Journal of Neuroscience Research 92:743–750 (2014)
Curcumin Ameliorates Rat Experimental Autoimmune Neuritis Fuyu Han, Bangwei Luo, Rongchen Shi, Changhao Han, Zhonghao Zhang, Jian Xiong, Man Jiang, and Zhiren Zhang* Institute of Immunology, Third Military Medical University of PLA, 30 Gaotanyan Mainstreet, Chongqing, People’s Republic of China
Experimental autoimmune neuritis (EAN) is a helper T cellmediated autoimmune demyelinating inflammatory disease of the peripheral nervous system that serves as an animal model for human Guillain-Barre syndrome. Curcumin, a naturally occurring polyphenolic phytochemical isolated from the medicinal plant Curcuma longa, has anti-inflammatory activities. Here we investigated the therapeutic effects and potential mechanisms of curcumin in EAN rats. Exogenous curcumin treatment (100 mg/ kg/day) significantly delayed the onset of EAN neurological signs, ameliorated EAN neurological severity, and reduced body weight loss of EAN rats. In EAN sciatic nerves, curcumin treatment suppressed the inflammatory cell accumulation and the expression of interferon (IFN)g, tumor necrosis factor-a, interleukin (IL)-1b, and IL-17. Furthermore, curcumin treatment significantly decreased the percentage of CD41 T helper cells in EAN spleen and suppressed concanavalin A-induced lymphocyte proliferation in vitro. In addition, curcumin altered helper T cell differentiation by decreasing IFN-g1CD41 Th1 cells in EAN lymph node and spleen. In summary, our data demonstrate that curcumin could effectively suppress EAN by attenuating inflammation, indicating that curcumin might be a candidate for treatment of autoimmune neuropathies. VC 2014 Wiley Periodicals, Inc. Key words: curcumin; experimental autoimmune neuritis; inflammation; T lymphocytes
Experimental autoimmune neuritis (EAN) is a helper T cell-mediated inflammatory demyelinating disease of the peripheral nervous system (PNS; Zhu et al., 2002) that mirrors many clinical and immunological features of the human acute inflammatory demyelinating polyradiculoneuropathies (AIDP) and has been widely applied as an animal model to investigate therapy and disease mechanisms of AIDP (Hughes and Cornblath, 2005). Patients with inflammatory polyneuropathies receive supportive management or active treatments, such as plasma exchange and intravenous immunoglobulin (Hughes, 2002; Aronovich et al., 2012). Although these treatments are beneficial to some patients (Lin et al., 2007), only about 65% patients with Guillain-Barre syndrome (GBS) respond to plasma exchange or intravenous immunoglobulin (Enders et al., 1997). About 8% of GBS patients die, C 2014 Wiley Periodicals, Inc. V
and up to 20% remain disabled (Kieseier et al., 2004). Even in those who recover well, residual weakness and loss of motor units can usually be detected (Winer, 2001). Therefore, it will be necessary to establish more effective therapeutic strategies for inflammatory polyneuropathies. EAN is characterized by the demyelination of peripheral nerves and the accumulation of autoreactive T cells in the PNS (Kieseier et al., 2004; Zhu et al., 2011). Reactive lymphocytes and macrophages cause a robust local inflammation that leads to demyelination and axon degeneration (Jung et al., 1996; Hughes and Cornblath, 2005). T helper cells are important for the pathogenesis of EAN, because EAN can be induced by adoptive transfer of PNS autoantigen-specific CD41 Th cells (Gold et al., 1999; Kieseier et al., 2004). Th cell polarization following autoantigen stimulation is essential for the determination of type and severity of autoimmune disorders (Wilson et al., 2009). EAN is considered to be mediated mainly by Th1 cells and Th1 cytokines such as interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), and interleukin (IL)-1b (Kieseier et al., 2004). In addition, Th17 cells are believed to contribute to the development of EAN pathogenesis (Zhang et al., 2009c), and the immune suppressive Treg cells help in the inflammation resolution and recovery of EAN (Zhang et al., 2009a). Curcumin is a naturally occurring yellow pigment isolated from the rhizomes of the plant Curcuma longa, which has antioxidant, antitumor, and anti-inflammatory properties (Srimal and Dhawan, 1973). Studies have shown that curcumin inhibits inflammation in animal models of such disorders as atherosclerosis, arthritis, and Contract grant sponsor: National Nature Science Foundation of China; Contract grant number: 31170851; Contract grant sponsor: National Science and Technology Major Projects of New Drugs; Contract grant number: 2012ZX09103301-035; Contract grant sponsor: SRF (for ROCS, SEM). *Correspondence to: Zhiren Zhang, Institute of Immunology, Third Military Medical University of PLA, 30 Gaotanyan Main Street, Chongqing 400038, People’s Republic of China. E-mail: [email protected] Received 18 September 2013; Revised 24 November 2013; Accepted 3 December 2013 Published online 31 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23357
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Alzheimer’s disease (Mukhopadhyay et al., 1982; Araujo and Leon, 2001; Ono et al., 2004). Although the exact mechanisms involved in the anti-inflammatory activity of curcumin remain elusive, recent reports indicate that curcumin can reduce proinflammatory cytokines such as TNF-a, IL-1, IL-8 through deactivation of nuclear factor-jB (NF-jB; Brouet and Ohshima, 1995; Araujo and Leon, 2001; Gaedeke et al., 2004). In addition, increasing data also shows that curcumin can inhibit Th1 differentiation in vitro by inhibiting IL-12 production from macrophages (Kang et al., 1999a,b). These findings suggest a potential role of curcumin in the treatment of autoimmune inflammatory diseases (Bao et al., 2002). Therefore, we investigated the therapeutic effects of curcumin in EAN and its underlying mechanisms. MATERIALS AND METHODS Animals Male Lewis rats (8–10 weeks, 200–220 g; Vital River, Beijing, China) were housed under a 12 hr light-12 hr dark cycle with free access to food and water. All animal procedures were in accordance with a protocol approved by the local Administration District Official Committee. All efforts were made to minimize the number of animals and their suffering. EAN Induction and Treatment Lewis rats were immunized by subcutaneous injection into both hind footpads of 100 ll of an inoculum containing 100 lg synthetic neuritogenic P257–81 peptide (Chinapeptide, Shanghai, China; Cavaletti et al., 2000). The peptide was dissolved in phosphate-buffered saline (PBS; 2 mg/ml) and then emulsified with an equal volume of complete Freund’s adjuvant (Sigma, St. Louis, MO) containing 