Journal of Neuroscience Research 92:1217–1226 (2014)
Ginsenoside Rd Ameliorates Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice Dongliang Zhu,1 Mei Liu,1 Yaowu Yang,2 Lili Ma,1 Ying Jiang,1 Linli Zhou,1 Qiling Huang,1 Rongbiao Pi,3 and Xiaohong Chen1* 1
Department of Neurology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China Department of Traditional Chinese Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China 3 Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China 2
Multiple sclerosis (MS) is a common disabling autoimmune disease without an effective treatment in young adults. Ginsenoside Rd, extracted from Panax notoginseng, has multiple pharmacological effects and potential therapeutic applications in diseases of the central nervous system. In this study, we explore the efficacy of ginsenoside Rd in experimental autoimmune encephalomyelitis (EAE), an established model of MS. EAE was induced by myelin oligodendrocyte glycoprotein 35–55amino-acid peptide. Ginsenoside Rd (10–80 mg/kg/day) or vehicle was intraperitoneally administered on the disease onset day, and the therapy persisted throughout the experiments. The dose of 40 mg/kg/day of ginsenoside Rd was selected as optimal. Ginsenoside Rd effectively ameliorated the clinical severity in EAE mice, reduced the permeability of the blood–brain barrier, regulated the secretion of interferon-gamma and interleukin-4, promoted the Th2 shift in vivo (cerebral cortex) and in vitro (splenocytes culture supernatants), and prevented the reduction in expression of brain-derived neurotrophic factor and nerve growth factor in both cerebral cortex and lumbar spinal cord of EAE mice. This study establishes the potency of ginsenoside Rd in inhibiting the clinical course of EAE. These findings suggest that ginsenoside Rd could be a promising agent for amelioration of neuroimmune dysfunction diseases such as MS. VC 2014 Wiley
derangement of cytokine and neurotrophin production were implicated in the neuroimmune dysfunction of MS and experimental autoimmune encephalomyelitis (EAE), an established model of MS (Noseworthy et al., 2000; Frischer et al., 2009; Stadelmann et al., 2011). Current drugs for MS management consist of classical antiinflammatory agents for acute relapses and immunosuppression or immunomodulatory medicines given as maintenance, to prevent relapses and slow disability progression (Tysiak et al., 2009). However, many patients have suboptimal responses to these therapies. Further improvement in MS therapeutics is needed (Virley. 2005). Ginsenoside Rd (dammer-24[25]-ene-3b,12b, 20[S]-triol-[20-O-b-D-glucopyranosyl]-3-O-b-D-glucopyranosyl-[1R2]-b-D-gluco-pyranoside [C48H82O18.3 H2O]; Fig. 1) is a dammarane-type steroid glycoside extracted from Pana notoginseng, a traditional Chinese herbal medicine, which has multiple pharmacological effects (Attele et al., 1999). It is highly lipophilic and capable of crossing biological membranes (Ye et al., 2008).Previous studies demonstrated that ginsenoside Rd possesses anti-inflammatory, antioxidative, antiapoptotic, and neuroprotective properties and has potential therapeutic applications in CNS diseases (Shang et al., 2007; Ye et al., 2009, 2011; Liu et al., 2012a,b). It has been reported that
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Key words: ginsenoside Rd; experimental autoimmune encephalomyelitis; therapy; multiple sclerosis
D. Zhu and M. Liu contributed equally to this work.
Multiple sclerosis (MS) is a widespread autoimmune-mediated neurodegenerative disease with characteristic inflammatory demyelination in the central nervous system (CNS) that causes significant disability in young adults (Frischer et al., 2009; Stadelmann et al., 2011). Although the pathogenesis of MS has not been fully elucidated, the disruption of the blood–brain barrier (BBB), migration of T lymphocytes into the CNS, and
*Correspondence to: Dr. Xiaohong Chen, Department of Neurology, The Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou, Guangdong 510630, China. E-mail: [email protected]
C 2014 Wiley Periodicals, Inc. V
Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: 81071068 (to X.C.).
Received 28 November 2013; Revised 26 February 2014; Accepted 1 April 2014 Published online 2 May 2014 in Wiley Online (wileyonlinelibrary.com). DOI: 10.1002/jnr.23397
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Fig. 1. Structure of ginsenoside Rd.
ginsenoside Rd suppresses lipopolysaccharide-induced nitric oxide formation and tumor necrosis factor-a production through inhibiting nuclear factor-jB (NF-jB) in N9 microglial cells and in rats (Wu et al., 2007; Wang et al., 2012a). It was also reported that Ginsenoside Rd could inhibit Ca21 influx, prevent glutamate-induced excitotoxicity in hippocampal neurons, ameliorate the outcome of focal cerebral ischemia in aged mice through attenuating redox imbalance, and improve scores of modified ranking scale and the National Institutes of Health Stroke Scale in patients with acute ischemic stroke (Ye et al., 2009, 2011; Liu et al., 2012b; Zhang et al., 2012). Recently, ginsenoside Rd was found to attenuate b-amyloid-induced tau phosphorylation by reducing glycogen synthase kinase-3b (GSK-3b) activity, enhance the activity of protein phosphatase 2A, downregulate caspase-3 proteins, and attenuate cognitive dysfunction in a rat model of Alzheimer’s disease (Liu et al., 2012a; Li et al., 2013). However, little is known about the effect of ginsenoside Rd on autoimmune inflammatory diseases of the CNS, such as MS and EAE. In this study, we tested the efficacy of ginsenoside Rd in a chronic EAE model induced by myelin oligodendrocyte glycoprotein 35–55-amino-acid peptide (MOG35–55) in C57 BL/6 mice. We further determined the permeability of the BBB, the alteration of interferong (IFN-g) and interleukin-4 (IL-4) in the cerebral cortex in vivo and the splenocytes culture supernatants in vitro, and the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the cerebral cortex and lumbar spinal cord of EAE mice. Our findings indicate that ginsenoside Rd attenuates the progression of clinical disease in EAE mice, which may be associated with preventing disruption of the BBB and modulating neuroimmune dysfunction. MATERIALS AND METHODS Animals and Regents Six- to eight-week-old female C57 BL/6 mice weighing 16–18 g were obtained from the Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China). Experiments were carried out according to the National Institutes of Health
Guide for the care and use of laboratory animals and were approved by the Bioethics Committee of Sun Yat-Sen University. Ginsenoside Rd with a purity of 98% was obtained from Nanjing Zelang Medical Technology (Nanjing, China). MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by CL BioScientific (Xi’an, China). Amino acid sequences were confirmed by amino acid analysis and mass spectroscopy. The purity of the peptide was greater than 95%. Mycobacterium tuberculosis H37RA was purchased from Difco (Detroit, MI). Pertussis toxin (PTX) was purchased from Alexis (San Diego, CA). Complete Freund’s adjuvant (CFA) and Evan’s blue (EB) were purchased from Sigma-Aldrich (St. Louis, MO). Polyclonal anti-BDNF and anti-NGF were purchased from Abcam (Cambridge, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. The cytokine assay by enzyme-linked immunosorbent assay (ELISA) kits for IFN-g and IL-4 were purchased from RayBiotech (Norcross, GA). Induction and Assessment of EAE The procedure used for the induction of EAE has been described previously (Chen et al., 2009). Briefly, mice received a subcutaneous injection in the flanks with 200 lg MOG35–55 peptide emulsified in CFA containing 500 lg Mycobacterium tuberculosis H37RA. Immediately thereafter and again 48 hr later, the mice received an intraperitoneal injection of 300 ng PTX in 100 ll phosphate-buffered saline (PBS). An additional injection of MOG35–55 peptide in CFA was delivered 7 days later. The animals were examined daily for disability. Clinical scores were defined as follows: 0, no signs; 1, loss of tail tonicity; 2, flaccid tail; 3, ataxia and/or paresis of hindlimbs; 4, complete paralysis of hindlimbs; 5, moribund or death. Dose-Finding Experiments and Treatment of Mice Mice were randomly assigned to three groups: control mice, vehicle-treated EAE mice, and ginsenoside Rd-treated EAE mice (n 5 8). The dose of ginsenoside Rd was chosen on the basis of previous data in vivo (Ye et al., 2011) and our preliminary dose-finding experiment. In our dose-finding experiment, ginsenoside Rd stock solutions were prepared in saline containing 10% 1,3-propanediol (v/v). Ginsenoside Rd was administered by intraperitoneal injections at dosages of 10, 20, 40, and 80 mg/kg/day, respectively (n 5 8). Treatment with ginsenoside Rd was initiated when the first mouse showed neurological symptoms, and medication was administered daily until mice were sacrificed at day 35 postimmunization (p.i.). Vehicle-treated EAE mice were treated with intraperitoneal administration of vehicle only. Each experiment was repeated three times. Histological Evaluation To assess the degree of CNS inflammation and demyelination, mice in the vehicle- and ginsenoside Rd-treated groups were anesthetized and perfused with ice-cold PBS, followed by 4% paraformaldehyde from the left ventricle at day 20 p.i. (n 5 6). Spinal cords were removed. Tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin for revealing inflammatory infiltration. Solochrome Journal of Neuroscience Research
