component of TLR signaling pathways (De Nardo et al., 2005). However, the roles of HSP-90 in modulating TLRs are still obscure in SLE. Cystamine is a transglutaminase-2 inhibitor (Folk, 1980; Lorand and Conrad, 1984) with neuron-protective effects in various nerve cell lines and a transgenic model of Huntington disease (Karpuj et al., 2002; Lesort et al., 2003; Ientile et al., 2003). Recently, we demonstrated the beneficial effects of cystamine on macrophage response (Hsu et al., 2007). Additionally, reduced hepatic abnormality and liver apoptosis were also
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observed in NZB/W F1 mice receiving cystamine (Tzang et al., 2008; Hsu et al., 2008b) as well as the reduced apoptosis in the brain of NZB/W F1 receiving cystamine (Hsu et al., 2008a). However, the involved effects of cystamine on brain abnormality in SLE are still unclear. Since cystamine has been indicated to have multiple potentials in a variety of disorders, herein we examine the effect of cystamine on NOS and TLR related molecules in the brain of NZB/W F1 mice and suggested the protective effects of cystamine on brain impairment in SLE. 2. Materials and methods 2.1. Mice and brain sample Female NZB/W F1 is a well-described lupus-prone mice strain and has been widely used as an SLE animal model, which reveals the symptoms of SLE spontaneously from the age of 12 weeks, including the production of various autoantibodies and neuropsychiatric involvement (Russell and Denman,1969; Carr et al.,1978; Hoffman et al.,1978). BALB/ c strain is used as a normal stain control to verify the effect of cystamine on brains. Female BALB/c and NZB/W F1 mice were purchased from the animal center, SINICA, Taiwan, and from the animal center, National Taiwan University, Taiwan, respectively. The mice were housed under supervision of the Institutional Animal Care and Use Committee at Chung Shan Medical University. Disease activity of NZB/W F1 mice was determined by monitoring the proteinuria biweekly with Albustix test strips from the age of 14 weeks for ten weeks as described previously (Karpuj et al., 2002) and scored according to the manufacturer's scoring system (Bayer Diagnostics, Hong Kong). BALB/c and NZB/W F1 mice of 188-day-old were divided into four groups (12 mice/group) and given PBS or cystamine for experiments, respectively. For cystamine (Sigma, Saint Louis Mo, USA) treatment, BALB/c and NZB/W F1 mice were
Fig. 2. Cystamine reduces the expression of p53 and p21 proteins. Brain samples of BALB/c and NZB/W F1 mice treated with PBS or cystamine were analyzed for the expression of (A), (B) p53 and (C), (D) p21 proteins by Western blot. The ratio of p53 or p21 to β-actin was shown in the lower panel and the P values are 0.477, 0.015, 0.762 and 0.039, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
injected intraperitoneally (i.p.) with 100 µl of 10 mM phosphate buffer saline (PBS) or 100 µl of 10 mM cystamine daily for 14 days (Hsu et al., 2007; Tzang et al., 2008; Hsu et al., 2008a; Hsu et al., 2008b). The dose of cystamine is 3.75 mg/kg after conversion and was recognized suitable for analysis. Mice were sacrificed by CO2 asphyxiation and rinsed in 70% ethanol solution. The whole brain samples of mice were obtained after CO2 sacrifice and stored at −80 °C until use. 2.2. Western blot
Fig. 1. Cystamine reduces the expression of nNOS and iNOS proteins. Brain samples of BALB/c and NZB/W F1 mice treated with PBS or cystamine were analyzed for the expression of (A), (B) nNOS and (C), (D) iNOS proteins by Western blot. The ratio of nNOS or iNOS to β-actin was shown in the lower panel and the P values are 0.237, 0.009, 0.197 and 0.036, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
The brain samples from the mice of each group were analyzed for immunoblotting and similar results were observed in the same group. The loading sample for each lane of Western blot was a pool of four random selected mice of the same group and the protein levels were assayed using a BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA) and were quantified by absorbance at 595 nm using a spectrophotometer (Beckman Coulter, Palo Alto, CA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% acrylamide gel, was performed as previously described (Laemmli,1970). Protein samples were denatured for 5 min in boiling water with sample buffer (0.0625 M Tris–HCl buffer, pH 6.8, containing 2.3% SDS, 5% 2-mercaptoethanol, and 10% glycerol). Thirty μg of loading samples for each lane was applied to the gel and run at 100–150 V for 1.5 h before electrophoretical transfer to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membrane was then soaked in PBS with 5% nonfat dry milk for 30 min at room temperature to saturate irrelevant protein binding sites. Antibodies against nNOS, iNOS, p53, p21WAF1/CIP1, heat shock protein (HSP)-90, TLR-4, TLR-5, TLR-7, TLR-9, and actin (Upstates, Charlottesville, Virginia, USA; Chemicon International, Temecula, CA, USA) were diluted in PBS with 2.5% BSA and incubated for 1.5 h with gentle agitation at room
