| Paper ⅠCurcumin ameliorates experimental autoimmune myasthenia gravis by diverse immune cellsBackground and objectives:Experimental autoimmune myasthenia gravis (EAMG) is a reliable animal model of human myasthenia gravis (MG), which can be induced in Lewis rats by immunization with torpedo acetylcholine receptor (TAChR) or a synthetic peptide corresponding to region 97-116 of the rat AChR a subunit (R97-116 peptide). MG/EAMG is an antibody-mediated, T-cell dependent autoimmune disease. The immunological mechanism underlying MG has advanced through immunology research in EAMG models. Both cellular and humoral immunity are thought to contribute to the pathogenesis of MG/EAMG. In MG/EAMG, auto-antibodies against the nicotinic acetylcholine receptor (AChR) attack AChR in the post-synaptic membrane of the neuromuscular junction, resulting in muscle fatigability and weakness.Curcumin, the active ingredient of the spice turmeric, is a polyphenolic compound derived from the rhizomes of the plant Curcuma longa and it has a long history as an herbal remedy. The medicinal value of curcumin has been well recognized because of its antioxidant, anti-tumor and anti-inflammatory activities. Previously, studies have demonstrated that curcumin plays immunomodulatory roles in many autoimmune and inflammatory diseases (such as atherosclerosis, arthritis, experimental autoimmune neuritis (EAN), and experimental autoimmune encephalomyelitis (EAE)) and is used as a means in the treatment of autoimmune diseases. Although the exact mechanisms involved in the anti-inflammatory activity of curcumin remain obscure, a large number of experiments reveals that the effects of curcumin on autoimmune diseases are associated with its ability to inhibit the production of pro-inflammatory cytokines, such as IL-1, IL-6, IL-12, tumor necrosis factor-a(TNF-a), and Interferon-y (IFN-y), which are associated with JAK-STAT, AP-1, and NF-κB signaling pathways in immune cells. Recent reports also indicate that curcumin treatment can attenuate autoimmune diseases by inhibiting Thl differentiation down-regulating Th17 production and shifting immune responses from Th1 to Th2 type responses. However, the immunomodulatory effects of curcumin in EAMG have not been reported, so in the present study, we investigated the effects of curcumin on the pathogenesis of EAMG.Objective:The aim of our present study was to explore the effect of curcumin on EAMG and further elucidate the underlying mechanisms.Methods:1.EAMG induction and assessmentFemale Lewis rats (6-8 weeks,160-180 g) was induced by subcutaneous injection into both hind foot pads with the R.97-116 peptide (200ul/rat) emulsified in incomplete Freund’s adjuvant combined with 1 mg of M. tuberculosis (day 0). A boost immunization was performed on day 11 with the same dose of R97-116 peptide in IFA with 1 mg M. tuberculosis. Rats were weighed and examined before immunization and thereafter every second day until day 45 post-immunization (p.i.).2.EAMG treatmentEAMG rats were randomly divided into three groups (n= 6 in each group):control group, low-dose group and high-dose group. For treatment with curcumin (Sigma), EAMG rats were intraperitoneally injected (i.p.) with 50 mg/kg (low-dose group) or 100mg/kg curcumin (high-dose group) dissolved in (Polyethylene glycol PEG) every other day from day 11 to day 45 p.i.. Rats in the control group received the same volume of PEG. The severity of clinical symptoms and body weight of EAMG was recorded from day 0 to day 45 p.i.3. Lymph node MNC preparationWe ultimately killed rats of every group and removed inguinal lymph nodes under aseptic conditions on day 45 p.i.. Then we grinded the organs through cell strainers in serum-free medium to obtain MNC suspensions and resuspended cells to 2 x 106/ml for the following experiments.4.Detection of intracellular cytokines by FACSSingle-cell suspensions of mononuclear cells (MNC) were incubated with cell stimulation cocktail (plus protein transport inhibitors) for 4 h, then collected and labeled as CD4+IFN-γ+(Thl), CD4+IL-4+(Th2), CD4+IL-17+(Th17) and