Ginsenoside Rd Ameliorates EAE cyanin technique was used for myelin staining. Histopathological examination was performed in a blinded fashion. The scale used to evaluate for inflammation (O’Neill et al., 2006) was: 0, no inflammatory cells; 1, a few scattered inflammatory cells; 2, organisation of inflammatory infiltrates around blood vessels; and 3, extensive perivascular cuffing with extension into adjacent parenchyma or parenchymal infiltration without obvious cuffing. Demyelination in the spinal cords was scored as previously described by Kuerten et al. (2007): 1, traces of subpial demyelination; 2, marked subpial and perivascular demyelination; 3, confluent perivascular or subpial demyelination; 4, massive perivascular and subpial demyelination involving one half of the spinal cord with presence of cellular infiltrates in the CNS parenchyma; 5, extensive perivascular and subpial demyelination involving the whole cord section with presence of cellular infiltrates in the CNS parenchyma. Evaluation of BBB Disruption The integrity of the BBB was detected by quantitative measurement for EB content at day 20 p.i. as described previously (Jiang et al., 2013). Briefly, sterilized 2% EB solution was administered intravenously at a dosage of 4 ml/kg per animal (n 5 6). Thirty minutes after injection, mice were perfused with saline to remove intravascular EB dye. Brains were rapidly removed and each sample was weighed and then homogenized with 2.5 ml PBS and mixed with 2.5 ml 60% trichloroacetic acid to precipitate protein. The samples were centrifuged for 30 min at 1,000g, and the supernatants were measured at 610 nm for absorbance of EB by using a spectrophotometer (Genesis 10 uv; Thermo Electron, Madison, WI). The content of EB was expressed as micrograms per gram of brain tissue. ELISA Assay Spleens of differently treated mice were aseptically harvested at day 20 p.i. Splenocytes (5 3 105 cells/well) from each group were incubated in 96-well flat-bottom plates in RPMI 1640 supplemented with 10% fetal calf serum, with MOG35–55 (15 lg/ml) used for the immunization. Culture supernatants were collected at 48 hr and stored at 280 C for assaying IFN-g and IL-4. Cerebral cortex was quickly dissected on a cold plate and frozen immediately in liquid nitrogen. All tissues were stored at 280 C until assay. Cerebral cortex was homogenized in 1 ml of ice-cold Tris buffer (pH 7.2, 4 C) containing 50 mM Tris, 1 mM EDTA, 6 mM MgCl2, and 5% (w/v) protease inhibitor cocktail. After homogenization, samples were sonicated for 10 sec by using an ultrasonic processor at a setting of 5, and then centrifuged at 20,800g for 20 min at 4 C (Jiang et al., 2013). Afterward, supernatants were collected for assaying IFN-g and IL-4. Total protein content was measured in each sample using a commercially available BCA protein assay kit (Key Gen Biotech, Nanjing, China). The IFN-g and IL-4 levels in the culture supernatants and cerebral cortex supernatants were measured according to the ELISA kit manual. IFN-g and IL-4 of cerebral cortex supernatants are expressed as picograms per milligram of total protein, whereas culture supernatant samples are expressed as picograms per milliliter of culture supernatant. The detection Journal of Neuroscience Research
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limits are 0.6–200 pg/ml for IL-4 and 5–2,000 pg/ml for IFNg in the cell supernatants and 1–200 pg/ml for IL-4 and 10– 2000 pg/ml for IFN-g in the brain tissues. Western Blot Analysis To investigate the expressions of BDNF and NGF in the cerebral cortex and lumbar spinal cord of differently treated mice, we performed Western blot analysis as described previously (Jiang et al., 2013). Samples from differently treated mice were loaded on 10% gradient sodium dodecyl sulfatepolyacrylamide gels (20 mg protein per lane). Proteins were transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked by 5% nonfat milk. Afterward, the membranes were incubated with polyclonal antiBDNF (1 lg/ml) and anti-NGF (1 lg/ml) overnight. After washing of the membranes three times with TBST buffer, the membranes were incubated with anti-mouse-HRP and goat anti-rabbit-HRP for 30 min. The experiment was repeated in triplicate and used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. The bands were quantified in Quantity One image analysis software (Bio-Rad). Statistical Analysis Data were expressed as mean 6 SEM and analyzed in SPSS 13.0 software (IBM, Armonk, NY). Quantitative data were processed using the Mann-Whitney U test, one-way analysis of variance (ANOVA), rank of one-way ANOVA, pairwise comparison among groups with the least significant difference (LSD), or Dunnett’s T3 test. P < 0.05 was considered statistically significant.
RESULTS Ginsenoside Rd Treatment Ameliorated Clinical Severity of EAE Mice In our experiment, “disease onset day” indicates the day when an individual mouse showed the first symptom. The mean clinical score represented the average score of each mouse during the experiment. The overall disease burden of each mouse was represented as a cumulative score, which was the sum of the disability scores obtained daily over the course of the 35-day experiment. First, we investigated the efficacy of ginsenoside Rd for the neurological behaviors of EAE mice. The therapy was started at disease onset day and persisted throughout the experiment. We found the mean clinical scores of 20, 40, and 80 mg/kg/day ginsenoside Rd-treated EAE mice were significantly reduced compared with both vehicle-treated mice (P < 0.05 for 20 and P < 0.01 for 40 and 80 mg/kg/ day ginsenoside Rd-treated EAE mice) and 10 mg/kg/ day ginsenoside Rd-treated EAE mice (P < 0.05 for 20 and P < 0.01 for 40 and 80 mg/kg/day ginsenoside Rdtreated EAE mice; Fig. 2B, Table I). Cumulative scores of 20, 40, and 80 mg/kg/day ginsenoside Rd-treated EAE mice were significantly reduced compared with both vehicle-treated (P < 0.01 for all) and 10 mg/kg/day ginsenoside Rd-treated EAE mice (P < 0.05 for 20 and P < 0.01 for 40 and 80 mg/kg/day ginsenoside Rd-
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Fig. 2. Ginsenoside Rd treatment ameliorated clinical severity of experimental autoimmune encephalomyelitis (EAE) mice. Daily clinical scores (A), mean clinical scores (B), and cumulative scores (C) of the vehicle- and ginsenoside Rd-treated (10–80 mg/kg/day) EAE mice. EAE was induced in female C57 BL/6 mice with myelin oligodendrocyte glycoprotein 35–55-amino-acid peptide, and treatment with ginsenoside Rd was performed as described in Materials and Methods. Each group had eight animals. Drug treatments were started from the day of disease onset. Doses of 20, 40, and 80 mg/kg/day
significantly ameliorated the clinical score of EAE, and 40 mg/kg/day was selected as the optimal dose (see also Table I). Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. vehicle-treated EAE mice; #P < 0.05, ##P < 0.01 vs. 10 mg/kg/day ginsenoside Rdtreated EAE mice; &P < 0.05 vs. 20 mg/kg/day ginsenoside Rdtreated EAE mice by rank of one-way ANOVA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
treated EAE mice; Fig. 2C, Table I). There were no significant differences in the mean clinical scores and cumulative scores between the vehicle- and 10 mg/kg/day ginsenoside Rd-treated EAE mice or between the 40 and 80 mg/kg/day ginsenoside Rd-treated EAE mice (P > 0.05). However, the mean clinical scores and cumulative scores of 40 and 80 mg/kg/day ginsenoside Rdtreated EAE mice were significantly reduced compared with 20 mg/kg/day ginsenoside Rd-treated EAE mice (P < 0.05 for both; Fig. 2, Table I). We did not find any obvious adverse effects of ginsenoside Rd at the dose of 10–80 mg/kg/day in EAE mice at 25 days after injection. From these results, we found that ginsenoside Rd suppressed the severity of EAE when used at dosages of 20, 40, and 80 mg/kg/day, although at a dose of 20 mg/ kg/day its efficacy was not as pronounced as at a dose of 40 or 80 mg/kg/day. There were no significant differences in disease severity between the 40 and 80 mg/kg/day
ginsenoside Rd-treated groups. Therefore, the dose of 40 mg/kg/day ginsenoside Rd was selected as the optimal dose in the following experiment. Ginsenoside Rd Treatment Improved Histopathology of EAE Mice To confirm the effects of ginsenoside Rd on inflammation and demyelination of EAE mice, lumbar spinal cords were removed for histopathologic assay at day 20 p.i. Blinded analyses revealed that the inflammatory scores, which indicated the diffuse infiltration of mixed macrophages T and B lymphocytes into CNS white matter in the lumbar spinal cords, were significantly reduced in ginsenoside Rd-treated EAE mice compared with vehicle-treated EAE mice (1.67 6 0.21 vs. 2.83 6 0.17, P < 0.01; Fig. 3B). Moreover, a large plaque of demyelination was seen in the vehicle-treated EAE mice, although Journal of Neuroscience Research
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TABLE I. Comparison of Clinical Characteristics in EAE Mice Treated With Different Dosages of Ginsenoside Rd* Treatment Vehicle 10 mg/kg/day 20 mg/kg/day 40 mg/kg/day 80 mg/kg/day
Ginsenoside Ginsenoside Ginsenoside Ginsenoside
Rd Rd Rd Rd
Disease onset day
Mean clinical scores
Cumulative scores
11.25 6 0.25 11.43 6 0.30 11.50 6 0.33 11.38 6 0.38 11.38 6 0.38
2.58 6 0.12 2.38 6 0.09 1.85 6 0.11†§ 1.15 6 0.16‡††k 1.24 6 0.10‡††k
64.00 6 2.35 58.50 6 2.66 45.25 6 2.91ठ28.38 6 3.85ࠠk 30.62 6 2.91ࠠk
*Data are expressed as mean 6 SEM. Treatment started from day 10 postinduction and lasted for various time periods. † P < 0.05, ‡ P < 0.01 vs. vehicle § P < 0.05, †† P < 0.01 vs. 10 mg/kg/day Ginsenoside Rd; k P < 0.05 vs. 20 mg/kg/day Ginsenoside Rd by rank one-way ANOVA.