4 mg/ml Mycobacterium tuberculosis for a final concentration of 2 mg/ml. Rats were weighed daily and inspected for disease severity. Clinical scores were given according to the following scale: 0 5 normal; 1 5 reduced tone of the tail, hanging tail tip; 2 5 limp tail, impaired righting; 3 5 absent righting; 4 5 gait ataxia, abnormal positioning; 5 5 mild paraparesis of the hind limbs; 6 5 moderate paraparesis; 7 5 severe paraparesis or paraplegia of the hind limbs; 8 5 tetraparesis; 9 5 moribund; 10 5 death. For treatment, rats in the test groups were treated (i.p.) with 50 mg/kg or 100 mg/kg curcumin (Sigma) in dimethyl sulfoxide (DMSO) every day from day 0 to day 25 or from day 7 to day 25 (6 rats/group) following the induction of EAN, and the same volume of DMSO was given as a vehicle control. Histological Analysis To assess the degree of PNS inflammation, three rats from each group were sacrificed under ether anesthesia on day 15. The sciatic nerves were removed quickly. A portion of sciatic nerves sample was fixed in 4% neutral buffered formalin and then cut into 3-lm-thick sections, as described previously. Tissues were stained with hematoxylin and eosin to assess inflammatory cell infiltration. Histological changes between different groups were compared by an established semiquantitative method. Briefly, four cross-sections from root and middle levels
of both sides of sciatic nerves of EAN rats were analyzed. All perivascular areas present in cross-sections were evaluated by two observers unaware of treatment, and the degree of pathological alteration was graded semiquantitatively on the following scale: 0 5 normal perivascular area; 1 5 mild cellular infiltrate adjacent to the vessel; 2 5 cellular infiltration plus demyelination in immediate proximity to the vessel; 3 5 cellular infiltration and demyelination throughout the section. Results were given as a mean histological score. Tissue Preparation, RNA Isolation, Reverse Transcription, and Real-Time Quantitative RT-PCR After treatment, EAN rats were perfused intracardially with 4 C PBS under anesthesia, and the sciatic nerves and inguinal lymph nodes were quickly removed and stored in liquid nitrogen until RNA isolation. Total RNA was isolated using Trizol LS reagent (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA with a Quantscript RT kit (Tiangen Biotech, Beijing, China). The resulting cDNA was used to measure quantitatively the expression of genes using SYBR Green qPCR Master Mix according to the manufacturer’s protocol (Tiangen Biotech). Real-time measurements of gene expression were performed with a DNA Engine Opticon 2 Real-Time Cycler PCR detection system (Bio-Rad, Richmond, CA). Primers used to measure gene expression were bactin (sense, CCG TCT TCC CCT CCA TCG T; antisense, ATC GTC CCA GTT GGT TAC AAT GC), T-bet (sense,AAC CAG TAT CCT GTT CCC AGC; antisense, TGT CGC CAC TGG AAG GAT AG-3), ROR-g (sense, CGC ACC AAC CTC TTC TCA CG; antisense, GAC TTC CAT TGC TCC TGC TTT C-3), IL-12p35 (sense, TGA AGA CCA CGG ACG ACA; antisense, TGT GAT TCA GAG ACC GCA TTA G), IL-6 (sense, GCC CTT CAG GAA CAG CTA TG; antisense, CAG AAT TGC CAT TGC ACA AC), GATA-3 (sense, CTC TCC TTT GCT CAC CTT TTC; antisense, AAG AGA TGC GGA CTG GAG TG-3), IL-4 (sense, TGA TGG GTC TCA GCC CCC ACC TTG C; antisense, CTT TCA GTG TTG TGA GCG TGG ACT C), Foxp3 (sense, GCA CAA GTG CTT TGT GCG AGT; antisense, TGT CTG TGG TTG CAG ACG TTG T), IFN-g (sense, AAA GAC AAC CAG GCC ATC AG; antisense, CTT TTC CGC TTC CTT AGG CT), TNF-a (sense, TGA TCG GTC CCA ACA AGG A; antisense, TGC TTG GTG GTT TGC TAC GA), IL-1b (sense, TGC TGA TGT ACC AGT TGG GG; antisense, CTC CAT GAG CTT TGT ACA AG), IL-17 (sense, TGG ACT CTG AGC CGC ATT GA; antisense, GAC GCA TGG CGG ACA ATA GA), TGF-b (sense, TGA ACC AAG GAG ACG GAA TAC AGG; antisense, TAC TGT GTG TCC AGG CTC CAA ATG), and IL-10 (sense, CCT GCT CTT ACT GGC TGG AG; antisense, TCT CCC AGG GAA TTC AAA TG). Flow Cytometric Analysis of Th1, Th2, and Treg Cells in Spleen Th1 cells were identified as CD41IFN-g1 cells, Th2 cells were identified as CD41IL-41 cells, and Treg cells were identified as CD41Foxp31 cells. In short, rat spleens were dissected under sterile conditions and passed through a 40-mm Journal of Neuroscience Research
Curcumin in EAN cell strainer followed by Red Blood Cell Lysis Buffer (Tiangen Biotech). For determination of Th1 and Th2 cells, splenocytes were incubated for 6 hr in the presence of 50 ng/ml PMA (Sigma), 1 mg/ml ionomycin (Sigma), and 1 lg/ml brefeldin A (Sigma) in flat-bottomed 96-well plates in RPMI 1640 medium (Gibco, Grand Island, NY) containing penicillin (100 U/ml), streptomycin (100 U/ml), and 10% fetal calf serum at a density of 106 cells/ml at 37 C and 5% CO2. Thereafter, splenocytes were fixed and permeabilized. FITC-labeled IFN-g antibody (Biolegend, San Diego, CA) and PE-labeled CD4 antibody (Biolegend) were used for staining of CD41IFN-g1 Th1 cells. PE-labeled IL-4 antibody (Biolegend) and FITC-labeled CD4 antibody (Biolegend) were used for staining of CD41IL-41 Th2 cells. AlexaFlour488-labeled Foxp3 antibody (Biolegend) and PE-labeled CD4 antibody were used for staining of CD41Foxp31 Treg cells. For all staining, isotype controls were used. After staining, cells were washed and suspended in PBS and then analyzed with a FACScan (BD Biosciences, Franklin Lakes, NJ). Mononuclear cells were gated by forward and sideward scatter. Mixed Lymphocyte Proliferation Assays Spleen peripheral blood mononuclear cell (PBMC) suspensions were prepared by gently homogenizing the aseptically dissected organs. The cells were washed and resuspended in culture medium. All lymphocyte proliferation assays were performed in triplicate in 96-well flat-bottomed microtiter plates. Lymphocyte activation was measured by seeding 3.5 3 105 lymphocytes per 100 ml medium per well. For some cultures, spleen cells were cultured with 20 lg/ml concanavalin A (ConA) in the presence of 0, 1, 10, 50, and 100 lg/ml curcumin. The plates were incubated at 37 C in a humified atmosphere of 5% CO. After 52 hr of culture, each well was pulsed with 0.25 lCi [3H]methylthymidine and 16 hr later harvested onto fiberglass filters by a 96-well harvester. [3H]thymidine incorporation was measured by liquid scintillation counting. The proliferative response was expressed as the average counts per minute (cpm) of triplicate cultures. Evaluation and Statistical Analysis The unpaired t-test or Mann-Whitney U test was performed to compare differences between curcumin-treated and DMSO control EAN rats (Graph Pad Prism 5.0 for Windows). For all statistical analysis, significance levels were set at P < 0.05.