CD4+Foxp3+(Treg). Intracellular cytokines IFN-y,TNF-a,IL-10 and 1L-17were also determined by FACS.5.Detection of levels and affinity of anti-R97-116 IgG by ELISASerum was collected on day 43 p.i. to detect anti-R97-116 IgG antibody by ELISA. The relative affinity of anti-R97-116 antibodies was determined by ELISA using thiocyanate elution.6.Detection of NKR-P1+cells by FACSSingle-cell suspensions of MNC were prepared from inguinal lymph nodes of individual rat on day 45 p.i. for FACS analysis. NK cells were defined by staining of anti-CD3, anti-CD161 antibodies, then cells were analyzed by a FACScan.7.Detection of regulatory B cells (Breg) by FACSSingle-cell suspensions of MNC were prepared from inguinal lymph nodes of individual rat on day 45 p.i. for FACS analysis. B10 cells were defined by staining of anti-CD 19 and anti-IL-10 antibodies.8.. Statistical analysisData was analyzed using SPSS 17.0 software (SPSS Inc., Chicago, USA). Results were expressed as mean ± standard deviation (SD). Differences between three groups were tested by single factor analysis of variance (ANOVA) followed by least significant difference (LSD) test as a post hoc test. A level of p< 0.05 was considered to be significant.Results:1. Curcumin treatment ameliorates clinical presentation of EAMGCurcumin, at doses of 50 mg/kg/day and 100 mg/kg/day, inhibited the development of EAMG, compared with control group. Statistically, the differences between control group and high-dose group were statistically significant on day 23,27,29,33,35,37, 39,41,43 and 45. When compared with control group, the clinical scores of EAMG symptoms in low-dose group were statistically significant on day 29,31,43 and 45.2. Curcumin suppresses the expression of CD80, CD86 and MHC class Ⅱ in lymphocytes of EAMGCompared with control group, curcumin treatment at dose of 100 mg/kg significantly decreased the expression of CD80 and MHC class Ⅱ in lymphocytes. The expression of CD86 in both low-and high-dose groups were both significantly reduced compared with that in the control group.3. Effects of curcumin on Thl/Th2/Th17/Treg imbalance and inflammatory cytokinesExamined by flow cytometry, curcumin attenuates EAMG via changed the proportions of Th1 (CD4+IFN-γ+), Treg (CD4+Foxp3+), Th17 (CD4+IL-17A+) and Th2 (CD4+IL-4+). The percentages of IFN-y-positive MNCs were significantly decreased in both low-and high-dose groups compared with control group. Compared with control group, the percentages of IL-10-positive MNCs were significantly increased in both low-dose group and high-dose group. In addition, the percentages of IL-17-positive MNCs and TNF-a-positive MNCs were significantly decreased in low-dose group compared with those in control group.4. Curcumin increases the numbers of NKcells and NKT cells in MNCsThe results showed that the percentage of NK cells was significantly increased in the high-dose group when compared with that in the control group. Compared with control group, the percentage of NKT cells was significantly increased in both low-dose group and high-dose group.5.Curcumin increases the numbers of B10 cells in MNCsThe results showed that the percentage of B10 cells was significantly increased in the high-dose group when compared with that in the control group. The percentage of B10 cells also increased in low-dose group, but there was no significant difference compared with control group.6. Curcumin increase the levels of anti-R97-116 peptide IgGl and decrease the relative affinity indexes of anti-R97-116 peptide IgGThe level of anti-R97-116 peptide IgGl was significantly increased in low-dose group compared to control group. The high-dose group showed an increase of IgGl, but this was not statistically significant. Meanwhile, the levels of IgG and IgG2b were not significantly changed among three groups. Lower affinity indexes of anti-R97-116 peptide IgG in both two groups were observed compared with that in EAMG rats.Conclusions:1. Curcumin ameliorated clinical scores of EAMG.2. Curcumin suppressed the expression of co-stimulatory molecules (CD80 and CD86) and MHC class II in EAMG rats.3. Curcumin