Ginsenoside Rd Treatment Reduced BBB Dysfunction in EAE Mice BBB destruction accompanied by large numbers of leucocytes infiltrating into the CNS plays an important role in the development and progression of MS and EAE (de Vries et al., 1997). In this experiment, BBB integrity was determined by EB extravasation at day 20 p.i. The concentrations of extravasated EB of different groups are shown in Figure 4. In the control mice, the baseline level of EB was 48.25 6 4.29 lg/g brain tissue. The content of EB in the brain of vehicle- and ginsenoside Rd-treated EAE mice was 141.12 6 3.68 and 107.39 6 5.54 lg/g brain tissue, respectively. Both vehicle- and ginsenoside Rd-treated EAE mice showed a significant increase in the content of EB in the brain compared with the control mice (P < 0.01). However, the increased content of EB in the brain of ginsenoside Rd-treated EAE mice was not as remarkable as that in the vehicle-treated EAE mice (P < 0.01). The results demonstrate that damage to BBB integrity is significantly improved by ginsenoside Rd treatment.
mice (P < 0.01 for vehicle-treated and P < 0.05 for ginsenoside Rd-treated EAE mice). The increased production of IFN-g in ginsenoside Rd-treated EAE mice was not as pronounced as that of the vehicle-treated EAE mice (P < 0.01). The IFN-g levels of cerebral cortex in vehicle-treated EAE mice were much higher than that of the control mice (P < 0.05), and treatment with ginsenoside Rd significantly reduced the production of IFN-g compared with vehicle-treated EAE mice (P < 0.05). Moreover, we found the IL-4 levels of splenocytes culture supernatants in vehicle-treated EAE mice were lower than in the control mice (P < 0.01), and ginsenoside Rd treatment increased the production of IL-4 compared with vehicle-treated EAE mice (P < 0.01; Fig. 5B). The IL-4 level of cerebral cortex in vehicle-treated EAE mice was much lower than that of the control mice (P < 0.05), and treatment with ginsenoside Rd significantly prevented the reduction of IL-4 secretion compared with vehicle-treated EAE mice (P < 0.05). The balance between Th1 and Th2, and the ratio of Th1:Th2, were correlated with cytokine (IFN-g and IL4) secretion level, which could be described by the ratio of IFN-g/IL-4 (Steinman, 2007; Valenzuela et al., 2007). In our experiment, the ratios of IFN-g/IL4 of both splenocytes culture supernatants and cerebral cortex were significantly increased in vehicle-treated EAE mice compared with the control mice (P < 0.01 for both; Fig. 5C). However, treatment with ginsenoside Rd led to a marked decrease in IFN-g/IL4 ratio (P < 0.01 for both; Fig. 5C). These results show that ginsenoside Rd regulates the Th1/Th2 balance and promotes the Th2 shift.
Ginsenoside Rd Treatment Modulated the Levels of IFN-c and IL-4 in the Splenocytes Culture Supernatants and Cerebral Cortex of EAE Mice IFN-g and IL-4, which are the hallmark cytokines that direct Th1 and Th2 development and play an important role in the pathogenesis of MS and EAE (McGeachy and Anderton, 2005; Oreja-Guevara et al., 2012), were detected by ELISA in the supernatants of splenocytes culture and cerebral cortex from different groups. As shown in Figure 5A, the IFN-g levels of splenocytes culture supernatants in both vehicle- and ginsenoside Rd-treated EAE mice increased more than the level in the control
Ginsenoside Rd Treatment Prevented the Reduction of BDNF and NGF Expression in the Cerebral Cortex and Lumbar Spinal Cord of EAE Mice Both BDNF and NGF are implicated in the pathogenesis of EAE and MS and have been hypothesized to be potential targets of neuroprotection in MS (Hohlfeld, 2008; Kerschensteiner et al., 2009). We further investigated the effects of ginsenoside Rd on the expressions of BDNF and NGF in the cerebral cortex and lumbar spinal cord of EAE mice by using Western blot analysis. We found the expression of BDNF was significantly lower in
the demyelination was markedly attenuated by ginsenoside Rd treatment (1.83 6 0.16 vs. 3.50 6 0.50, P < 0.01; Fig. 3B). Representative sections of lumbar spinal cords showing the effects of vehicle and ginsenoside Rd treatment on inflammation and demyelination in EAE mice are presented in Figure 3A. These results indicate that ginsenoside Rd treatment attenuates the inflammation and demyelination in lumbar spinal cords of EAE mice.
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Fig. 4. Ginsenoside Rd treatment reduced blood–brain barrier (BBB) dysfunction in experimental autoimmune encephalomyelitis (EAE) mice. The integrity of the BBB was detected by quantitative measurement for Evan’s blue (EB) content at day 20 postimmunization in six animals per group. EB is expressed as micrograms per gram of brain tissue. Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. control mice; ##P < 0.01 vs. vehicle-treated EAE mice by ANOVA with least significant difference test.
treated EAE mice compared with the control mice (P < 0.01; Fig. 6B), and ginsenoside Rd increased the expression of NGF in both cerebral cortex (P < 0.01 compared with both control mice and vehicle-treated mice) and lumbar spinal cord (P < 0.01 compared with the vehicle-treated EAE mice).
Fig. 3. Ginsenoside Rd treatment improved histopathology of experimental autoimmune encephalomyelitis (EAE) mice. Mice were subjected to histopathological assay at day 20 postimmunization (p.i.). Each group had six animals. Consecutive sections were analyzed for hematoxylin and eosin staining and solochrome cyanin staining to detect inflammatory infiltration and demyelination, respectively. Inflammatory infiltration with extensive perivascular cuffing (Aa1, 340), which was widespread in the vehicle-treated EAE mice, was attenuated in ginsenoside Rd-treated EAE mice (Aa2). A large plaque of demyelination occurred in the vehicle-treated EAE mice (Aa3). Demyelination was markedly attenuated by ginsenoside Rd-treated EAE mice (Aa4). The degree of histopathology of lumbar spinal cords was decreased by ginsenoside Rd-treated EAE mice (B). Values represent the mean 6 SEM. **P < 0.01 vs. vehicle-treated EAE mice by Mann-Whitney U test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the both cerebral cortex and lumbar spinal cord of the vehicle-treated EAE mice compared with the control mice (P < 0.01; Fig. 6A). Ginsenoside Rd prevented the reduction of BDNF expression in the two regions of EAE mice compared with the vehicle-treated EAE mice (P < 0.01; Fig. 6A). The expression of NGF decreased in both cerebral cortex and lumbar spinal cord of vehicle-
DISCUSSION MS is a common disabling autoimmune disease without an effective treatment in young adults. Here we provide evidence for a potential role of ginsenoside Rd, which can reduce the severity and progression of EAE in C57 BL/6 mice. In this study, 40 mg/kg/day ginsenoside Rd was found to be the optimal dose and attenuated disease severity, reducing both inflammatory infiltrates and demyelination in lumbar spinal cords of EAE mice. The cytokines produced by inflammatory cells are believed to lead to BBB impairment (Abbott, 2002). The Th1 cytokines, particularly IFN-g, have been shown to alter and even diminish the architectural organization of the tight and adherens junctions, which are subcellular structures that maintain the restrictive properties of the BBB (Minagar and Alexander, 2003). Other studies have shown that IL-4, one of Th2 cytokines, leads to increased stabilization of the cerebral microvasculature tight junction proteins (Poliani et al., 2001). Moreover, previous studies have shown that overexpression of IFN-g was encephalitogenic in the CNS of MS and EAE, and upregulation of IL-4 has been shown to be important for preventing or ameliorating the disease severity of EAE (Frischer et al., 2009; Stadelmann et al., 2011; OrejaGuevara et al. 2012). In this study, we found ginsenoside Rd could decrease the IFN-g/IL-4 ratio (the reduced production of IFN-g and the increased secretion of IL-4) of EAE mice in vivo (cerebral cortex) and in vitro (splenocytes culture supernatants). This ratio is commonly used to describe the balance in the immune system between proinflammatory IFN-g1Th1 cells and antiJournal of Neuroscience Research
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inflammatory IL-41Th2 cells (Steinman, 2007; Valenzuela et al., 2007). Similar effects of ginsenoside Rd have been reported in a rat skin transplantation model (Wang et al., 2012b). These findings provide support that ginsenoside Rd can promote the Th2 shift for the treatment of Th1-driven diseases, including EAE. The results suggest that the attenuation of ginsenoside Rd on EAE may be due to its ability to modulate the immune response.