RESULTS Curcumin Treatment Ameliorated Neurological Signs in EAN Rats were immunized with neuritogenic synthetic P2 peptide and grouped randomly. The preventive groups were treated (i.p.) with high-dosage of curcumin (100 mg/kg) or low-dosage of curcumin (50 mg/kg) once daily from day 0 to day 25. The therapeutic groups were treated (i.p.) in the same manner every day from day 7 to day 25 following the induction of EAN. For DMSO control groups, the same volume of diluted DMSO was Journal of Neuroscience Research
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given. For DMSO groups, the first neurological sign of EAN rats was observed on day 6, the neurological severity of EAN reached a maximum on day 15 (7.33 6 0.33) and had disappeared by day 24. Neither preventive nor therapeutic low-dosage curcumin treatment attenuated EAN severity compared with DMSO control group (Fig. 1B). However, both preventive and therapeutic high-dosage curcumin treatment greatly delayed disease onset (Fig. 1A), decreased neurologic severity (Fig. 1A), and reduced EAN body weight loss (Fig. 1C) during the early period of EAN compared with DMSO control group. Effects of Curcumin Treatment on Inflammatory Cell Accumulation and Expression of Inflammation-Related Molecules in Sciatic Nerves of EAN Rats EAN is characterized by the accumulation of inflammatory cells in the PNS, which leads to the demyelination and axon degeneration of EAN sciatic nerves. To determine the effects of curcumin on inflammatory cell accumulation, sciatic nerves of EAN rats preventively or therapeutically treated with high-dosage curcumin were taken on day 15 for histological analysis. As shown in Figure 2A, profound inflammatory cell infiltration was observed in the sciatic nerves of DMSO control EAN rats. Conversely, both therapeutic and preventive treatment with curcumin obviously decreased sciatic nerve inflammatory cell infiltration (Fig. 2B,C). It is known that inflammatory cytokines such as IL1b, IFN-g, IL-17, and TNF-a have disease-promoting roles in EAN. Therefore, we further analyzed the effects of curcumin on mRNA level of certain inflammatoryrelated molecules in the sciatic nerves of EAN rats. As shown in Figure 2E, mRNA expression of IL-1b, IFN-g, IL-17, and TNF-a in day 15 EAN sciatic nerves was greatly reduced by curcumin. However, increases of IL-4, TGF-b, and IL-10 were not detected, indicating that curcumins help to ameliorate EAN by reducing proinflammatory cytokine. Curcumin Inhibited Spleen Cell Proliferation EAN is an inflammatory autoimmune disease mediated mainly by CD41 helper T cell. To study the influence of curcumin on T-cell proliferation in EAN, we examined the cell number change of CD41 Th cells in EAN spleen. EAN rats were preventively treated with high-dosage curcumin or vehicle control as described; on day 15 and day 23, rats were sacrificed to take the spleen for flow cytometry analysis. As shown in Figure 3A,B, the percentages and numbers of CD41 helper T cells in day 15 EAN spleen were greatly reduced after curcumin treatment, suggesting that curcumin may inhibit lymphocyte proliferation in vivo. Furthermore, the mixed lymphocyte proliferation assay was applied in vitro to determine the effects of curcumin on T-cell proliferation. Compared with the DMSO control group, curcumin treatment significantly suppressed ConA-induced spleen PBMC proliferation in a dose-dependent way (Fig. 3C).
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Fig. 1. Curcumin treatment attenuates the severity of neurologic disease in EAN. Rats (n 5 6) were injected intraperitoneally once daily with 50 mg/kg (low-dosage) or 100 mg/kg (high-dosage) curcumin dissolved in DMSO or vehicle from day 0 to day 25 (preventive treatment) or day 7 to day 25 (therapeutic treatment). All rats were monitored daily for body weight and neurological signs of EAN. A: High-dosage curcumin treatment decreased neurologic severity of EAN and delayed EAN onset in comparison with the DMSO control
group. B: Low-dosage curcumin failed to attenuate EAN neurologic signs compared with the DMSO control group. C: High-dosage of curcumin treatment educed body weight loss in comparison to the DMSO control group. D: Low-dosage curcumin treatment failed to reduce EAN body weight loss compared with the DMSO control group. *P < 0.05 compared with DMSO control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
Curcumin Treatment Altered T Helper Cell Differentiation in EAN One of the most important immune cells in EAN is the T lymphocyte. The polarization of T lymphocytes following autoantigen stimulation is a central process defining the nature and severity of autoimmune disorders. EAN rats were preventively treated with high-dosage curcumin or vehicle as described previously and were sacrificed on day 15. The inguinal lymph node and spleen were taken for real-time PCR or flow cytometric analysis. As shown in Figure 4A, in day 15 EAN lymph nodes, curcumin treatment significantly reduced the mRNA level of T-bet and ROR-gt, the master transcription factors for Th1 and Th17 cells, respectively, but the expression of Foxp-3, the master transcription factor for regulatory T cells, was not significantly changed. Correspondingly, expression of IL-12p35 and IL-6, which favors Th1 and Th17 differentiation, was reduced after curcumin treatment, but the expression of IL-4, IL-10,
and TGF-b, implying the Th2 and Treg differentiation, was not changed (Fig. 4A). In addition, flow cytometric analysis was applied to detect the percentage change of Th1, Th2, and Treg cells in curcumin-treated EAN spleen. As shown in Figure 4B, the percentage of CD41IFNg1 Th1 cell was reduced after curcumin treatment compared with DMSO control group on day 15. However, percentage of Foxp31CD41 Treg cell (Fig. 4C) and IL-41CD41 Th2 cell (Fig. 4D) was not significantly influenced by curcumin treatment. DISCUSSION EAN is a classic animal model for inflammatory demyelinating polyneuropathies and is useful in investigating new therapeutic approaches (Hahn, 1996). Here we have studied the therapeutic effects of curcumin in EAN rats. Our findings demonstrate that curcumin greatly delayed the onset of EAN neurological signs and reduced paraparesis of EAN rats. Furthermore, curcumin treatment Journal of Neuroscience Research
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Fig. 2. Curcumin treatment reduced inflammatory cell infiltration and altered inflammatory cytokine expression in EAN sciatic nerves. Curcumin (100 mg/kg/day) or vehicle was preventively or therapeutically given to EAN rats from day 0 or day 7, and rats were sacrificed to take sciatic nerves for HE staining or RT-PCR analysis. A–D: HE staining was applied to show inflammatory infiltration of day 15 in EAN sciatic nerves. Representative microimages showed that inflammatory cell infiltration was significantly suppressed by therapeutic and
preventive curcumin treatment compared with DMSO control group (n 5 3). E,F: Sciatic nerves from day 15 and day 23 preventively curcumin-treated EAN rats were taken for RT-PCR analysis (n 5 3). The mRNA levels of IFN-g, TNF-a, IL-1b, and IL-17 levels were significantly reduced by curcumin treatment, but mRNA levels of IL4, IL-10, and TGF-b were not significantly changed. *P < 0.05 compared with DMSO control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 3. Curcumin suppressed lymphocyte proliferation in vitro and in vivo. Curcumin (100 mg/kg/day) was given to EAN rats from day 0 to day 25. A: Percentage of CD41 Th cells in EAN spleen was detected by flow cytometric analysis (n 5 3). Curcumin treatment obviously reduced the percentage of CD41 Th cells in EAN spleen on day 15. B: Cell number of CD41 Th cells in EAN spleen was cal-
culated. Curcumin treatment had reduced CD41 Th cell numbers in EAN spleen by day 15. C: In the lymphocyte proliferation assay, spleen PBMCs were stimulated by ConA, and, after a total of 72 hr culture, the cell proliferation of spleenocytes was significantly inhibited by curcumin. *P < 0.05 compared with vehicle control.