down-regulated the levels of pro-inflammatory cytokines (IL-17, IFN-y and TNF-a), up-regulated the levels of anti-inflammatory cytokines IL-10, shifted the balance from Thl/Th17 toward Th2/Treg in EAMG rats.4. Curcumin increased the numbers of NKR-P1+cells (natural killer cell receptor protein 1 positive cells, including NK and NKT cells) in EAMG rats..5. Curcumin promoted the differentiation of B cells into B10 subsets in EAMG rats.6. Curcumin increased the anti-R97-166 peptide IgGl levels and decreased the relative affinity indexes of anti-R97-116 peptide IgG in EAMG rats.paper ⅡAnatabine Ameliorates Experimental Autoimmune NeuritisGuillain-Barre syndrome (GBS) is an acute inflammatory polyradiculoneuropathy in humans. Acute inflammatory demyelinating polyneuropathy (AIDP), is a CD4+T cell-mediated disease, which is characterized by inflammatory demyelination and axon damage in the peripheral nervous system (PNS), the most common form of the Guillain-Barre syndrome (GBS). Experimental autoimmune neuritis (EAN) is an established animal model for AIDP in humans, its clinical symptoms, electrophysiological signs, pathogenesis, neuropathological changes and immunological parameters are similar to human GBS. Previous investigations have demonstrated the anti-inflammatory effects of nicotine, but it cannot be recommended because it is addictive. Anatabine, a minor tobacco alkaloid which is also present in plants of the Solanacea family including green tomatoes, peppers and eggplants. It displays a chemical structural similarity with nicotine, has shown certain anti-inflammatory properties and substantial therapeutically functions in many autoimmune disease. Anatabine can inhibits nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling and readily crosses the blood-brain barrier, suggesting it could represent a suitable agent to treat neuro-inflammatory disorders. More recently, anatabine have showned that it prevents the release of inflammatory cytokines induced by an intraperitoneal injection of lipopolysaccharide in mice by blocking the activation of NFkB and signal transducer and activator of transcription 3 (STAT3). Anatabine has also recently been shown to ameliorate experimental autoimmune thyroiditis by reducing inflammatory genes expression in the thyroid including cytokines. Our present study was designed to determine whether anatabine had beneficial effects on the development of EAN in rat and to precisely determine its underlying mechanism of action in this rat model of Guillain-Barre syndrome. we evaluated the effect of anatabine on EAN and found that anatabine can ameliorated EAN disease severity, which was associated with decreases the numbers of inflammatory cells in the PNS, down-regulation of IL-10 cytokines, up-regulates the number of CD4+Foxp3+ Treg cells. Collectively, our data suggest that anatabine may be effective in the treatment of Guillain-Barre syndrome.Methods:1.Induction and assessment of EANFemale Lewis rats, SPF standard,6-8 weeks old, weighing 150-170g, was induced by immunization in both hind footpads with 200μl inoculums containing 200μl P0 peptide 180-199,2 mg Mycobacterium tuberculosis in 100Oμl saline and 100μl Freund’s incomplete adjuvant.2.Administration of anatabine15 rats were randomly assigned to three groups:control group, low-dose group and high-dose group, there were five rats in each group. Rats in low-and high-dose anatabine treatment groups were treated intragastrically with anatabine dissolve in purified water at doses of 2 mg/kg/day and 20 mg/kg/day from day 5 to 16 p.i. respectively. Control EAN rats received purified water intragastrically of the same volume.3.Histopathological assessmentThe rats were killed on day 16 p.i. just at the fastigium of clinical symptoms of EAN. Sciatic nerve segments were fixed in 10% paraformaldehyde and then interbedded in paraffin. Sciatic nerves (5μm) were stained with hematoxylinandeosin for evaluation of the inflammatory cells by light microscopy. The quantity of inflammatory cells was counted at 400 × magnification. The average results were expressed as cells