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panel of Fig. 1D. However, no significant variations of both nNOS and iNOS expression were observed between the brain samples from BALB/c mice that were treated with PBS or cystamine (Fig. 1A and C). 3.2. Cystamine reduces the expressions of p53, p21WAF1/CIP1, and HSP90 To further clarify the effect of cystamine on the mechanism involving the brain impairment of NZB/W F1 mice, Western blots were performed to detect the expression of p53, p21WAF1/CIP1, and HSP90 proteins. Fig. 2B reveals the significant reduction of p53 protein in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS. The ratio of p53/β-actin was indicated in the lower panel of Fig. 2B. Moreover, similar result was observed in the expression of p21WAF1/CIP1, a downstream molecule of p53 protein. Significant reduction of p21WAF1/CIP1 protein was detected in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that
Fig. 3. Cystamine reduces the expression of HSP90 protein. Brain samples of (A) BALB/c and (B) NZB/W F1 mice treated with PBS or cystamine were analyzed for the expression of HSP90 protein by Western blot. The ratio of HSP90 to β-actin was shown in the lower panel and the P values are 0.626 and 0.025, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
temperature. The membranes were washed twice with PBS-Tween for 1 h and secondary antibody conjugated with horseradish peroxidase (HRP) was added. Pierce's Supersignal West Dura HRP Detection Kit (Pierce Biotechnology Inc., Rockford, IL) was used to detect antigen–antibody complexes. The blots were also scanned and quantified by densitometry (Appraise, Beckman Coulter, Brea, California, USA). 2.3. Statistical analysis All the statistical analyses were performed using SPSS 10.0 software (SPSS Inc, Chicago, IL). Three independent experiments were repeated. Statistical analyses were performed using the Student's t test or one-way ANOVA. P b 0.05 was considered statistically significant. 3. Results 3.1. Cystamine reduces the expressions of nNOS and iNOS Nitric Oxide Synthase is known to play crucial roles in brain disorders. To investigate the effect of cystamine in the brain of NZB/W F1 mice, the expressions of nNOS and iNOS proteins were detected by Western blot. Significant reduction of nNOS protein was detected in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS (Fig. 1B). The ratio of nNOS/β-actin was indicated in the lower panel of Fig. 1B. Additionally, the presence of iNOS was also examined. Fig. 1D shows the significant reduction of iNOS in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS. The ratio of iNOS/β-actin was indicated in the lower
Fig. 4. Cystamine reduces the expressions of TLR-4, TLR-5 and TLR-7 proteins. Brain sample of (A) BALB/c and NZB/W F1 mice treated with PBS or cystamine was analyzed for the expression of TLR-4, TLR-5, TLR-7, and TLR-9 protein by Western blot. The ratios of (B) TLR-4, (C) TLR-5, and (D) TLR-7 to β-actin were shown and the P values are 0.489, 0.002, 0.125, 0.012, 0.833, and 0.016, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
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were treated with PBS (Fig. 2D). However, no significant variation of both p53 and p21WAF1/CIP1 expressions was detected between the brain samples from BALB/c mice that were treated with PBS or cystamine (Fig. 2A and C). Since HSP90 is known to be associated with the presence of nNOS, the expression of HSP90 was also detected. As shown in Fig. 3B, significant reduction of HSP90 was observed in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS. However, no significant variation was detected between brain samples from BALB/c mice that were treated with PBS or cystamine (Fig. 3A). 3.3. Cystamine reduces the expressions of TLR-4, TLR-5 and TLR-7 Since innate immunity plays important roles in the pathogenesis of SLE, the expressions of various lupus-related TLR molecules were examined. The expressions of TLR-4, TLR-5, TLR-7, and TLR-9 in the brain samples from both BALB/c and NZB/W F1 mice were detected by Western blots. No variation of TLR-4, TLR-5, TLR-7, and TLR-9 proteins was observed between brain samples from BALB/c that were treated with PBS or cystamine (Fig. 4A). Similar result in the expression of TLR-9 proteins was also observed in the brain of NZB/W F1 mice that were treated with PBS or cystamine (Fig. 4A). However, significant reductions of TLR-4, TLR5 and TLR-7 proteins were detected in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS (Fig. 4A). The quantified results of TLR-4, TLR-5 and TLR-7 were shown in the lower panels of Fig. 4B, C, and D. 4. Discussion The impairment of CNS and