suppressed inflammatory cell infiltration and expression of proinflammatory cytokines in EAN peripheral nerves. In lymph nodes, curcumin reduced transcription factors of Th1 and Th17 cells without increasing those of Th2 or Treg cells. In EAN spleen, curcumin reduced the percentage of IFN-g1CD41 Th1 cells, whereas the percentages of Foxp31CD41 Treg cells and IL-41CD41 Th2 cells were not changed. In this investigation, curcumin treatment significantly improved EAN outcome and suppressed accu-
mulation of proinflammatory cytokines in peripheral nerves of EAN. In peripheral nerves of EAN rats, cytokines are produced and released by many cell types and regulate inflammation and immunity (Zhu et al., 1997). Proinflammatory cytokines such as TNF-a (Bao et al., 2003; Lu et al., 2007; Mao et al., 2010), IFN-g (Zhang et al., 2012), IL-1b (Skundric et al., 2001), and IL-17 (Pelidou et al., 2000) have disease-promoting roles in EAN (Lu and Zhu, 2011). IFN-g augments both inflammation and subsequent immune responses
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Fig. 4. Curcumin treatment altered Th cell differentiation in EAN lymph nodes and spleen. Curcumin (100 mg/kg/day) was preventively given to EAN rats from day 0 to day 25, and on day 15 rats were sacrificed to take inguinal lymph nodes for real-time PCR analysis (n 5 3) and flow cytometric analysis (n 5 3). A: In day 15 EAN inguinal lymph nodes, curcumin treatment had significantly decreased mRNA levels of T-bet, IL-12p35, RORgt, and IL-6 compared with the DMSO control group, but levels of IL-4, GATA-3, and Foxp-3 were not significantly changed. *P < 0.05 compared with DMSO control. B: Curcumin
treatment had reduced the percentage of CD41IFN-g1 Th1 cell compared with DMSO control in day 15 EAN spleen. C: Percentage of CD41IL-41 Th2 cell had not changed after curcumin treatment compared with DMSO control group on day 15. D: Percentage of CD41Foxp31 regulatory T cells in EAN spleen had not changed after curcumin treatment on day 15 compared with DMSO control group. *P < 0.05 compared with their DMSO control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.] Journal of Neuroscience Research
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in EAN by activation of macrophages (Sobue et al., 1982) to release oxygen radicals, promoting T-cell and macrophage homing to the PNS. IL-1 is considered to participate in the initiation of the autoimmune response in EAN. IL-17 is produced by Th17 cells and stimulates production of IL-6, nitric oxide and prostaglandin E2 to amplify local inflammation, mediates chemotaxis of neutrophils and monocytes to sites of inflammation and augments the induction of costimulatory molecules to support T cell activation (Pelidou et al., 2000; Zhang et al., 2009b). Curcumin attenuated accumulation of inflammatory cells, and expression of inflammation-related molecules including IFN-g, TNFa, IL-1b, and IL-17 in PNS could reduce local inflammation to contribute EAN outcome. Helper T cell differentiation, which can be differentiated from na€ıve T cells by different cytokines and by the induction of independent gene programs, is the central process defining the nature of the developing immune responses (Luckheeram et al., 2012). Th1 cells produce large quantities of IFN-g, TNF-a, and IL-1b, whereas Th2 cells produce IL-4, IL-10, and TGF-b. Th1 cells elicit delayed-type hypersensitivity responses (Taylor and Pollard, 2001), activate macrophages, and are highly effective in clearing intracellular pathogens or causing tissue damage (Spies et al., 1995). Th2 cells, in contrast, are immune-suppressive in EAN, and this suppression of EAN is associated with altered Th1/Th2 balance (decreased Th1/Th2 ratio; Kiefer et al., 1993; Bai et al., 1997; Gillen et al., 1998; Zou et al., 2002). Th17 are a distinct T helper cell subset, producing mainly IL-17, and are also key players in autoimmune diseases; recently, it has been shown to participate in the progression of EAN (Rautajoki et al., 2008). Furthermore, regulatory T cells suppress the activation of the immune system and thereby prevent excessive inflammation and autoimmunity of EAN. We observed that curcumin reduced IFN-g and IL-17 mRNA levels in sciatic nerves of EAN rats (Andre et al., 2009). In EAN spleen, flow cytometric analysis showed that IFN-g1CD41 Th1 cell was decreased after curcumin treatment, whereas Foxp31CD41 regulatory T helper cells and IL-41CD41 Th2 cells were not significantly changed, indicating that curcumin suppressed the autoimmune response in EAN by inhibiting Th1 and Th17 polarization but not through increasing Th2 or Treg cell polarization. In summary, we have investigated the effects of curcumin in EAN, an animal model of human GBS. Our data show that curcumin greatly reduced inflammation accumulation and attenuated paraparesis in EAN. Curcumin exerts the inflammation-inhibitory role by suppressing lymphocyte proliferation and altering CD41 T helper cell differentiation. Therefore, our data demonstrate that curcumin could effectively suppress EAN, suggesting its potential role in treatment of inflammatory neuropathies. ACKNOWLEDGMENTS The authors have no financial conflicts of interest. Journal of Neuroscience Research
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Srimal RC, Dhawan BN. 1973. Pharmacology of diferuloyl methane (curcumin), a non-steroidal anti-inflammatory agent. J Pharm Pharmacol 25:447–452. Taylor JM, Pollard JD. 2001. Dominance of autoreactive T cell-mediated delayed-type hypersensitivity or antibody-mediated demyelination results in distinct forms of experimental autoimmune neuritis in the Lewis rat. J Neuropathol Exp Neurol 60:637–646. Wilson CB, Rowell E, Sekimata M. 2009. Epigenetic control of Thelper-cell differentiation. Nat Rev Immunol 9:91–105. Winer JB. 2001. Guillain Barre syndrome. Mol Pathol 54:381–385. Zhang H-L, Azimullah S, Zheng X-Y, Wang X-K, Amir N, MensahBrown EP, Al Shamsi M, Shahin A, Press R, Zhu J, Adem A. 2012. IFN-gamma deficiency exacerbates experimental autoimmune neuritis in mice despite a mitigated systemic Th1 immune response. J Neuroimmunol 246:18–26. Zhang Z, Zhang Z-Y, Fauser U, Schluesener HJ. 2009a. Distribution of Foxp31 T-regulatory cells in experimental autoimmune neuritis rats. Exp Neurol 216:75–82. Zhang ZR, Zhang ZY, Schluesener HJ. 2009b. Compound A, a plant origin ligand of glucocorticoid receptors, increases regulatory T cells and M2 macrophages to attenuate experimental autoimmune neuritis with reduced side effects. J Immunol 183:3081–3091. Zhang ZY, Zhang Z, Schluesener HJ. 2009c. FTY720 attenuates lesional interleukin-171 cell accumulation in rat experimental autoimmune neuritis. Neuropathol Appl Neurobiol 35:487–495. Zhu J, Bai XF, Mix E, Link H. 1997. Cytokine dichotomy in peripheral nervous system influences the outcome of experimental allergic neuritis: dynamics of mRNA expression for IL-1 beta, IL-6, IL-10, IL-12, TNF-alpha, TNF-beta, and cytolysin. Clin Immunol Immunopathol 84:85–94. Zhu W, Zhang K, Mix E, Wang X, Adem A, Zhu J. 2011. Differential susceptibility to experimental autoimmune neuritis in Lewis rat strains is associated with T-cell immunity to myelin antigens. J Neurosci Res 89: 448–456. Zhu Y, Bao L, Zhu S, Chen Z, van der Meide P, Nennesmo I, Winblad B, Ljunggren H-G, Zhu J. 2002. CD4 and CD8 T cells, but not B cells, are critical to the control of murine experimental autoimmune neuritis. Exp Neurol 177:314–320. Zou LP, Abbas N, Volkmann I, Nennesmo I, Levi M, Wahren B, Winblad B, Hedlund G, Zhu J. 2002. Suppression of experimental autoimmune neuritis by ABR-215062 is associated with altered Th1/Th2 balance and inhibited migration of inflammatory cells into the peripheral nerve tissue. Neuropharmacology 42:731– 739.