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Moreover, both IFN-g and IL-4 were implicated in derangement of the BBB, and the permeability of the BBB also was reduced by ginsenoside Rd treatment. Impairment of the BBB is thought to be a crucial step in the pathology of EAE (de Vries et al., 1997), so the reconstitution of BBB integrity by ginsenoside Rd is an important therapeutic effect in EAE mice. In addition to cytokines, derangement of neurotrophin production has been implicated in the pathogenesis of MS and EAE (Noseworthy et al., 2000; Hohlfeld, 2008; Kerschensteiner et al., 2009). Among neurotrophins, BDNF and NGF in the CNS were strictly correlated with clinical outcome of MS. BDNF and NGF levels detected in patients with full recovery from relapse symptoms were significantly higher than that in patients with partial recovery (Caggiula et al., 2005). Moreover, BDNF and NGF have been shown to regulate inflammation, repair, oligodendrocyte proliferation and remyelination, and regeneration of neurons and have been proposed as therapeutic strategies in controlling the disease severity of EAE and MS (McTigue et al., 1998; Kerschensteiner et al., 2009). In this study, we found that BDNF and NGF expressions were reduced in the target organs (cerebral cortex and lumbar spinal cord) of the vehicle-treated EAE mice, which meant the reduction of their protection or that there was an increased consumption of BDNF and NGF in the CNS due to the damaged tissue in EAE (D’Intino et al., 2005). The results provide support that BDNF and NGF participate in the protective mechanisms against tissue damage in EAE (D’Intino et al., 2005; Kerschensteiner et al., 2009). Ginsenoside Rd treatment prevented the reduction of BDNF and NGF expression in both cerebral cortex and lumbar spinal cord. These findings provide additional support for ginsenoside Rd’s neuroprotective effects in EAE. The alteration of BDNF, NGF, IFN-g, and IL-4 in EAE mice might reflect the result of a cross-talk between the nervous and the immune systems in EAE development to some degree (Hohlfeld, 2008; Kerschensteiner et al., 2009). It has been reported that the altertion of BDNF and NGF appears to be mediated by skewing the Th1/Th2 balance toward Th2 type by inhibition of IFNg and enhanced IL-4 production in the CNS of EAE (Villoslada et al., 2000; Arredondo et al., 2001; Makar et al., 2009). IL-4 treatment increases BDNF expression
Fig. 5. Ginsenoside Rd treatment modulated the levels of IFN-g and IL-4 and induced a Th2 bias in the splenocytes culture supernatants and cerebral cortex of experimental autoimmune encephalomyelitis (EAE) mice. Spleens of differently treated mice were aseptically harvested at day 20 postimmunization. Splenocytes (5 3 105) were incubated for 48 hr with myelin oligodendrocyte glycoprotein 35–55 amino acid peptide used for immunization. Cerebral cortex and culture supernatants were collected and analyzed by ELISA to detect levels of IFN-g (A), IL-4 (B), and the ratio of IFN-g/IL-4 (C). Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. control mice; # P < 0.05, ##P < 0.01 vs. vehicle-treated EAE mice by ANOVA with least significant difference test or Dunnett’s T3 test. Journal of Neuroscience Research
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Fig. 6. Ginsenoside Rd treatment prevented the reduction of BDNF and NGF expression in the cerebral cortex and lumbar spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. BDNF (A) and NGF (B) expressions were analyzed by Western blot at day 20 postimmunization. Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. control mice; #P < 0.05, ##P < 0.01 vs. vehicle-treated EAE mice by rank of one-way ANOVA.
in retinal ganglion cells of newborn rat culture and there is an increased production of mRNA for BDNF when astrocytes are incubated with IL-4, which suggest that BDNF is the neurotrophin involved in IL-4 effect (Kerschensteiner et al., 2009; Derecki et al., 2010; de AraujoMartins et al., 2013). IL-4 has also been shown to increase the synthesis of NGF by astrocytes, lymphocytes, and C6 glial cells (Brodie and Goldreich, 1994; Brodie et al., 1998; Arredondo et al., 2001). However, IFN-g can inhibit NGF secretion by astrocytes (Brodie, 1996; Hohlfeld, 2008; Kerschensteiner et al., 2009). In this study, ginsenoside Rd regulated the production/expression of both cytokines (IFN-g and IL-4) and neurotrophins (BDNF and NGF), suggesting that it could modulate the neuroimmune dysfunction in EAE mice. It has been reported that ginsenoside Rd can regulate the NF-jB signal pathway in in vitro and in vivo animal models (Wu et al., 2007; Hu et al., 2013). NF-jB plays a pivotal role in regulating the production/expression of inflammatory cytokines, including IFN-g and IL-4, and neurotrophins, including BDNF and NGF (Yoshimura et al., 2003; Heese et al., 2006). Ginsenoside Rd also can regulate other pathways, including mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERKs) and Akt/GSK-3b signaling that mediate inflammatory and neuroprotective effects (Li et al., 2013; Wang
et al., 2013; Zhang et al., 2013). Defining the exact signal pathway of IFN-g/IL-4 and BDNF/NGF by ginsenoside Rd in EAE mice is attractive and merits further exploration. In conclusion, the data presented here demonstrate that ginsenoside Rd can effectively ameliorate the clinical severity of EAE mice through reducing the permeability of the BBB and regulating the production/expression of IFN-g/IL-4 and BDNF/NGF. These findings suggest that ginsenoside Rd could be a potential therapeutic agent for the treatment of MS. ACKNOWLEDGMENTS The authors have no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. REFERENCES Abbott NJ. 2002. Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat 200:629–638. Arredondo LR, Deng C, Ratts RB, Lovett-Racke AE, Holtzman DM, Racke MK. 2001. Role of nerve growth factor in experimental autoimmune encephalomyelitis. Eur J Immunol 31:625–633. Attele AS, Wu JA, Yuan CS. 1999. Ginseng pharmacology: multiple constituents and multiple actions. Biochem Pharmacol 58:1685–1693. Journal of Neuroscience Research
Ginsenoside Rd Ameliorates EAE Brodie C. 1996. Differential effects of Th1 and Th2 derived cytokines on NGF synthesis by mouse astrocytes. FEBS Lett 394:117–120. Brodie C, Goldreich N. 1994. Interleukin-4 modulates the proliferation and differentiation of glial cells. J Neuroimmunol 55:91–97. Brodie C, Goldreich N, Haiman T, Kazimirsky G. 1998. Functional IL4 receptors on mouse astrocytes: IL-4 inhibits astrocyte activation and induces NGF secretion. J Neuroimmunol 81:20–30. Caggiula M, Batocchi AP, Frisullo G, Angelucci F, Patanella AK, Sancricca C, Nociti V, Tonali PA, Mirabella M. 2005. Neurotrophic factors and clinical recovery in relapsing-remitting multiple sclerosis. Scand J Immunol 62:176–182. Chen X, Hu X, Zou Y, Pi R, Liu M, Wang T, Zheng X, Lin M, Liu P, Tao L. 2009. Combined treatment with minocycline and prednisone attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice. J Neuroimmunol 210:22–29. de Araujo-Martins L, de Oliveira RM, dos Santos GV, dos Santos RC, dos Santos AA, Giestal de Araujo E. 2013. Treatment in vitro of retinal cells with IL-4 increases the survival of retinal ganglion cells: the involvement of BDNF. Neurochem Res 38:162–173. Derecki NC, Cardani AN, Yang CH, Quinnies KM, Crihfield A, Lynch KR, Kipnis J. 2010. Regulation of learning and memory by meningeal immunity: a key role for IL-4. J Exp Med 207:1067–1080. de Vries HE, Kuiper J, de Boer AG, Van Berkel TJ, Breimer DD. 1997. The blood-brain barrier in neuroinflammatory diseases. Pharmacol Rev 49:143–155. D’Intino G, Paradisi M, Fernandez M, Giuliani A, Aloe L, Giardino L, Calza L. 2005. Cognitive deficit associated with cholinergic and nerve growth factor downregulation in experimental allergic encephalomyelitis in rats. Proc Natl Acad Sci U S A 102:3070–3075. Frischer JM, Bramow S, Dal-Bianco A, Lucchinetti CF, Rauschka H, Schmidbauer M, Laursen H, Sorensen PS, Lassmann H. 2009. The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain 132:1175–1189. Heese K, Inoue N, Sawada T. 2006. NF-jB regulates B-cell-derived nerve growth factor expression. Cell Mol Immunol 3:63–66. Hohlfeld R. 2008. Neurotrophic cross-talk between the nervous and immune systems: relevance for repair strategies in multiple sclerosis? J Neurol Sci 265:93–96. Hu G, Wu Z, Yang F, Zhao H, Liu X, Deng Y, Shi M, Zhao G. 2013. Ginsenoside Rd blocks AIF mitochondrio-nuclear translocation and NF-jB nuclear accumulation by inhibiting poly(ADP-ribose) polymerase-1 after focal cerebral ischemia in rats. Neurol Sci 34:2101– 2106. Jiang Y, Zou Y, Chen S, Zhu C, Wu A, Liu Y, Ma L, Zhu D, Ma X, Liu M, Kang Z, Pi R, Peng F, Wang Q, Chen X. 2013. The antiinflammatory effect of donepezil on experimental autoimmune encephalomyelitis in C57 BL/6 mice. Neuropharmacology 73:415–424. Kerschensteiner M, Meinl E, Hohlfeld R. 2009. Neuro-immune crosstalk in CNS diseases. Neuroscience 158:1122–1132. Kuerten S, Kostova-Bales DA, Frenzel LP, Tigno JT, Tary-Lehmann M, Angelov DN, Lehmann PV. 2007. MP4- and MOG:35–55-induced EAE in C57BL/6 mice differentially targets brain, spinal cord and cerebellum. J Neuroimmunol 189:31–40. Li L, Liu Z, Liu J, Tai X, Hu X, Liu X, Wu Z, Zhang G, Shi M, Zhao G. 2013. Ginsenoside Rd attenuates beta-amyloid-induced tau phosphorylation by altering the functional balance of glycogen synthase kinase 3beta and protein phosphatase 2A. Neurolbiol Dis 54:320–328. Liu J, Yan X, Li L, Zhu Y, Qin K, Zhou L, Sun D, Zhang X, Ye R, Zhao G. 2012a. Ginsennoside rd attenuates cognitive dysfunction in a rat model of Alzheimer’s disease. Neurochem Res 37:2738–2747. Liu X, Wang L, Wen A, Yang J, Yan Y, Song Y, Liu X, Ren H, Wu Y, Li Z, Chen W, Xu Y, Li L, Xia J, Zhao G. 2012b. Ginsenoside-Rd improves outcome of acute ischaemic stroke-a randomized, doubleblind, placebo-controlled, multicenter trial. Eur J Neurol 19:855–863. Journal of Neuroscience Research