neuropsychiatric disorder has been considered a major cause of long-standing dysfunction and mortality in patients with SLE (Kaell et al., 1986; Jonsson et al., 1989). Although our recent study indicated the anti-apoptotic effect of cystamine in the brain of NZB/W F1 mice, the precise mechanism is still unclear. In the current study, we further demonstrated the protective effect of cystamine in the brain of NZB/W F1 mice by reducing nNOS, iNOS, p53, p21WAF1/CIP1, HSP90, TLR-4, TLR-5, and TLR-7 expressions, whereas no variation was detected between brain samples from BALB/c mice that were treated with PBS or cystamine. NOS has been associated with the pathogenesis of SLE. A previous study indicated that significant increase of apoptosis was detected in human umbilical vein endothelial cells (HUVEC) incubated with antidsDNA-containing-IgG, a prominent autoantibody of SLE, and has been associated with the increased expression NOS (Lai et al., 1997). Another study reported that significant increase of apoptosis was observed on spleen lymphocytes of MRL/lpr mice with active disease and was reduced by pharmacologic inhibition of NOS (Oates and Gilkeson, 2004). Moreover, in an iron-induced hippocampal neurotoxicity rat model, mean neuron loss was markedly reduced by treatment with 7-nitroindazole, a well-known inhibitor of nNOS (Babbedge et al., 1993), from 43% to 11% (Bostanci and Bağirici, 2007). In clinical studies, increased NO synthase activity or expression was detected in rheumatoid arthritis (RA) patients with high disease activity and in patients with SLE (Belmont et al., 1997; Wigand et al., 1997). Indeed, these results did emphasize the crucial roles of NOS in the pathogenesis of SLE. Herein, current study reveals the reduced nNOS and iNOS expression in the brain of NZB/W F1 mice treated with cystamine and probably suggests a beneficial effect of cystamine on neuroprotection in SLE. P53 is known as a tumor suppressor gene with diverse functions. Recently, p53 is demonstrated to play a role in the pathogenesis of SLE and has been associated with the disease activity. Patients with active SLE had higher levels of p53 expression and resulted in the impaired regulation of autoreactive lymphocytes (Miret et al., 2003). Besides, generation of anti-p53 autoantibody was also found in patients with SLE (Kuhn et al., 1999; Herkel et al., 2000). A recent study indicated
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that p53 plays an important role in brain edema by up-regulating the MMP-9 expression (Yan et al., 2008). Another study reported the protective effect of anthocyanins in a rat cerebral ischemia model by suppressing the activation of p53 (Shin et al., 2006). Additionally, our recent investigation demonstrated the beneficial effect of cystamine on hepatic apoptosis in the liver from NZB/W F1 mice by reducing the p53 expression (Hsu et al., 2008b). These experimental results strongly suggest the pathological role of p53 in SLE. In the current study, significant reduction of p53 and its downstream molecule p21 was detected in the brain of NZB/W F1 mice treated with cystamine, which probably indicates the protective effect of cystamine in the brain in SLE. Recently, HSP90 is recognized as an evolving therapeutic target in various disorders and known to act as a signaling mediator of NOdependent responses (Bender et al., 1999; Shah et al., 1999; Song et al., 2001). HSP90 is a molecular chaperone and plays diverse roles in both physiological and pathological processes. HSP90 was reported to associate with eNOS as an allosteric enhancer and also facilitated nNOS activation in nNOS-transfected cells (García-Cardeña et al., 1998; Bender et al., 1999). Heat shock-induced HSP90 protein forms a complex with eNOS and leads to excess NO accumulation in H9C2 cells (Ilangovan et al., 2004). In a rat portal hypertension model, HSP90 is demonstrated to be involved in the excessive NOS production (Shah et al., 1999). Additionally, direct evidence demonstrated that HSP90 enhances nNOS catalytic function in vitro and in intact cells (Song et al., 2001). In the current study, significant reduction of nNOS, iNOS, and HSP90 expression was observed in the brain of NZB/W F1 mice that were treated with cystamine and suggested the protective effect of cystamine on neuroprotection in SLE probably through NOS-HSP90 related signaling pathway. HSP-90 is also known to play important roles in modulating TLR activation (Bandholtz et al., 2003; De Nardo et al., 2005). Besides, various studies have connected the association between TLRs and SLE. In a collagen-induced arthritis mice model, inhibition of TLR-4 suppresses the severity of experimental arthritis and reduces IL-1 expression in arthritic joints (Abdollahi-Roodsaz et al., 2007). In pristane-induced murine lupus, experimental results demonstrated that TLR-7 is specifically