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Curcumin Ameliorates Rat Experimental Autoimmune Neuritis Fuyu Han, Bangwei Luo, Rongchen Shi, Changhao Han, Zhonghao Zhang, Jian Xiong, Man Jiang, and Zhiren Zhang* Institute of Immunology, Third Military Medical University of PLA, 30 Gaotanyan Mainstreet, Chongqing, People’s Republic of China
Experimental autoimmune neuritis (EAN) is a helper T cellmediated autoimmune demyelinating inflammatory disease of the peripheral nervous system that serves as an animal model for human Guillain-Barre syndrome. Curcumin, a naturally occurring polyphenolic phytochemical isolated from the medicinal plant Curcuma longa, has anti-inflammatory activities. Here we investigated the therapeutic effects and potential mechanisms of curcumin in EAN rats. Exogenous curcumin treatment (100 mg/ kg/day) significantly delayed the onset of EAN neurological signs, ameliorated EAN neurological severity, and reduced body weight loss of EAN rats. In EAN sciatic nerves, curcumin treatment suppressed the inflammatory cell accumulation and the expression of interferon (IFN)g, tumor necrosis factor-a, interleukin (IL)-1b, and IL-17. Furthermore, curcumin treatment significantly decreased the percentage of CD41 T helper cells in EAN spleen and suppressed concanavalin A-induced lymphocyte proliferation in vitro. In addition, curcumin altered helper T cell differentiation by decreasing IFN-g1CD41 Th1 cells in EAN lymph node and spleen. In summary, our data demonstrate that curcumin could effectively suppress EAN by attenuating inflammation, indicating that curcumin might be a candidate for treatment of autoimmune neuropathies. VC 2014 Wiley Periodicals, Inc. Key words: curcumin; experimental autoimmune neuritis; inflammation; T lymphocytes
Experimental autoimmune neuritis (EAN) is a helper T cell-mediated inflammatory demyelinating disease of the peripheral nervous system (PNS; Zhu et al., 2002) that mirrors many clinical and immunological features of the human acute inflammatory demyelinating polyradiculoneuropathies (AIDP) and has been widely applied as an animal model to investigate therapy and disease mechanisms of AIDP (Hughes and Cornblath, 2005). Patients with inflammatory polyneuropathies receive supportive management or active treatments, such as plasma exchange and intravenous immunoglobulin (Hughes, 2002; Aronovich et al., 2012). Although these treatments are beneficial to some patients (Lin et al., 2007), only about 65% patients with Guillain-Barre syndrome (GBS) respond to plasma exchange or intravenous immunoglobulin (Enders et al., 1997). About 8% of GBS patients die, C 2014 Wiley Periodicals, Inc. V
and up to 20% remain disabled (Kieseier et al., 2004). Even in those who recover well, residual weakness and loss of motor units can usually be detected (Winer, 2001). Therefore, it will be necessary to establish more effective therapeutic strategies for inflammatory polyneuropathies. EAN is characterized by the demyelination of peripheral nerves and the accumulation of autoreactive T cells in the PNS (Kieseier et al., 2004; Zhu et al., 2011). Reactive lymphocytes and macrophages cause a robust local inflammation that leads to demyelination and axon degeneration (Jung et al., 1996; Hughes and Cornblath, 2005). T helper cells are important for the pathogenesis of EAN, because EAN can be induced by adoptive transfer of PNS autoantigen-specific CD41 Th cells (Gold et al., 1999; Kieseier et al., 2004). Th cell polarization following autoantigen stimulation is essential for the determination of type and severity of autoimmune disorders (Wilson et al., 2009). EAN is considered to be mediated mainly by Th1 cells and Th1 cytokines such as interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), and interleukin (IL)-1b (Kieseier et al., 2004). In addition, Th17 cells are believed to contribute to the development of EAN pathogenesis (Zhang et al., 2009c), and the immune suppressive Treg cells help in the inflammation resolution and recovery of EAN (Zhang et al., 2009a). Curcumin is a naturally occurring yellow pigment isolated from the rhizomes of the plant Curcuma longa, which has antioxidant, antitumor, and anti-inflammatory properties (Srimal and Dhawan, 1973). Studies have shown that curcumin inhibits inflammation in animal models of such disorders as atherosclerosis, arthritis, and Contract grant sponsor: National Nature Science Foundation of China; Contract grant number: 31170851; Contract grant sponsor: National Science and Technology Major Projects of New Drugs; Contract grant number: 2012ZX09103301-035; Contract grant sponsor: SRF (for ROCS, SEM). *Correspondence to: Zhiren Zhang, Institute of Immunology, Third Military Medical University of PLA, 30 Gaotanyan Main Street, Chongqing 400038, People’s Republic of China. E-mail: [email protected] Received 18 September 2013; Revised 24 November 2013; Accepted 3 December 2013 Published online 31 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23357
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Alzheimer’s disease (Mukhopadhyay et al., 1982; Araujo and Leon, 2001; Ono et al., 2004). Although the exact mechanisms involved in the anti-inflammatory activity of curcumin remain elusive, recent reports indicate that curcumin can reduce proinflammatory cytokines such as TNF-a, IL-1, IL-8 through deactivation of nuclear factor-jB (NF-jB; Brouet and Ohshima, 1995; Araujo and Leon, 2001; Gaedeke et al., 2004). In addition, increasing data also shows that curcumin can inhibit Th1 differentiation in vitro by inhibiting IL-12 production from macrophages (Kang et al., 1999a,b). These findings suggest a potential role of curcumin in the treatment of autoimmune inflammatory diseases (Bao et al., 2002). Therefore, we investigated the therapeutic effects of curcumin in EAN and its underlying mechanisms. MATERIALS AND METHODS Animals Male Lewis rats (8–10 weeks, 200–220 g; Vital River, Beijing, China) were housed under a 12 hr light-12 hr dark cycle with free access to food and water. All animal procedures were in accordance with a protocol approved by the local Administration District Official Committee. All efforts were made to minimize the number of animals and their suffering. EAN Induction and Treatment Lewis rats were immunized by subcutaneous injection into both hind footpads of 100 ll of an inoculum containing 100 lg synthetic neuritogenic P257–81 peptide (Chinapeptide, Shanghai, China; Cavaletti et al., 2000). The peptide was dissolved in phosphate-buffered saline (PBS; 2 mg/ml) and then emulsified with an equal volume of complete Freund’s adjuvant (Sigma, St. Louis, MO) containing 