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Makar TK, Bever CT, Singh IS, Royal W, Sahu SN, Sura TP, Sultana S, Sura KT, Patel N, Dhib-Jalbut S, Trisler D. 2009. Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle. J Neuroimmunol 210:40–51. McGeachy MJ, Anderton SM. 2005. Cytokines in the induction and resolution of experimental autoimmune encephalomyelitis. Cytokine 32:81–84. McTigue DM, Horner PJ, Stokes BT, Gage FH. 1998. Neurotrophin-3 and brain-derived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord. J Neurosci 18:5354–5365. Minagar A, Alexander JS. 2003. Blood–brain barrier disruption in multiple sclerosis. Mult Scler 9:540–549. Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG. 2000. Multiple sclerosis. N Engl J Med 343:938–952. O’Neill EJ, Day MJ, Wraith DC. 2006. IL-10 is essential for disease protection following intranasal peptide administration in the C57BL/6 model of EAE. J Neuroimmunol 178:1–8. Oreja-Guevara C, Ramos-Cejudo J, Aroeira LS, Chamorro B, DiezTejedor E. 2012. TH1/TH2 Cytokine profile in relapsing-remitting multiple sclerosis patients treated with Glatiramer acetate or Natalizumab. BMC Neurol 12:95. Poliana PL, Brok H, Furlan R, Ruffini F, Bergami A, Desina G, Marconi PC, Rovaris M, Uccelli A, Glorioso JC, Penna G, Adorini L, Comi G, ‘t Hart B, Martino G. 2001. Delivery to the central nervous system of a nonreplicative herpes simplex type 1 vector engineered with the interleukin 4 gene protects rhesus monkeys from hyperacute autoimmune encephalomyelitis. Hum Gene Ther 12:905–920. Shang W, Yang Y, Jiang B, Jin H, Zhou L, Liu S, Chen M. 2007. Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression. Life Sci 80:618–625. Stadelmann C, Wegner C, Bruck W. 2011. Inflammation, demyelination, and degeneration—recent insights from MS pathology. Biochim Biophys Acta 1812:275–282. Steinman L. 2007. A brief history of T(h)17, the first major revision in the T(h)1/T(h)2 hypothesis of T cell-mediated tissue damage. Nat Med 13:139–145. Tysiak E, Asbach P, Aktas O, Waiczies H, Smyth M, Schnorr J, Taupitz M, Wuerfel J. 2009. Beyond blood–brain barrier breakdown—in vivo detection of occult neuroinflammatory foci by magnetic nanoparticles in high field MRI. J Neuroinflammation 6:20. Valenzuela RM, Costello K, Chen M, Said A, Johnson KP, Dhib-Jalbut S. 2007. Clinical response to glatiramer acetate correlates with modulation of IFN-gamma and IL-4 expression in multiple sclerosis. Mult Scler 13:754–762. Villoslada P, Hauser SL, Bartke I, Unger J, Heald N, Rosenberg D, Cheung SW, Mobley WC, Fisher S, Genain CP. 2000. Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system. J Exp Med 191:1799– 1806. Virley DJ. 2005. Developing therapeutics for the treatment of multiple sclerosis. NeuroRx 2:638–649. Wang L, Zhang Y, Wang Z, Li S, Min G, Wang L, Chen J, Cheng J, Wu Y. 2012a. Inhibitory effect of ginsenoside-Rd on carrageenaninduced inflammation in rats. Can J Physiol Pharmacol 90:229–236. Wang L, Zhang Y, Chen J, Li S, Wang Y, Hu L, Wang L, Wu Y. 2012b. Immunosuppressive effects of ginsenoside-Rd on skin allograft rejection in rats. J Surg Res 176:267–274. Wang Y, Li X, Wang X, Lau W, Wang Y, Xing Y, Zhang X, Ma X, Gao F. 2013. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/GSK-3b signaling and inhibition of the mitochondria-dependent apoptptic pathway. PLoS One 8:e70956. Wu CF, Bi XL, Yang JY, Zhan JY, Dong YX, Wang JH, Wang JM, Zhang R, Li X. 2007. Differential effects of ginsenosides on NO and
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Yoshimura A, Mori H, Ohishi M, Aki D, Hanada T. 2003. Negative regulation of cytokine signaling influences inflammation. Curr Opin Immunol 15:704–708. Zhang C, Du F, Shi M, Ye R, Cheng H, Han J, Ma L, Cao R, Rao Z, Zhao G. 2012. Ginsenoside Rd protects neurons against glutamateinduced excitotoxicity by inhibiting Ca21 influx. Cell Mol Neurobiol 32:121–128. Zhang YX, Wang L, Xiao EL, Li SJ, Chen JJ, Gao B, Min GN, Wang ZP, Wu YJ. 2013. Ginsenoside Rd exhibits anti-inflammatory activities thriugh elevation of antioxidant enzyme activities and inhibition of JNK and ERK activation in vivo. Int Immunopharmacol 17:1094–1100.
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Ginsenoside Rd Ameliorates Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice Dongliang Zhu,1 Mei Liu,1 Yaowu Yang,2 Lili Ma,1 Ying Jiang,1 Linli Zhou,1 Qiling Huang,1 Rongbiao Pi,3 and Xiaohong Chen1* 1
Department of Neurology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China Department of Traditional Chinese Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China 3 Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China 2
Multiple sclerosis (MS) is a common disabling autoimmune disease without an effective treatment in young adults. Ginsenoside Rd, extracted from Panax notoginseng, has multiple pharmacological effects and potential therapeutic applications in diseases of the central nervous system. In this study, we explore the efficacy of ginsenoside Rd in experimental autoimmune encephalomyelitis (EAE), an established model of MS. EAE was induced by myelin oligodendrocyte glycoprotein 35–55amino-acid peptide. Ginsenoside Rd (10–80 mg/kg/day) or vehicle was intraperitoneally administered on the disease onset day, and the therapy persisted throughout the experiments. The dose of 40 mg/kg/day of ginsenoside Rd was selected as optimal. Ginsenoside Rd effectively ameliorated the clinical severity in EAE mice, reduced the permeability of the blood–brain barrier, regulated the secretion of interferon-gamma and interleukin-4, promoted the Th2 shift in vivo (cerebral cortex) and in vitro (splenocytes culture supernatants), and prevented the reduction in expression of brain-derived neurotrophic factor and nerve growth factor in both cerebral cortex and lumbar spinal cord of EAE mice. This study establishes the potency of ginsenoside Rd in inhibiting the clinical course of EAE. These findings suggest that ginsenoside Rd could be a promising agent for amelioration of neuroimmune dysfunction diseases such as MS. VC 2014 Wiley
derangement of cytokine and neurotrophin production were implicated in the neuroimmune dysfunction of MS and experimental autoimmune encephalomyelitis (EAE), an established model of MS (Noseworthy et al., 2000; Frischer et al., 2009; Stadelmann et al., 2011). Current drugs for MS management consist of classical antiinflammatory agents for acute relapses and immunosuppression or immunomodulatory medicines given as maintenance, to prevent relapses and slow disability progression (Tysiak et al., 2009). However, many patients have suboptimal responses to these therapies. Further improvement in MS therapeutics is needed (Virley. 2005). Ginsenoside Rd (dammer-24[25]-ene-3b,12b, 20[S]-triol-[20-O-b-D-glucopyranosyl]-3-O-b-D-glucopyranosyl-[1R2]-b-D-gluco-pyranoside [C48H82O18.3 H2O]; Fig. 1) is a dammarane-type steroid glycoside extracted from Pana notoginseng, a traditional Chinese herbal medicine, which has multiple pharmacological effects (Attele et al., 1999). It is highly lipophilic and capable of crossing biological membranes (Ye et al., 2008).Previous studies demonstrated that ginsenoside Rd possesses anti-inflammatory, antioxidative, antiapoptotic, and neuroprotective properties and has potential therapeutic applications in CNS diseases (Shang et al., 2007; Ye et al., 2009, 2011; Liu et al., 2012a,b). It has been reported that
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Key words: ginsenoside Rd; experimental autoimmune encephalomyelitis; therapy; multiple sclerosis
D. Zhu and M. Liu contributed equally to this work.
Multiple sclerosis (MS) is a widespread autoimmune-mediated neurodegenerative disease with characteristic inflammatory demyelination in the central nervous system (CNS) that causes significant disability in young adults (Frischer et al., 2009; Stadelmann et al., 2011). Although the pathogenesis of MS has not been fully elucidated, the disruption of the blood–brain barrier (BBB), migration of T lymphocytes into the CNS, and
*Correspondence to: Dr. Xiaohong Chen, Department of Neurology, The Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou, Guangdong 510630, China. E-mail: [email protected]
C 2014 Wiley Periodicals, Inc. V
Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: 81071068 (to X.C.).