required for the production of RNA-reactive autoantibodies and the development of glomerulonephritis (Savarese et al., 2008). Another study suggested the opposing effects of TLR-9 and TLR-7 on clinical disease in SLE. In gene-knocked lupus-prone mice, the absence of TLR-9 led to exacerbated autoimmune disease including activated lymphocytes, plasmacytoid dentric cells, increased serum IgG and IFN-alpha. In contrast, TLR-7-deficient mice revealed ameliorated disease and failed to generate antibody against Smith (Sm) Ag (Christensen et al., 2006). In the current study, significant reductions of TLR-4, TLR-5 and TLR-7 expressions were observed in the brain of NZB/W F1 mice that were treated with cystamine as compared to those that were treated with PBS, whereas no variation of TLR-9 was detected. Although the precise mechanisms of cystamine on reducing TLR-4, TLR-5 and TLR-7 are still unclear and needed further investigations, these findings did suggest the alleviating effects of cystamine on TLR-4, TLR-5 and TLR-7 expression in the brain of NZB/W F1 mice. In summary, this study revealed the protective effects of cystamine on brain abnormality in NZB/W F1 mice by reducing the expressions of nNOS, iNOS, p53, p21WAF1/CIP1, HSP-90, TLR-4, TLR-5, and TLR-7 proteins. Although the administration of cystamine on clinical treatment needs further evaluation, this study did provide a clue in understanding the action of cystamine and therapeutic potential for neuropsychiatric SLE. Acknowledgements This study was supported by the grants CSMU-CHA-097-002 from Chung Shan Medical University and Chia-Yi Christian Hospital.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Cystamine attenuates the expressions of NOS- and TLR-associated molecules in the brain of NZB/W F1 mice Hon-Pin Wang a,1, Tsai-Ching Hsu b,1, Gwo-Jong Hsu c, Sin-Lun Li d, Bor-Show Tzang d,⁎ a
Division of Allergy, Rheumatology and Immunology, Department of Internal Medicine, Chia-Yi Christian Hospital, Chia-Yi, Taiwan Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan Division of Infectious disease and Department of Internal Medicine, Chia-Yi Christian Hospital, Chia-Yi, Taiwan d Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 7 January 2009 Received in revised form 12 February 2009 Accepted 12 February 2009 Available online 4 March 2009 Keywords: SLE (systemic lupus erythematosus) NOS (Nitric Oxide Synthase) Neuropsychiatric TLR (toll-like receptor)
a b s t r a c t Evidence have indicated the impairment of central nervous system (CNS) and neuropsychiatric disorder in patients with systemic lupus erythematosus (SLE). However, little is known to improve the brain abnormality in SLE. To investigate the effect of cystamine on brain abnormality in SLE, NZB/W F1 mice were used as the animal model. Notably, significantly reduced neural Nitric Oxide Synthase (nNOS), inducible Nitric Oxide Synthase (iNOS), p53, p21WAF1/CIP1, and heat shock protein (HSP)-90 proteins were detected in the brain of NZB/W F1 mice that were treated with cystamine. In contrast, no variation was observed between the brain samples of BALB/c mice that were treated with PBS or cystamine. Moreover, significantly reduced Toll-like receptors- (TLR-) 4, 5 and 7 were detected in the brain samples of NZB/W F1 mice that were treated with cystamine whereas no variation of TLR-4, TLR-5, TLR-7, and TLR-9 was observed in BALB/c mice that were treated with PBS or cystamine. These findings demonstrated the beneficial effects of cystamine on brain abnormality in NZB/W F1 mice and probably suggested the potential of cystamine on treating patients with neuropsychiatric SLE. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Many clinical manifestations of systemic lupus erythematosus (SLE), including CNS involvement, lead to neuropsychiatric disease and mortality (Kaell et al., 1986; Jonsson et al., 1989). Indeed, various studies have indicated the increased involvement of central nervous system (CNS) or neuropsychosis in SLE patients (Feinglass et al., 1976; McCune and Golbus, 1988; Futrell et al., 1992; Sibley et al., 1992; Hanly and Liang, 1997), which is recognized as a major cause of longstanding functional impairment and mortality (Jonsson et al., 1989). However, little is known about the mechanism and treatment for brain abnormality in SLE. A number of studies have associated the nitric oxide (NO)associated tissue injury with different autoimmune diseases, including SLE (Belmont et al., 1997; Abramson et al., 2001). NO is produced by a group of enzymes denominated Nitric Oxide Synthases (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS), inducible NOS (iNOS) and mitochondrial NOS (mtNOS), which play important roles in both physiological and pathological processes (Elfering et al., 2002;