4 mg/ml Mycobacterium tuberculosis for a final concentration of 2 mg/ml. Rats were weighed daily and inspected for disease severity. Clinical scores were given according to the following scale: 0 5 normal; 1 5 reduced tone of the tail, hanging tail tip; 2 5 limp tail, impaired righting; 3 5 absent righting; 4 5 gait ataxia, abnormal positioning; 5 5 mild paraparesis of the hind limbs; 6 5 moderate paraparesis; 7 5 severe paraparesis or paraplegia of the hind limbs; 8 5 tetraparesis; 9 5 moribund; 10 5 death. For treatment, rats in the test groups were treated (i.p.) with 50 mg/kg or 100 mg/kg curcumin (Sigma) in dimethyl sulfoxide (DMSO) every day from day 0 to day 25 or from day 7 to day 25 (6 rats/group) following the induction of EAN, and the same volume of DMSO was given as a vehicle control. Histological Analysis To assess the degree of PNS inflammation, three rats from each group were sacrificed under ether anesthesia on day 15. The sciatic nerves were removed quickly. A portion of sciatic nerves sample was fixed in 4% neutral buffered formalin and then cut into 3-lm-thick sections, as described previously. Tissues were stained with hematoxylin and eosin to assess inflammatory cell infiltration. Histological changes between different groups were compared by an established semiquantitative method. Briefly, four cross-sections from root and middle levels
of both sides of sciatic nerves of EAN rats were analyzed. All perivascular areas present in cross-sections were evaluated by two observers unaware of treatment, and the degree of pathological alteration was graded semiquantitatively on the following scale: 0 5 normal perivascular area; 1 5 mild cellular infiltrate adjacent to the vessel; 2 5 cellular infiltration plus demyelination in immediate proximity to the vessel; 3 5 cellular infiltration and demyelination throughout the section. Results were given as a mean histological score. Tissue Preparation, RNA Isolation, Reverse Transcription, and Real-Time Quantitative RT-PCR After treatment, EAN rats were perfused intracardially with 4 C PBS under anesthesia, and the sciatic nerves and inguinal lymph nodes were quickly removed and stored in liquid nitrogen until RNA isolation. Total RNA was isolated using Trizol LS reagent (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA with a Quantscript RT kit (Tiangen Biotech, Beijing, China). The resulting cDNA was used to measure quantitatively the expression of genes using SYBR Green qPCR Master Mix according to the manufacturer’s protocol (Tiangen Biotech). Real-time measurements of gene expression were performed with a DNA Engine Opticon 2 Real-Time Cycler PCR detection system (Bio-Rad, Richmond, CA). Primers used to measure gene expression were bactin (sense, CCG TCT TCC CCT CCA TCG T; antisense, ATC GTC CCA GTT GGT TAC AAT GC), T-bet (sense,AAC CAG TAT CCT GTT CCC AGC; antisense, TGT CGC CAC TGG AAG GAT AG-3), ROR-g (sense, CGC ACC AAC CTC TTC TCA CG; antisense, GAC TTC CAT TGC TCC TGC TTT C-3), IL-12p35 (sense, TGA AGA CCA CGG ACG ACA; antisense, TGT GAT TCA GAG ACC GCA TTA G), IL-6 (sense, GCC CTT CAG GAA CAG CTA TG; antisense, CAG AAT TGC CAT TGC ACA AC), GATA-3 (sense, CTC TCC TTT GCT CAC CTT TTC; antisense, AAG AGA TGC GGA CTG GAG TG-3), IL-4 (sense, TGA TGG GTC TCA GCC CCC ACC TTG C; antisense, CTT TCA GTG TTG TGA GCG TGG ACT C), Foxp3 (sense, GCA CAA GTG CTT TGT GCG AGT; antisense, TGT CTG TGG TTG CAG ACG TTG T), IFN-g (sense, AAA GAC AAC CAG GCC ATC AG; antisense, CTT TTC CGC TTC CTT AGG CT), TNF-a (sense, TGA TCG GTC CCA ACA AGG A; antisense, TGC TTG GTG GTT TGC TAC GA), IL-1b (sense, TGC TGA TGT ACC AGT TGG GG; antisense, CTC CAT GAG CTT TGT ACA AG), IL-17 (sense, TGG ACT CTG AGC CGC ATT GA; antisense, GAC GCA TGG CGG ACA ATA GA), TGF-b (sense, TGA ACC AAG GAG ACG GAA TAC AGG; antisense, TAC TGT GTG TCC AGG CTC CAA ATG), and IL-10 (sense, CCT GCT CTT ACT GGC TGG AG; antisense, TCT CCC AGG GAA TTC AAA TG). Flow Cytometric Analysis of Th1, Th2, and Treg Cells in Spleen Th1 cells were identified as CD41IFN-g1 cells, Th2 cells were identified as CD41IL-41 cells, and Treg cells were identified as CD41Foxp31 cells. In short, rat spleens were dissected under sterile conditions and passed through a 40-mm Journal of Neuroscience Research
Curcumin in EAN cell strainer followed by Red Blood Cell Lysis Buffer (Tiangen Biotech). For determination of Th1 and Th2 cells, splenocytes were incubated for 6 hr in the presence of 50 ng/ml PMA (Sigma), 1 mg/ml ionomycin (Sigma), and 1 lg/ml brefeldin A (Sigma) in flat-bottomed 96-well plates in RPMI 1640 medium (Gibco, Grand Island, NY) containing penicillin (100 U/ml), streptomycin (100 U/ml), and 10% fetal calf serum at a density of 106 cells/ml at 37 C and 5% CO2. Thereafter, splenocytes were fixed and permeabilized. FITC-labeled IFN-g antibody (Biolegend, San Diego, CA) and PE-labeled CD4 antibody (Biolegend) were used for staining of CD41IFN-g1 Th1 cells. PE-labeled IL-4 antibody (Biolegend) and FITC-labeled CD4 antibody (Biolegend) were used for staining of CD41IL-41 Th2 cells. AlexaFlour488-labeled Foxp3 antibody (Biolegend) and PE-labeled CD4 antibody were used for staining of CD41Foxp31 Treg cells. For all staining, isotype controls were used. After staining, cells were washed and suspended in PBS and then analyzed with a FACScan (BD Biosciences, Franklin Lakes, NJ). Mononuclear cells were gated by forward and sideward scatter. Mixed Lymphocyte Proliferation Assays Spleen peripheral blood mononuclear cell (PBMC) suspensions were prepared by gently homogenizing the aseptically dissected organs. The cells were washed and resuspended in culture medium. All lymphocyte proliferation assays were performed in triplicate in 96-well flat-bottomed microtiter plates. Lymphocyte activation was measured by seeding 3.5 3 105 lymphocytes per 100 ml medium per well. For some cultures, spleen cells were cultured with 20 lg/ml concanavalin A (ConA) in the presence of 0, 1, 10, 50, and 100 lg/ml curcumin. The plates were incubated at 37 C in a humified atmosphere of 5% CO. After 52 hr of culture, each well was pulsed with 0.25 lCi [3H]methylthymidine and 16 hr later harvested onto fiberglass filters by a 96-well harvester. [3H]thymidine incorporation was measured by liquid scintillation counting. The proliferative response was expressed as the average counts per minute (cpm) of triplicate cultures. Evaluation and Statistical Analysis The unpaired t-test or Mann-Whitney U test was performed to compare differences between curcumin-treated and DMSO control EAN rats (Graph Pad Prism 5.0 for Windows). For all statistical analysis, significance levels were set at P < 0.05.