Received 28 November 2013; Revised 26 February 2014; Accepted 1 April 2014 Published online 2 May 2014 in Wiley Online (wileyonlinelibrary.com). DOI: 10.1002/jnr.23397
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Fig. 1. Structure of ginsenoside Rd.
ginsenoside Rd suppresses lipopolysaccharide-induced nitric oxide formation and tumor necrosis factor-a production through inhibiting nuclear factor-jB (NF-jB) in N9 microglial cells and in rats (Wu et al., 2007; Wang et al., 2012a). It was also reported that Ginsenoside Rd could inhibit Ca21 influx, prevent glutamate-induced excitotoxicity in hippocampal neurons, ameliorate the outcome of focal cerebral ischemia in aged mice through attenuating redox imbalance, and improve scores of modified ranking scale and the National Institutes of Health Stroke Scale in patients with acute ischemic stroke (Ye et al., 2009, 2011; Liu et al., 2012b; Zhang et al., 2012). Recently, ginsenoside Rd was found to attenuate b-amyloid-induced tau phosphorylation by reducing glycogen synthase kinase-3b (GSK-3b) activity, enhance the activity of protein phosphatase 2A, downregulate caspase-3 proteins, and attenuate cognitive dysfunction in a rat model of Alzheimer’s disease (Liu et al., 2012a; Li et al., 2013). However, little is known about the effect of ginsenoside Rd on autoimmune inflammatory diseases of the CNS, such as MS and EAE. In this study, we tested the efficacy of ginsenoside Rd in a chronic EAE model induced by myelin oligodendrocyte glycoprotein 35–55-amino-acid peptide (MOG35–55) in C57 BL/6 mice. We further determined the permeability of the BBB, the alteration of interferong (IFN-g) and interleukin-4 (IL-4) in the cerebral cortex in vivo and the splenocytes culture supernatants in vitro, and the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the cerebral cortex and lumbar spinal cord of EAE mice. Our findings indicate that ginsenoside Rd attenuates the progression of clinical disease in EAE mice, which may be associated with preventing disruption of the BBB and modulating neuroimmune dysfunction. MATERIALS AND METHODS Animals and Regents Six- to eight-week-old female C57 BL/6 mice weighing 16–18 g were obtained from the Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China). Experiments were carried out according to the National Institutes of Health
Guide for the care and use of laboratory animals and were approved by the Bioethics Committee of Sun Yat-Sen University. Ginsenoside Rd with a purity of 98% was obtained from Nanjing Zelang Medical Technology (Nanjing, China). MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by CL BioScientific (Xi’an, China). Amino acid sequences were confirmed by amino acid analysis and mass spectroscopy. The purity of the peptide was greater than 95%. Mycobacterium tuberculosis H37RA was purchased from Difco (Detroit, MI). Pertussis toxin (PTX) was purchased from Alexis (San Diego, CA). Complete Freund’s adjuvant (CFA) and Evan’s blue (EB) were purchased from Sigma-Aldrich (St. Louis, MO). Polyclonal anti-BDNF and anti-NGF were purchased from Abcam (Cambridge, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. The cytokine assay by enzyme-linked immunosorbent assay (ELISA) kits for IFN-g and IL-4 were purchased from RayBiotech (Norcross, GA). Induction and Assessment of EAE The procedure used for the induction of EAE has been described previously (Chen et al., 2009). Briefly, mice received a subcutaneous injection in the flanks with 200 lg MOG35–55 peptide emulsified in CFA containing 500 lg Mycobacterium tuberculosis H37RA. Immediately thereafter and again 48 hr later, the mice received an intraperitoneal injection of 300 ng PTX in 100 ll phosphate-buffered saline (PBS). An additional injection of MOG35–55 peptide in CFA was delivered 7 days later. The animals were examined daily for disability. Clinical scores were defined as follows: 0, no signs; 1, loss of tail tonicity; 2, flaccid tail; 3, ataxia and/or paresis of hindlimbs; 4, complete paralysis of hindlimbs; 5, moribund or death. Dose-Finding Experiments and Treatment of Mice Mice were randomly assigned to three groups: control mice, vehicle-treated EAE mice, and ginsenoside Rd-treated EAE mice (n 5 8). The dose of ginsenoside Rd was chosen on the basis of previous data in vivo (Ye et al., 2011) and our preliminary dose-finding experiment. In our dose-finding experiment, ginsenoside Rd stock solutions were prepared in saline containing 10% 1,3-propanediol (v/v). Ginsenoside Rd was administered by intraperitoneal injections at dosages of 10, 20, 40, and 80 mg/kg/day, respectively (n 5 8). Treatment with ginsenoside Rd was initiated when the first mouse showed neurological symptoms, and medication was administered daily until mice were sacrificed at day 35 postimmunization (p.i.). Vehicle-treated EAE mice were treated with intraperitoneal administration of vehicle only. Each experiment was repeated three times. Histological Evaluation To assess the degree of CNS inflammation and demyelination, mice in the vehicle- and ginsenoside Rd-treated groups were anesthetized and perfused with ice-cold PBS, followed by 4% paraformaldehyde from the left ventricle at day 20 p.i. (n 5 6). Spinal cords were removed. Tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin for revealing inflammatory infiltration. Solochrome Journal of Neuroscience Research
Ginsenoside Rd Ameliorates EAE cyanin technique was used for myelin staining. Histopathological examination was performed in a blinded fashion. The scale used to evaluate for inflammation (O’Neill et al., 2006) was: 0, no inflammatory cells; 1, a few scattered inflammatory cells; 2, organisation of inflammatory infiltrates around blood vessels; and 3, extensive perivascular cuffing with extension into adjacent parenchyma or parenchymal infiltration without obvious cuffing. Demyelination in the spinal cords was scored as previously described by Kuerten et al. (2007): 1, traces of subpial demyelination; 2, marked subpial and perivascular demyelination; 3, confluent perivascular or subpial demyelination; 4, massive perivascular and subpial demyelination involving one half of the spinal cord with presence of cellular infiltrates in the CNS parenchyma; 5, extensive perivascular and subpial demyelination involving the whole cord section with presence of cellular infiltrates in the CNS parenchyma. Evaluation of BBB Disruption The integrity of the BBB was detected by quantitative measurement for EB content at day 20 p.i. as described previously (Jiang et al., 2013). Briefly, sterilized 2% EB solution was administered intravenously at a dosage of 4 ml/kg per animal (n 5 6). Thirty minutes after injection, mice were perfused with saline to remove intravascular EB dye. Brains were rapidly removed and each sample was weighed and then homogenized with 2.5 ml PBS and mixed with 2.5 ml 60% trichloroacetic acid to precipitate protein. The samples were centrifuged for 30 min at 1,000g, and the supernatants were measured at 610 nm for absorbance of EB by using a spectrophotometer (Genesis 10 uv; Thermo Electron, Madison, WI). The content of EB was expressed as micrograms per gram of brain tissue. ELISA Assay Spleens of differently treated mice were aseptically harvested at day 20 p.i. Splenocytes (5 3 105 cells/well) from each group were incubated in 96-well flat-bottom plates in RPMI 1640 supplemented with 10% fetal calf serum, with MOG35–55 (15 lg/ml) used for the immunization. Culture supernatants were collected at 48 hr and stored at 280 C for assaying IFN-g and IL-4. Cerebral cortex was quickly dissected on a cold plate and frozen immediately in liquid nitrogen. All tissues were stored at 280 C until assay. Cerebral cortex was homogenized in 1 ml of ice-cold Tris buffer (pH 7.2, 4 C) containing 50 mM Tris, 1 mM EDTA, 6 mM MgCl2, and 5% (w/v) protease inhibitor cocktail. After homogenization, samples were sonicated for 10 sec by using an ultrasonic processor at a setting of 5, and then centrifuged at 20,800g for 20 min at 4 C (Jiang et al., 2013). Afterward, supernatants were collected for assaying IFN-g and IL-4. Total protein content was measured in each sample using a commercially available BCA protein assay kit (Key Gen Biotech, Nanjing, China). The IFN-g and IL-4 levels in the culture supernatants and cerebral cortex supernatants were measured according to the ELISA kit manual. IFN-g and IL-4 of cerebral cortex supernatants are expressed as picograms per milligram of total protein, whereas culture supernatant samples are expressed as picograms per milliliter of culture supernatant. The detection Journal of Neuroscience Research
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limits are 0.6–200 pg/ml for IL-4 and 5–2,000 pg/ml for IFNg in the cell supernatants and 1–200 pg/ml for IL-4 and 10– 2000 pg/ml for IFN-g in the brain tissues. Western Blot Analysis To investigate the expressions of BDNF and NGF in the cerebral cortex and lumbar spinal cord of differently treated mice, we performed Western blot analysis as described previously (Jiang et al., 2013). Samples from differently treated mice were loaded on 10% gradient sodium dodecyl sulfatepolyacrylamide gels (20 mg protein per lane). Proteins were transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked by 5% nonfat milk. Afterward, the membranes were incubated with polyclonal antiBDNF (1 lg/ml) and anti-NGF (1 lg/ml) overnight. After washing of the membranes three times with TBST buffer, the membranes were incubated with anti-mouse-HRP and goat anti-rabbit-HRP for 30 min. The experiment was repeated in triplicate and used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. The bands were quantified in Quantity One image analysis software (Bio-Rad). Statistical Analysis Data were expressed as mean 6 SEM and analyzed in SPSS 13.0 software (IBM, Armonk, NY). Quantitative data were processed using the Mann-Whitney U test, one-way analysis of variance (ANOVA), rank of one-way ANOVA, pairwise comparison among groups with the least significant difference (LSD), or Dunnett’s T3 test. P < 0.05 was considered statistically significant.