⁎ Corresponding author. Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung Taiwan, NO. 110, Jianguo N. Rd. Taichung 402, Taiwan. Tel.: +886 4 23248168; fax: +886 4 23248195. E-mail address: [email protected] (B.-S. Tzang). 1 H.P. Wang and T.C. Hsu contributed equally to this paper. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.02.039
Guix et al., 2005). In an iNOS-knockout MRL-lpr/lpr animal model, iNOS (−/−) and iNOS(+/−) mice had significantly less vasculitis and lower IgG rheumatoid factor levels as compared to iNOS(+/+) mice (Gilkeson et al., 1997). Additionally, increased NOS is related to the disease activity in SLE patients (Belmont et al., 1997; Wigand et al., 1997). Toll-like receptors (TLRs) play crucial roles in innate immunity and pathogenesis of autoimmune diseases. Sterile inflammation or autoimmunity can be induced by inappropriate activation of TLRs (Abdollahi-Roodsaz et al., 2007; Barrat and Coffman, 2008). Evidence have indicated the genetic variation in TLR-5 and TLR-9 that relates to the disease activity of SLE (Hawn et al., 2005; De Jager et al., 2006). Recently, various studies have indicated that HSP-90 could bind CpG oligonucleotides directly and act as a ligand transfer molecule in TLR-9 signaling cascade (Bandholtz et al., 2003). Similar result was reported in another study that HSP90 (heat shock protein 90)-Cdc37 chaperone module directly influences the magnitude of TLR-mediated macrophage activation by binding interleukin-1 receptor associated kinase (IRAK-1), a key component of TLR signaling pathways (De Nardo et al., 2005). However, the roles of HSP-90 in modulating TLRs are still obscure in SLE. Cystamine is a transglutaminase-2 inhibitor (Folk, 1980; Lorand and Conrad, 1984) with neuron-protective effects in various nerve cell lines and a transgenic model of Huntington disease (Karpuj et al., 2002; Lesort et al., 2003; Ientile et al., 2003). Recently, we demonstrated the beneficial effects of cystamine on macrophage response (Hsu et al., 2007). Additionally, reduced hepatic abnormality and liver apoptosis were also
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observed in NZB/W F1 mice receiving cystamine (Tzang et al., 2008; Hsu et al., 2008b) as well as the reduced apoptosis in the brain of NZB/W F1 receiving cystamine (Hsu et al., 2008a). However, the involved effects of cystamine on brain abnormality in SLE are still unclear. Since cystamine has been indicated to have multiple potentials in a variety of disorders, herein we examine the effect of cystamine on NOS and TLR related molecules in the brain of NZB/W F1 mice and suggested the protective effects of cystamine on brain impairment in SLE. 2. Materials and methods 2.1. Mice and brain sample Female NZB/W F1 is a well-described lupus-prone mice strain and has been widely used as an SLE animal model, which reveals the symptoms of SLE spontaneously from the age of 12 weeks, including the production of various autoantibodies and neuropsychiatric involvement (Russell and Denman,1969; Carr et al.,1978; Hoffman et al.,1978). BALB/ c strain is used as a normal stain control to verify the effect of cystamine on brains. Female BALB/c and NZB/W F1 mice were purchased from the animal center, SINICA, Taiwan, and from the animal center, National Taiwan University, Taiwan, respectively. The mice were housed under supervision of the Institutional Animal Care and Use Committee at Chung Shan Medical University. Disease activity of NZB/W F1 mice was determined by monitoring the proteinuria biweekly with Albustix test strips from the age of 14 weeks for ten weeks as described previously (Karpuj et al., 2002) and scored according to the manufacturer's scoring system (Bayer Diagnostics, Hong Kong). BALB/c and NZB/W F1 mice of 188-day-old were divided into four groups (12 mice/group) and given PBS or cystamine for experiments, respectively. For cystamine (Sigma, Saint Louis Mo, USA) treatment, BALB/c and NZB/W F1 mice were
Fig. 2. Cystamine reduces the expression of p53 and p21 proteins. Brain samples of BALB/c and NZB/W F1 mice treated with PBS or cystamine were analyzed for the expression of (A), (B) p53 and (C), (D) p21 proteins by Western blot. The ratio of p53 or p21 to β-actin was shown in the lower panel and the P values are 0.477, 0.015, 0.762 and 0.039, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
injected intraperitoneally (i.p.) with 100 µl of 10 mM phosphate buffer saline (PBS) or 100 µl of 10 mM cystamine daily for 14 days (Hsu et al., 2007; Tzang et al., 2008; Hsu et al., 2008a; Hsu et al., 2008b). The dose of cystamine is 3.75 mg/kg after conversion and was recognized suitable for analysis. Mice were sacrificed by CO2 asphyxiation and rinsed in 70% ethanol solution. The whole brain samples of mice were obtained after CO2 sacrifice and stored at −80 °C until use. 2.2. Western blot