RESULTS Curcumin Treatment Ameliorated Neurological Signs in EAN Rats were immunized with neuritogenic synthetic P2 peptide and grouped randomly. The preventive groups were treated (i.p.) with high-dosage of curcumin (100 mg/kg) or low-dosage of curcumin (50 mg/kg) once daily from day 0 to day 25. The therapeutic groups were treated (i.p.) in the same manner every day from day 7 to day 25 following the induction of EAN. For DMSO control groups, the same volume of diluted DMSO was Journal of Neuroscience Research
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given. For DMSO groups, the first neurological sign of EAN rats was observed on day 6, the neurological severity of EAN reached a maximum on day 15 (7.33 6 0.33) and had disappeared by day 24. Neither preventive nor therapeutic low-dosage curcumin treatment attenuated EAN severity compared with DMSO control group (Fig. 1B). However, both preventive and therapeutic high-dosage curcumin treatment greatly delayed disease onset (Fig. 1A), decreased neurologic severity (Fig. 1A), and reduced EAN body weight loss (Fig. 1C) during the early period of EAN compared with DMSO control group. Effects of Curcumin Treatment on Inflammatory Cell Accumulation and Expression of Inflammation-Related Molecules in Sciatic Nerves of EAN Rats EAN is characterized by the accumulation of inflammatory cells in the PNS, which leads to the demyelination and axon degeneration of EAN sciatic nerves. To determine the effects of curcumin on inflammatory cell accumulation, sciatic nerves of EAN rats preventively or therapeutically treated with high-dosage curcumin were taken on day 15 for histological analysis. As shown in Figure 2A, profound inflammatory cell infiltration was observed in the sciatic nerves of DMSO control EAN rats. Conversely, both therapeutic and preventive treatment with curcumin obviously decreased sciatic nerve inflammatory cell infiltration (Fig. 2B,C). It is known that inflammatory cytokines such as IL1b, IFN-g, IL-17, and TNF-a have disease-promoting roles in EAN. Therefore, we further analyzed the effects of curcumin on mRNA level of certain inflammatoryrelated molecules in the sciatic nerves of EAN rats. As shown in Figure 2E, mRNA expression of IL-1b, IFN-g, IL-17, and TNF-a in day 15 EAN sciatic nerves was greatly reduced by curcumin. However, increases of IL-4, TGF-b, and IL-10 were not detected, indicating that curcumins help to ameliorate EAN by reducing proinflammatory cytokine. Curcumin Inhibited Spleen Cell Proliferation EAN is an inflammatory autoimmune disease mediated mainly by CD41 helper T cell. To study the influence of curcumin on T-cell proliferation in EAN, we examined the cell number change of CD41 Th cells in EAN spleen. EAN rats were preventively treated with high-dosage curcumin or vehicle control as described; on day 15 and day 23, rats were sacrificed to take the spleen for flow cytometry analysis. As shown in Figure 3A,B, the percentages and numbers of CD41 helper T cells in day 15 EAN spleen were greatly reduced after curcumin treatment, suggesting that curcumin may inhibit lymphocyte proliferation in vivo. Furthermore, the mixed lymphocyte proliferation assay was applied in vitro to determine the effects of curcumin on T-cell proliferation. Compared with the DMSO control group, curcumin treatment significantly suppressed ConA-induced spleen PBMC proliferation in a dose-dependent way (Fig. 3C).
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Fig. 1. Curcumin treatment attenuates the severity of neurologic disease in EAN. Rats (n 5 6) were injected intraperitoneally once daily with 50 mg/kg (low-dosage) or 100 mg/kg (high-dosage) curcumin dissolved in DMSO or vehicle from day 0 to day 25 (preventive treatment) or day 7 to day 25 (therapeutic treatment). All rats were monitored daily for body weight and neurological signs of EAN. A: High-dosage curcumin treatment decreased neurologic severity of EAN and delayed EAN onset in comparison with the DMSO control
group. B: Low-dosage curcumin failed to attenuate EAN neurologic signs compared with the DMSO control group. C: High-dosage of curcumin treatment educed body weight loss in comparison to the DMSO control group. D: Low-dosage curcumin treatment failed to reduce EAN body weight loss compared with the DMSO control group. *P < 0.05 compared with DMSO control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
Curcumin Treatment Altered T Helper Cell Differentiation in EAN One of the most important immune cells in EAN is the T lymphocyte. The polarization of T lymphocytes following autoantigen stimulation is a central process defining the nature and severity of autoimmune disorders. EAN rats were preventively treated with high-dosage curcumin or vehicle as described previously and were sacrificed on day 15. The inguinal lymph node and spleen were taken for real-time PCR or flow cytometric analysis. As shown in Figure 4A, in day 15 EAN lymph nodes, curcumin treatment significantly reduced the mRNA level of T-bet and ROR-gt, the master transcription factors for Th1 and Th17 cells, respectively, but the expression of Foxp-3, the master transcription factor for regulatory T cells, was not significantly changed. Correspondingly, expression of IL-12p35 and IL-6, which favors Th1 and Th17 differentiation, was reduced after curcumin treatment, but the expression of IL-4, IL-10,
and TGF-b, implying the Th2 and Treg differentiation, was not changed (Fig. 4A). In addition, flow cytometric analysis was applied to detect the percentage change of Th1, Th2, and Treg cells in curcumin-treated EAN spleen. As shown in Figure 4B, the percentage of CD41IFNg1 Th1 cell was reduced after curcumin treatment compared with DMSO control group on day 15. However, percentage of Foxp31CD41 Treg cell (Fig. 4C) and IL-41CD41 Th2 cell (Fig. 4D) was not significantly influenced by curcumin treatment. DISCUSSION EAN is a classic animal model for inflammatory demyelinating polyneuropathies and is useful in investigating new therapeutic approaches (Hahn, 1996). Here we have studied the therapeutic effects of curcumin in EAN rats. Our findings demonstrate that curcumin greatly delayed the onset of EAN neurological signs and reduced paraparesis of EAN rats. Furthermore, curcumin treatment Journal of Neuroscience Research
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Fig. 2. Curcumin treatment reduced inflammatory cell infiltration and altered inflammatory cytokine expression in EAN sciatic nerves. Curcumin (100 mg/kg/day) or vehicle was preventively or therapeutically given to EAN rats from day 0 or day 7, and rats were sacrificed to take sciatic nerves for HE staining or RT-PCR analysis. A–D: HE staining was applied to show inflammatory infiltration of day 15 in EAN sciatic nerves. Representative microimages showed that inflammatory cell infiltration was significantly suppressed by therapeutic and
preventive curcumin treatment compared with DMSO control group (n 5 3). E,F: Sciatic nerves from day 15 and day 23 preventively curcumin-treated EAN rats were taken for RT-PCR analysis (n 5 3). The mRNA levels of IFN-g, TNF-a, IL-1b, and IL-17 levels were significantly reduced by curcumin treatment, but mRNA levels of IL4, IL-10, and TGF-b were not significantly changed. *P < 0.05 compared with DMSO control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 3. Curcumin suppressed lymphocyte proliferation in vitro and in vivo. Curcumin (100 mg/kg/day) was given to EAN rats from day 0 to day 25. A: Percentage of CD41 Th cells in EAN spleen was detected by flow cytometric analysis (n 5 3). Curcumin treatment obviously reduced the percentage of CD41 Th cells in EAN spleen on day 15. B: Cell number of CD41 Th cells in EAN spleen was cal-
culated. Curcumin treatment had reduced CD41 Th cell numbers in EAN spleen by day 15. C: In the lymphocyte proliferation assay, spleen PBMCs were stimulated by ConA, and, after a total of 72 hr culture, the cell proliferation of spleenocytes was significantly inhibited by curcumin. *P < 0.05 compared with vehicle control.