per mm2 tissue section.4.Preparation of lymph node MNCOn day 16, We ultimately killed rats of every group and removed inguinal lymph nodes under aseptic conditions. Then we grinded the organs through cell strainers in serum-free medium to obtain MNC suspensions and resuspended cells to 2×106/ml for the following experiments.5.Flow cytometric analysis of Treg cellsSingle-cell suspensions of MNC were prepared from inguinal lymph nodes of individual rat on day 16 p.i. for FACS analysis. Treg cells were defined by staining of anti-CD3, anti-foxp3 antibodies, then cells were analyzed by a FACScan.6.Detection of NK cells by FACSSingle-cell suspensions of MNC were prepared from inguinal lymph nodes of individual rat on day 16 p.i. for FACS analysis. NK cells were defined by staining of anti-CD3, anti-CD161 antibodies, then cells were analyzed by a FACScan.7.Detection of cytokines by FACSSingle-cell suspensions of mononuclear cells (MNC) were incubated with cell stimulation cocktail (plus protein transport inhibitors) for 4h, then collected and labeled as intracellular cytokines IFN-γ, IL-10, IL-17 were also determined by FACS.8.Statistical analysisData was analyzed using SPSS17.0 software (SPSS Inc., Chicago, USA). Results were expressed as mean±standard deviation (SD). Differences between three groups were tested by single factor analysis of variance (ANOVA) followed by least significant difference (LSD) test as a post hoc test. A level of p< 0.05 was considered to be significant.Results:1. Anatabine inhibits the development of EANBoth low-dosage anatabine treatment attenuated EAN severity compared with water control group. Compared with control group, differences were statistically significant on day 11,12,13,14,15,16 (P<0.05) in high-dose group; While In low-dose group, differences were statistically significant on day 11,14,15,16 (P<0.05). These data suggested that anatabine can ameliorate disease severity of EAN.2. Anatabine decreases the number of inflammatory cells in the PNSCompared with control group,20 mg/kg anatabine treatedand group and 2mg/kg anatabine treated group Presented fewer inflammatory cells in sciatic nerves. In addition, the rats in high dose group had fewer inflammatory cells than the rats in low dose group.3. Anatabine down-regulates IL-10 and IFN-γIntracellular cytokines of lymph nodes MNC were determined by FACS, anatabine treatment groups significantly reduced the expression of IL-10 compared with control group. The percentagesof IFN-y-positive cells decreased in High-dose group compared with the control group. IL-17-positive cells was decreased in low-dose group as well as in high-dose groupbut, but without statistical significance.4. Anatabine up-regulates the number of CD4+Foxp3+T cellsHigh-dose anatabine-treated groupincreased the percentages of CD4+Foxp3+T cells compared with the control group. The percentages of CD4+Foxp3+T cells increased in high-dose group compared with the control group. The level of CD4+CD25+Foxp3+cells increased in both anatabine treatment groups compared with control group. However, there were no significant differences.5. Anatabine increase the numbers of NK cells in MNC from lymph nodesTo further examine whether anatabine influence NK cells in EAN rats, we analyzed CD3 and CD161a in MNC from lymph nodes. The results showed that the percentage of CD3-CD161a+NK cells increased in high-dose group compared with the control group, NK cells in low-dose group were also increased, but there were no significant statistical differences.Conclusions:Data presented in this study demonstrated that anatbine ameliorated clinical scores of EAN,, down-regulated the levels of pro-inflammatory cytokines IL-10 and IFN-γ, increased the numbers of natural killer (NK) cells. Moreover, increased the numbers of Treg cells. In conclusion, our results showed that both low and high doses of anatabine inhibited EAN development, which provides new experimental evidence that anatabine exerts diverse immune regulatory property and suggests that anatabine might be a potential therapeutic agent for GBS. |
PDF Full Text Request