Fig. 1. Cystamine reduces the expression of nNOS and iNOS proteins. Brain samples of BALB/c and NZB/W F1 mice treated with PBS or cystamine were analyzed for the expression of (A), (B) nNOS and (C), (D) iNOS proteins by Western blot. The ratio of nNOS or iNOS to β-actin was shown in the lower panel and the P values are 0.237, 0.009, 0.197 and 0.036, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
The brain samples from the mice of each group were analyzed for immunoblotting and similar results were observed in the same group. The loading sample for each lane of Western blot was a pool of four random selected mice of the same group and the protein levels were assayed using a BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA) and were quantified by absorbance at 595 nm using a spectrophotometer (Beckman Coulter, Palo Alto, CA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% acrylamide gel, was performed as previously described (Laemmli,1970). Protein samples were denatured for 5 min in boiling water with sample buffer (0.0625 M Tris–HCl buffer, pH 6.8, containing 2.3% SDS, 5% 2-mercaptoethanol, and 10% glycerol). Thirty μg of loading samples for each lane was applied to the gel and run at 100–150 V for 1.5 h before electrophoretical transfer to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membrane was then soaked in PBS with 5% nonfat dry milk for 30 min at room temperature to saturate irrelevant protein binding sites. Antibodies against nNOS, iNOS, p53, p21WAF1/CIP1, heat shock protein (HSP)-90, TLR-4, TLR-5, TLR-7, TLR-9, and actin (Upstates, Charlottesville, Virginia, USA; Chemicon International, Temecula, CA, USA) were diluted in PBS with 2.5% BSA and incubated for 1.5 h with gentle agitation at room
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panel of Fig. 1D. However, no significant variations of both nNOS and iNOS expression were observed between the brain samples from BALB/c mice that were treated with PBS or cystamine (Fig. 1A and C). 3.2. Cystamine reduces the expressions of p53, p21WAF1/CIP1, and HSP90 To further clarify the effect of cystamine on the mechanism involving the brain impairment of NZB/W F1 mice, Western blots were performed to detect the expression of p53, p21WAF1/CIP1, and HSP90 proteins. Fig. 2B reveals the significant reduction of p53 protein in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS. The ratio of p53/β-actin was indicated in the lower panel of Fig. 2B. Moreover, similar result was observed in the expression of p21WAF1/CIP1, a downstream molecule of p53 protein. Significant reduction of p21WAF1/CIP1 protein was detected in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that
Fig. 3. Cystamine reduces the expression of HSP90 protein. Brain samples of (A) BALB/c and (B) NZB/W F1 mice treated with PBS or cystamine were analyzed for the expression of HSP90 protein by Western blot. The ratio of HSP90 to β-actin was shown in the lower panel and the P values are 0.626 and 0.025, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
temperature. The membranes were washed twice with PBS-Tween for 1 h and secondary antibody conjugated with horseradish peroxidase (HRP) was added. Pierce's Supersignal West Dura HRP Detection Kit (Pierce Biotechnology Inc., Rockford, IL) was used to detect antigen–antibody complexes. The blots were also scanned and quantified by densitometry (Appraise, Beckman Coulter, Brea, California, USA). 2.3. Statistical analysis All the statistical analyses were performed using SPSS 10.0 software (SPSS Inc, Chicago, IL). Three independent experiments were repeated. Statistical analyses were performed using the Student's t test or one-way ANOVA. P b 0.05 was considered statistically significant. 3. Results 3.1. Cystamine reduces the expressions of nNOS and iNOS Nitric Oxide Synthase is known to play crucial roles in brain disorders. To investigate the effect of cystamine in the brain of NZB/W F1 mice, the expressions of nNOS and iNOS proteins were detected by Western blot. Significant reduction of nNOS protein was detected in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS (Fig. 1B). The ratio of nNOS/β-actin was indicated in the lower panel of Fig. 1B. Additionally, the presence of iNOS was also examined. Fig. 1D shows the significant reduction of iNOS in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS. The ratio of iNOS/β-actin was indicated in the lower
Fig. 4. Cystamine reduces the expressions of TLR-4, TLR-5 and TLR-7 proteins. Brain sample of (A) BALB/c and NZB/W F1 mice treated with PBS or cystamine was analyzed for the expression of TLR-4, TLR-5, TLR-7, and TLR-9 protein by Western blot. The ratios of (B) TLR-4, (C) TLR-5, and (D) TLR-7 to β-actin were shown and the P values are 0.489, 0.002, 0.125, 0.012, 0.833, and 0.016, respectively. The loading sample for each lane was a pool of three different mice from the same group. Similar results were obtained in three independent experiments and ⁎indicates significant difference.