suppressed inflammatory cell infiltration and expression of proinflammatory cytokines in EAN peripheral nerves. In lymph nodes, curcumin reduced transcription factors of Th1 and Th17 cells without increasing those of Th2 or Treg cells. In EAN spleen, curcumin reduced the percentage of IFN-g1CD41 Th1 cells, whereas the percentages of Foxp31CD41 Treg cells and IL-41CD41 Th2 cells were not changed. In this investigation, curcumin treatment significantly improved EAN outcome and suppressed accu-
mulation of proinflammatory cytokines in peripheral nerves of EAN. In peripheral nerves of EAN rats, cytokines are produced and released by many cell types and regulate inflammation and immunity (Zhu et al., 1997). Proinflammatory cytokines such as TNF-a (Bao et al., 2003; Lu et al., 2007; Mao et al., 2010), IFN-g (Zhang et al., 2012), IL-1b (Skundric et al., 2001), and IL-17 (Pelidou et al., 2000) have disease-promoting roles in EAN (Lu and Zhu, 2011). IFN-g augments both inflammation and subsequent immune responses
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Fig. 4. Curcumin treatment altered Th cell differentiation in EAN lymph nodes and spleen. Curcumin (100 mg/kg/day) was preventively given to EAN rats from day 0 to day 25, and on day 15 rats were sacrificed to take inguinal lymph nodes for real-time PCR analysis (n 5 3) and flow cytometric analysis (n 5 3). A: In day 15 EAN inguinal lymph nodes, curcumin treatment had significantly decreased mRNA levels of T-bet, IL-12p35, RORgt, and IL-6 compared with the DMSO control group, but levels of IL-4, GATA-3, and Foxp-3 were not significantly changed. *P < 0.05 compared with DMSO control. B: Curcumin
treatment had reduced the percentage of CD41IFN-g1 Th1 cell compared with DMSO control in day 15 EAN spleen. C: Percentage of CD41IL-41 Th2 cell had not changed after curcumin treatment compared with DMSO control group on day 15. D: Percentage of CD41Foxp31 regulatory T cells in EAN spleen had not changed after curcumin treatment on day 15 compared with DMSO control group. *P < 0.05 compared with their DMSO control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.] Journal of Neuroscience Research
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in EAN by activation of macrophages (Sobue et al., 1982) to release oxygen radicals, promoting T-cell and macrophage homing to the PNS. IL-1 is considered to participate in the initiation of the autoimmune response in EAN. IL-17 is produced by Th17 cells and stimulates production of IL-6, nitric oxide and prostaglandin E2 to amplify local inflammation, mediates chemotaxis of neutrophils and monocytes to sites of inflammation and augments the induction of costimulatory molecules to support T cell activation (Pelidou et al., 2000; Zhang et al., 2009b). Curcumin attenuated accumulation of inflammatory cells, and expression of inflammation-related molecules including IFN-g, TNFa, IL-1b, and IL-17 in PNS could reduce local inflammation to contribute EAN outcome. Helper T cell differentiation, which can be differentiated from na€ıve T cells by different cytokines and by the induction of independent gene programs, is the central process defining the nature of the developing immune responses (Luckheeram et al., 2012). Th1 cells produce large quantities of IFN-g, TNF-a, and IL-1b, whereas Th2 cells produce IL-4, IL-10, and TGF-b. Th1 cells elicit delayed-type hypersensitivity responses (Taylor and Pollard, 2001), activate macrophages, and are highly effective in clearing intracellular pathogens or causing tissue damage (Spies et al., 1995). Th2 cells, in contrast, are immune-suppressive in EAN, and this suppression of EAN is associated with altered Th1/Th2 balance (decreased Th1/Th2 ratio; Kiefer et al., 1993; Bai et al., 1997; Gillen et al., 1998; Zou et al., 2002). Th17 are a distinct T helper cell subset, producing mainly IL-17, and are also key players in autoimmune diseases; recently, it has been shown to participate in the progression of EAN (Rautajoki et al., 2008). Furthermore, regulatory T cells suppress the activation of the immune system and thereby prevent excessive inflammation and autoimmunity of EAN. We observed that curcumin reduced IFN-g and IL-17 mRNA levels in sciatic nerves of EAN rats (Andre et al., 2009). In EAN spleen, flow cytometric analysis showed that IFN-g1CD41 Th1 cell was decreased after curcumin treatment, whereas Foxp31CD41 regulatory T helper cells and IL-41CD41 Th2 cells were not significantly changed, indicating that curcumin suppressed the autoimmune response in EAN by inhibiting Th1 and Th17 polarization but not through increasing Th2 or Treg cell polarization. In summary, we have investigated the effects of curcumin in EAN, an animal model of human GBS. Our data show that curcumin greatly reduced inflammation accumulation and attenuated paraparesis in EAN. Curcumin exerts the inflammation-inhibitory role by suppressing lymphocyte proliferation and altering CD41 T helper cell differentiation. Therefore, our data demonstrate that curcumin could effectively suppress EAN, suggesting its potential role in treatment of inflammatory neuropathies. ACKNOWLEDGMENTS The authors have no financial conflicts of interest. Journal of Neuroscience Research
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