RESULTS Ginsenoside Rd Treatment Ameliorated Clinical Severity of EAE Mice In our experiment, “disease onset day” indicates the day when an individual mouse showed the first symptom. The mean clinical score represented the average score of each mouse during the experiment. The overall disease burden of each mouse was represented as a cumulative score, which was the sum of the disability scores obtained daily over the course of the 35-day experiment. First, we investigated the efficacy of ginsenoside Rd for the neurological behaviors of EAE mice. The therapy was started at disease onset day and persisted throughout the experiment. We found the mean clinical scores of 20, 40, and 80 mg/kg/day ginsenoside Rd-treated EAE mice were significantly reduced compared with both vehicle-treated mice (P < 0.05 for 20 and P < 0.01 for 40 and 80 mg/kg/ day ginsenoside Rd-treated EAE mice) and 10 mg/kg/ day ginsenoside Rd-treated EAE mice (P < 0.05 for 20 and P < 0.01 for 40 and 80 mg/kg/day ginsenoside Rdtreated EAE mice; Fig. 2B, Table I). Cumulative scores of 20, 40, and 80 mg/kg/day ginsenoside Rd-treated EAE mice were significantly reduced compared with both vehicle-treated (P < 0.01 for all) and 10 mg/kg/day ginsenoside Rd-treated EAE mice (P < 0.05 for 20 and P < 0.01 for 40 and 80 mg/kg/day ginsenoside Rd-
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Fig. 2. Ginsenoside Rd treatment ameliorated clinical severity of experimental autoimmune encephalomyelitis (EAE) mice. Daily clinical scores (A), mean clinical scores (B), and cumulative scores (C) of the vehicle- and ginsenoside Rd-treated (10–80 mg/kg/day) EAE mice. EAE was induced in female C57 BL/6 mice with myelin oligodendrocyte glycoprotein 35–55-amino-acid peptide, and treatment with ginsenoside Rd was performed as described in Materials and Methods. Each group had eight animals. Drug treatments were started from the day of disease onset. Doses of 20, 40, and 80 mg/kg/day
significantly ameliorated the clinical score of EAE, and 40 mg/kg/day was selected as the optimal dose (see also Table I). Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. vehicle-treated EAE mice; #P < 0.05, ##P < 0.01 vs. 10 mg/kg/day ginsenoside Rdtreated EAE mice; &P < 0.05 vs. 20 mg/kg/day ginsenoside Rdtreated EAE mice by rank of one-way ANOVA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
treated EAE mice; Fig. 2C, Table I). There were no significant differences in the mean clinical scores and cumulative scores between the vehicle- and 10 mg/kg/day ginsenoside Rd-treated EAE mice or between the 40 and 80 mg/kg/day ginsenoside Rd-treated EAE mice (P > 0.05). However, the mean clinical scores and cumulative scores of 40 and 80 mg/kg/day ginsenoside Rdtreated EAE mice were significantly reduced compared with 20 mg/kg/day ginsenoside Rd-treated EAE mice (P < 0.05 for both; Fig. 2, Table I). We did not find any obvious adverse effects of ginsenoside Rd at the dose of 10–80 mg/kg/day in EAE mice at 25 days after injection. From these results, we found that ginsenoside Rd suppressed the severity of EAE when used at dosages of 20, 40, and 80 mg/kg/day, although at a dose of 20 mg/ kg/day its efficacy was not as pronounced as at a dose of 40 or 80 mg/kg/day. There were no significant differences in disease severity between the 40 and 80 mg/kg/day
ginsenoside Rd-treated groups. Therefore, the dose of 40 mg/kg/day ginsenoside Rd was selected as the optimal dose in the following experiment. Ginsenoside Rd Treatment Improved Histopathology of EAE Mice To confirm the effects of ginsenoside Rd on inflammation and demyelination of EAE mice, lumbar spinal cords were removed for histopathologic assay at day 20 p.i. Blinded analyses revealed that the inflammatory scores, which indicated the diffuse infiltration of mixed macrophages T and B lymphocytes into CNS white matter in the lumbar spinal cords, were significantly reduced in ginsenoside Rd-treated EAE mice compared with vehicle-treated EAE mice (1.67 6 0.21 vs. 2.83 6 0.17, P < 0.01; Fig. 3B). Moreover, a large plaque of demyelination was seen in the vehicle-treated EAE mice, although Journal of Neuroscience Research
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TABLE I. Comparison of Clinical Characteristics in EAE Mice Treated With Different Dosages of Ginsenoside Rd* Treatment Vehicle 10 mg/kg/day 20 mg/kg/day 40 mg/kg/day 80 mg/kg/day
Ginsenoside Ginsenoside Ginsenoside Ginsenoside
Rd Rd Rd Rd
Disease onset day
Mean clinical scores
Cumulative scores
11.25 6 0.25 11.43 6 0.30 11.50 6 0.33 11.38 6 0.38 11.38 6 0.38
2.58 6 0.12 2.38 6 0.09 1.85 6 0.11†§ 1.15 6 0.16‡††k 1.24 6 0.10‡††k
64.00 6 2.35 58.50 6 2.66 45.25 6 2.91ठ28.38 6 3.85ࠠk 30.62 6 2.91ࠠk
*Data are expressed as mean 6 SEM. Treatment started from day 10 postinduction and lasted for various time periods. † P < 0.05, ‡ P < 0.01 vs. vehicle § P < 0.05, †† P < 0.01 vs. 10 mg/kg/day Ginsenoside Rd; k P < 0.05 vs. 20 mg/kg/day Ginsenoside Rd by rank one-way ANOVA.
Ginsenoside Rd Treatment Reduced BBB Dysfunction in EAE Mice BBB destruction accompanied by large numbers of leucocytes infiltrating into the CNS plays an important role in the development and progression of MS and EAE (de Vries et al., 1997). In this experiment, BBB integrity was determined by EB extravasation at day 20 p.i. The concentrations of extravasated EB of different groups are shown in Figure 4. In the control mice, the baseline level of EB was 48.25 6 4.29 lg/g brain tissue. The content of EB in the brain of vehicle- and ginsenoside Rd-treated EAE mice was 141.12 6 3.68 and 107.39 6 5.54 lg/g brain tissue, respectively. Both vehicle- and ginsenoside Rd-treated EAE mice showed a significant increase in the content of EB in the brain compared with the control mice (P < 0.01). However, the increased content of EB in the brain of ginsenoside Rd-treated EAE mice was not as remarkable as that in the vehicle-treated EAE mice (P < 0.01). The results demonstrate that damage to BBB integrity is significantly improved by ginsenoside Rd treatment.
mice (P < 0.01 for vehicle-treated and P < 0.05 for ginsenoside Rd-treated EAE mice). The increased production of IFN-g in ginsenoside Rd-treated EAE mice was not as pronounced as that of the vehicle-treated EAE mice (P < 0.01). The IFN-g levels of cerebral cortex in vehicle-treated EAE mice were much higher than that of the control mice (P < 0.05), and treatment with ginsenoside Rd significantly reduced the production of IFN-g compared with vehicle-treated EAE mice (P < 0.05). Moreover, we found the IL-4 levels of splenocytes culture supernatants in vehicle-treated EAE mice were lower than in the control mice (P < 0.01), and ginsenoside Rd treatment increased the production of IL-4 compared with vehicle-treated EAE mice (P < 0.01; Fig. 5B). The IL-4 level of cerebral cortex in vehicle-treated EAE mice was much lower than that of the control mice (P < 0.05), and treatment with ginsenoside Rd significantly prevented the reduction of IL-4 secretion compared with vehicle-treated EAE mice (P < 0.05). The balance between Th1 and Th2, and the ratio of Th1:Th2, were correlated with cytokine (IFN-g and IL4) secretion level, which could be described by the ratio of IFN-g/IL-4 (Steinman, 2007; Valenzuela et al., 2007). In our experiment, the ratios of IFN-g/IL4 of both splenocytes culture supernatants and cerebral cortex were significantly increased in vehicle-treated EAE mice compared with the control mice (P < 0.01 for both; Fig. 5C). However, treatment with ginsenoside Rd led to a marked decrease in IFN-g/IL4 ratio (P < 0.01 for both; Fig. 5C). These results show that ginsenoside Rd regulates the Th1/Th2 balance and promotes the Th2 shift.
Ginsenoside Rd Treatment Modulated the Levels of IFN-c and IL-4 in the Splenocytes Culture Supernatants and Cerebral Cortex of EAE Mice IFN-g and IL-4, which are the hallmark cytokines that direct Th1 and Th2 development and play an important role in the pathogenesis of MS and EAE (McGeachy and Anderton, 2005; Oreja-Guevara et al., 2012), were detected by ELISA in the supernatants of splenocytes culture and cerebral cortex from different groups. As shown in Figure 5A, the IFN-g levels of splenocytes culture supernatants in both vehicle- and ginsenoside Rd-treated EAE mice increased more than the level in the control
Ginsenoside Rd Treatment Prevented the Reduction of BDNF and NGF Expression in the Cerebral Cortex and Lumbar Spinal Cord of EAE Mice Both BDNF and NGF are implicated in the pathogenesis of EAE and MS and have been hypothesized to be potential targets of neuroprotection in MS (Hohlfeld, 2008; Kerschensteiner et al., 2009). We further investigated the effects of ginsenoside Rd on the expressions of BDNF and NGF in the cerebral cortex and lumbar spinal cord of EAE mice by using Western blot analysis. We found the expression of BDNF was significantly lower in
the demyelination was markedly attenuated by ginsenoside Rd treatment (1.83 6 0.16 vs. 3.50 6 0.50, P < 0.01; Fig. 3B). Representative sections of lumbar spinal cords showing the effects of vehicle and ginsenoside Rd treatment on inflammation and demyelination in EAE mice are presented in Figure 3A. These results indicate that ginsenoside Rd treatment attenuates the inflammation and demyelination in lumbar spinal cords of EAE mice.
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Fig. 4. Ginsenoside Rd treatment reduced blood–brain barrier (BBB) dysfunction in experimental autoimmune encephalomyelitis (EAE) mice. The integrity of the BBB was detected by quantitative measurement for Evan’s blue (EB) content at day 20 postimmunization in six animals per group. EB is expressed as micrograms per gram of brain tissue. Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. control mice; ##P < 0.01 vs. vehicle-treated EAE mice by ANOVA with least significant difference test.
treated EAE mice compared with the control mice (P < 0.01; Fig. 6B), and ginsenoside Rd increased the expression of NGF in both cerebral cortex (P < 0.01 compared with both control mice and vehicle-treated mice) and lumbar spinal cord (P < 0.01 compared with the vehicle-treated EAE mice).