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were treated with PBS (Fig. 2D). However, no significant variation of both p53 and p21WAF1/CIP1 expressions was detected between the brain samples from BALB/c mice that were treated with PBS or cystamine (Fig. 2A and C). Since HSP90 is known to be associated with the presence of nNOS, the expression of HSP90 was also detected. As shown in Fig. 3B, significant reduction of HSP90 was observed in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS. However, no significant variation was detected between brain samples from BALB/c mice that were treated with PBS or cystamine (Fig. 3A). 3.3. Cystamine reduces the expressions of TLR-4, TLR-5 and TLR-7 Since innate immunity plays important roles in the pathogenesis of SLE, the expressions of various lupus-related TLR molecules were examined. The expressions of TLR-4, TLR-5, TLR-7, and TLR-9 in the brain samples from both BALB/c and NZB/W F1 mice were detected by Western blots. No variation of TLR-4, TLR-5, TLR-7, and TLR-9 proteins was observed between brain samples from BALB/c that were treated with PBS or cystamine (Fig. 4A). Similar result in the expression of TLR-9 proteins was also observed in the brain of NZB/W F1 mice that were treated with PBS or cystamine (Fig. 4A). However, significant reductions of TLR-4, TLR5 and TLR-7 proteins were detected in the brain of NZB/W F1 mice that were treated with cystamine as compared to those mice that were treated with PBS (Fig. 4A). The quantified results of TLR-4, TLR-5 and TLR-7 were shown in the lower panels of Fig. 4B, C, and D. 4. Discussion The impairment of CNS and neuropsychiatric disorder has been considered a major cause of long-standing dysfunction and mortality in patients with SLE (Kaell et al., 1986; Jonsson et al., 1989). Although our recent study indicated the anti-apoptotic effect of cystamine in the brain of NZB/W F1 mice, the precise mechanism is still unclear. In the current study, we further demonstrated the protective effect of cystamine in the brain of NZB/W F1 mice by reducing nNOS, iNOS, p53, p21WAF1/CIP1, HSP90, TLR-4, TLR-5, and TLR-7 expressions, whereas no variation was detected between brain samples from BALB/c mice that were treated with PBS or cystamine. NOS has been associated with the pathogenesis of SLE. A previous study indicated that significant increase of apoptosis was detected in human umbilical vein endothelial cells (HUVEC) incubated with antidsDNA-containing-IgG, a prominent autoantibody of SLE, and has been associated with the increased expression NOS (Lai et al., 1997). Another study reported that significant increase of apoptosis was observed on spleen lymphocytes of MRL/lpr mice with active disease and was reduced by pharmacologic inhibition of NOS (Oates and Gilkeson, 2004). Moreover, in an iron-induced hippocampal neurotoxicity rat model, mean neuron loss was markedly reduced by treatment with 7-nitroindazole, a well-known inhibitor of nNOS (Babbedge et al., 1993), from 43% to 11% (Bostanci and Bağirici, 2007). In clinical studies, increased NO synthase activity or expression was detected in rheumatoid arthritis (RA) patients with high disease activity and in patients with SLE (Belmont et al., 1997; Wigand et al., 1997). Indeed, these results did emphasize the crucial roles of NOS in the pathogenesis of SLE. Herein, current study reveals the reduced nNOS and iNOS expression in the brain of NZB/W F1 mice treated with cystamine and probably suggests a beneficial effect of cystamine on neuroprotection in SLE. P53 is known as a tumor suppressor gene with diverse functions. Recently, p53 is demonstrated to play a role in the pathogenesis of SLE and has been associated with the disease activity. Patients with active SLE had higher levels of p53 expression and resulted in the impaired regulation of autoreactive lymphocytes (Miret et al., 2003). Besides, generation of anti-p53 autoantibody was also found in patients with SLE (Kuhn et al., 1999; Herkel et al., 2000). A recent study indicated
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that p53 plays an important role in brain edema by up-regulating the MMP-9 expression (Yan et al., 2008). Another study reported the protective effect of anthocyanins in a rat cerebral ischemia model by suppressing the activation of p53 (Shin et al., 2006). Additionally, our recent investigation demonstrated the beneficial effect of cystamine on hepatic apoptosis in the liver from NZB/W F1 mice by reducing the p53 expression (Hsu et al., 2008b). These experimental results strongly suggest the pathological role of p53 in SLE. In the current study, significant reduction of p53 and its downstream molecule p21 was detected in the brain of NZB/W F1 mice treated with cystamine, which probably indicates the protective effect of cystamine in the brain in SLE. Recently, HSP90 is recognized as an evolving therapeutic target in various disorders and known to act as a signaling mediator of NOdependent responses (Bender et al., 1999; Shah et al., 1999; Song et al., 2001). HSP90 is a molecular chaperone and plays diverse roles in both physiological and pathological processes. HSP90 was reported to associate with eNOS as an allosteric enhancer and also facilitated nNOS activation in nNOS-transfected cells (García-Cardeña et al., 1998; Bender et al., 1999). Heat shock-induced HSP90 protein forms a complex with eNOS and leads to excess NO accumulation in H9C2 cells (Ilangovan et al., 2004). In a rat portal hypertension model, HSP90 is demonstrated to be involved in the excessive NOS production (Shah et al., 1999). Additionally, direct evidence demonstrated that HSP90 enhances nNOS catalytic function in vitro and in intact cells (Song et al., 2001). In the current study, significant reduction of nNOS, iNOS, and HSP90 expression was observed in the brain of NZB/W F1 mice that were treated with cystamine and suggested the protective effect of cystamine on neuroprotection in SLE probably through NOS-HSP90 related signaling pathway. HSP-90 is also known to play important roles in modulating TLR activation (Bandholtz et al., 2003; De Nardo et al., 2005). Besides, various studies have connected the association between TLRs and SLE. In a collagen-induced arthritis mice model, inhibition of TLR-4 suppresses the severity of experimental arthritis and reduces IL-1 expression in arthritic joints (Abdollahi-Roodsaz et al., 2007). In pristane-induced murine lupus, experimental results demonstrated that TLR-7 is specifically required for the production of RNA-reactive autoantibodies and the development of glomerulonephritis (Savarese et al., 2008). Another study suggested the opposing effects of TLR-9 and TLR-7 on clinical disease in SLE. In gene-knocked lupus-prone mice, the absence of TLR-9 led to exacerbated autoimmune disease including activated lymphocytes, plasmacytoid dentric cells, increased serum IgG and IFN-alpha. In contrast, TLR-7-deficient mice revealed ameliorated disease and failed to generate antibody against Smith (Sm) Ag (Christensen et al., 2006). In the current study, significant reductions of TLR-4, TLR-5 and TLR-7 expressions were observed in the brain of NZB/W F1 mice that were treated with cystamine as compared to those that were treated with PBS, whereas no variation of TLR-9 was detected. Although the precise mechanisms of cystamine on reducing TLR-4, TLR-5 and TLR-7 are still unclear and needed further investigations, these findings did suggest the alleviating effects of cystamine on TLR-4, TLR-5 and TLR-7 expression in the brain of NZB/W F1 mice. In summary, this study revealed the protective effects of cystamine on brain abnormality in NZB/W F1 mice by reducing the expressions of nNOS, iNOS, p53, p21WAF1/CIP1, HSP-90, TLR-4, TLR-5, and TLR-7 proteins. Although the administration of cystamine on clinical treatment needs further evaluation, this study did provide a clue in understanding the action of cystamine and therapeutic potential for neuropsychiatric SLE. Acknowledgements This study was supported by the grants CSMU-CHA-097-002 from Chung Shan Medical University and Chia-Yi Christian Hospital.
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