Fig. 3. Ginsenoside Rd treatment improved histopathology of experimental autoimmune encephalomyelitis (EAE) mice. Mice were subjected to histopathological assay at day 20 postimmunization (p.i.). Each group had six animals. Consecutive sections were analyzed for hematoxylin and eosin staining and solochrome cyanin staining to detect inflammatory infiltration and demyelination, respectively. Inflammatory infiltration with extensive perivascular cuffing (Aa1, 340), which was widespread in the vehicle-treated EAE mice, was attenuated in ginsenoside Rd-treated EAE mice (Aa2). A large plaque of demyelination occurred in the vehicle-treated EAE mice (Aa3). Demyelination was markedly attenuated by ginsenoside Rd-treated EAE mice (Aa4). The degree of histopathology of lumbar spinal cords was decreased by ginsenoside Rd-treated EAE mice (B). Values represent the mean 6 SEM. **P < 0.01 vs. vehicle-treated EAE mice by Mann-Whitney U test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the both cerebral cortex and lumbar spinal cord of the vehicle-treated EAE mice compared with the control mice (P < 0.01; Fig. 6A). Ginsenoside Rd prevented the reduction of BDNF expression in the two regions of EAE mice compared with the vehicle-treated EAE mice (P < 0.01; Fig. 6A). The expression of NGF decreased in both cerebral cortex and lumbar spinal cord of vehicle-
DISCUSSION MS is a common disabling autoimmune disease without an effective treatment in young adults. Here we provide evidence for a potential role of ginsenoside Rd, which can reduce the severity and progression of EAE in C57 BL/6 mice. In this study, 40 mg/kg/day ginsenoside Rd was found to be the optimal dose and attenuated disease severity, reducing both inflammatory infiltrates and demyelination in lumbar spinal cords of EAE mice. The cytokines produced by inflammatory cells are believed to lead to BBB impairment (Abbott, 2002). The Th1 cytokines, particularly IFN-g, have been shown to alter and even diminish the architectural organization of the tight and adherens junctions, which are subcellular structures that maintain the restrictive properties of the BBB (Minagar and Alexander, 2003). Other studies have shown that IL-4, one of Th2 cytokines, leads to increased stabilization of the cerebral microvasculature tight junction proteins (Poliani et al., 2001). Moreover, previous studies have shown that overexpression of IFN-g was encephalitogenic in the CNS of MS and EAE, and upregulation of IL-4 has been shown to be important for preventing or ameliorating the disease severity of EAE (Frischer et al., 2009; Stadelmann et al., 2011; OrejaGuevara et al. 2012). In this study, we found ginsenoside Rd could decrease the IFN-g/IL-4 ratio (the reduced production of IFN-g and the increased secretion of IL-4) of EAE mice in vivo (cerebral cortex) and in vitro (splenocytes culture supernatants). This ratio is commonly used to describe the balance in the immune system between proinflammatory IFN-g1Th1 cells and antiJournal of Neuroscience Research
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inflammatory IL-41Th2 cells (Steinman, 2007; Valenzuela et al., 2007). Similar effects of ginsenoside Rd have been reported in a rat skin transplantation model (Wang et al., 2012b). These findings provide support that ginsenoside Rd can promote the Th2 shift for the treatment of Th1-driven diseases, including EAE. The results suggest that the attenuation of ginsenoside Rd on EAE may be due to its ability to modulate the immune response.
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Moreover, both IFN-g and IL-4 were implicated in derangement of the BBB, and the permeability of the BBB also was reduced by ginsenoside Rd treatment. Impairment of the BBB is thought to be a crucial step in the pathology of EAE (de Vries et al., 1997), so the reconstitution of BBB integrity by ginsenoside Rd is an important therapeutic effect in EAE mice. In addition to cytokines, derangement of neurotrophin production has been implicated in the pathogenesis of MS and EAE (Noseworthy et al., 2000; Hohlfeld, 2008; Kerschensteiner et al., 2009). Among neurotrophins, BDNF and NGF in the CNS were strictly correlated with clinical outcome of MS. BDNF and NGF levels detected in patients with full recovery from relapse symptoms were significantly higher than that in patients with partial recovery (Caggiula et al., 2005). Moreover, BDNF and NGF have been shown to regulate inflammation, repair, oligodendrocyte proliferation and remyelination, and regeneration of neurons and have been proposed as therapeutic strategies in controlling the disease severity of EAE and MS (McTigue et al., 1998; Kerschensteiner et al., 2009). In this study, we found that BDNF and NGF expressions were reduced in the target organs (cerebral cortex and lumbar spinal cord) of the vehicle-treated EAE mice, which meant the reduction of their protection or that there was an increased consumption of BDNF and NGF in the CNS due to the damaged tissue in EAE (D’Intino et al., 2005). The results provide support that BDNF and NGF participate in the protective mechanisms against tissue damage in EAE (D’Intino et al., 2005; Kerschensteiner et al., 2009). Ginsenoside Rd treatment prevented the reduction of BDNF and NGF expression in both cerebral cortex and lumbar spinal cord. These findings provide additional support for ginsenoside Rd’s neuroprotective effects in EAE. The alteration of BDNF, NGF, IFN-g, and IL-4 in EAE mice might reflect the result of a cross-talk between the nervous and the immune systems in EAE development to some degree (Hohlfeld, 2008; Kerschensteiner et al., 2009). It has been reported that the altertion of BDNF and NGF appears to be mediated by skewing the Th1/Th2 balance toward Th2 type by inhibition of IFNg and enhanced IL-4 production in the CNS of EAE (Villoslada et al., 2000; Arredondo et al., 2001; Makar et al., 2009). IL-4 treatment increases BDNF expression
Fig. 5. Ginsenoside Rd treatment modulated the levels of IFN-g and IL-4 and induced a Th2 bias in the splenocytes culture supernatants and cerebral cortex of experimental autoimmune encephalomyelitis (EAE) mice. Spleens of differently treated mice were aseptically harvested at day 20 postimmunization. Splenocytes (5 3 105) were incubated for 48 hr with myelin oligodendrocyte glycoprotein 35–55 amino acid peptide used for immunization. Cerebral cortex and culture supernatants were collected and analyzed by ELISA to detect levels of IFN-g (A), IL-4 (B), and the ratio of IFN-g/IL-4 (C). Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. control mice; # P < 0.05, ##P < 0.01 vs. vehicle-treated EAE mice by ANOVA with least significant difference test or Dunnett’s T3 test. Journal of Neuroscience Research
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Fig. 6. Ginsenoside Rd treatment prevented the reduction of BDNF and NGF expression in the cerebral cortex and lumbar spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. BDNF (A) and NGF (B) expressions were analyzed by Western blot at day 20 postimmunization. Values represent the mean 6 SEM. *P < 0.05, **P < 0.01 vs. control mice; #P < 0.05, ##P < 0.01 vs. vehicle-treated EAE mice by rank of one-way ANOVA.
in retinal ganglion cells of newborn rat culture and there is an increased production of mRNA for BDNF when astrocytes are incubated with IL-4, which suggest that BDNF is the neurotrophin involved in IL-4 effect (Kerschensteiner et al., 2009; Derecki et al., 2010; de AraujoMartins et al., 2013). IL-4 has also been shown to increase the synthesis of NGF by astrocytes, lymphocytes, and C6 glial cells (Brodie and Goldreich, 1994; Brodie et al., 1998; Arredondo et al., 2001). However, IFN-g can inhibit NGF secretion by astrocytes (Brodie, 1996; Hohlfeld, 2008; Kerschensteiner et al., 2009). In this study, ginsenoside Rd regulated the production/expression of both cytokines (IFN-g and IL-4) and neurotrophins (BDNF and NGF), suggesting that it could modulate the neuroimmune dysfunction in EAE mice. It has been reported that ginsenoside Rd can regulate the NF-jB signal pathway in in vitro and in vivo animal models (Wu et al., 2007; Hu et al., 2013). NF-jB plays a pivotal role in regulating the production/expression of inflammatory cytokines, including IFN-g and IL-4, and neurotrophins, including BDNF and NGF (Yoshimura et al., 2003; Heese et al., 2006). Ginsenoside Rd also can regulate other pathways, including mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERKs) and Akt/GSK-3b signaling that mediate inflammatory and neuroprotective effects (Li et al., 2013; Wang
et al., 2013; Zhang et al., 2013). Defining the exact signal pathway of IFN-g/IL-4 and BDNF/NGF by ginsenoside Rd in EAE mice is attractive and merits further exploration. In conclusion, the data presented here demonstrate that ginsenoside Rd can effectively ameliorate the clinical severity of EAE mice through reducing the permeability of the BBB and regulating the production/expression of IFN-g/IL-4 and BDNF/NGF. These findings suggest that ginsenoside Rd could be a potential therapeutic agent for the treatment of MS. ACKNOWLEDGMENTS The authors have no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. REFERENCES Abbott NJ. 2002. Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat 200:629–638. Arredondo LR, Deng C, Ratts RB, Lovett-Racke AE, Holtzman DM, Racke MK. 2001. Role of nerve growth factor in experimental autoimmune encephalomyelitis. Eur J Immunol 31:625–633. Attele AS, Wu JA, Yuan CS. 1999. Ginseng pharmacology: multiple constituents and multiple actions. Biochem Pharmacol 58:1685–1693. Journal of Neuroscience Research
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