Amelioration of Experimental Autoimmune Encephalomyelitis by

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Amelioration of Experimental Autoimmune Encephalomyelitis by Isogarcinol Extracted from Garcinia mangostana L. Mengqi Wang, Yufei Xie, Youxiu Zhong, Juren Cen, Lei Wang, Yuanyuan Liu, Ying Zhu, Li Tong, and Qun Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04145 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 14, 2016

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Journal of Agricultural and Food Chemistry

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Amelioration of Experimental Autoimmune Encephalomyelitis by Isogarcinol

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Extracted from Garcinia mangostana L.

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Mengqi Wang†, Yufei Xie†, Youxiu Zhong†‡, Juren Cen†§, Lei Wang†, Yuanyuan Liu†,

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Ying Zhu†, Li Tong†, Qun Wei†*

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† Department of Biochemistry and Molecular Biology, Beijing Normal University,

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Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing 100875,

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People’s Republic of China

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‡ National Vaccine and Serum Institute, Beijing 100024, People’s Republic of China

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§ Key Laboratory of Protection and Development Utilization of Tropical Crop

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Germplasm Resources, Ministry of Education, College of Landscape and Horticulture,

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Hainan University, Haikou 570228, People’s Republic of China

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* Corresponding author

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Tel: 86-010-58807365. Fax: 86-010-58807365. E-mail: [email protected].

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1

ACS Paragon Plus Environment


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ABSTRACT

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Isogarcinol is a new natural immunosuppressant that was extracted from Garcinia

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mangostana L. in our laboratory. Knowledge of its effects on treatable diseases and its

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mechanism of action is still very limited. In this study, we explored the therapeutic

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effect of isogarcinol in experimental autoimmune encephalomyelitis (EAE), a murine

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model of multiple sclerosis (MS). Treatment with oral 100 mg/kg isogarcinol

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markedly ameliorated clinical scores, alleviated inflammation and demyelination of

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the spinal cord, and reduced intracranial lesions in EAE mice. The percentages of Th

26

cells and macrophages were also strongly reduced. Isogarcinol appeared to act by

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inhibiting T helper (Th) 1 and Th17 cell differentiation via the Jak/STAT pathway, and

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impairing macrophage function. Our data suggest that isogarcinol has the potential to

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be an effective therapeutic agent of low toxicity for treating MS and other

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autoimmune diseases.

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KEYWORDS: Garcinia mangostana L., isogarcinol, experimental autoimmune

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encephalomyelitis, multiple sclerosis, cytokines, Jak/STAT pathway

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INTRODUCTION

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Multiple sclerosis (MS) is a common and disabling autoimmune disease of the central

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nervous system (CNS) in young adults.1 Underling MS is chronic multifocal

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inflammatory demyelination of the white matter of the CNS, and dysfunction of the

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blood brain barrier (BBB), which results in a range of clinical symptoms, including

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physical and mental disabilities.1-3 There were about 2.3 million people suffering from

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MS in 2013, and the ratio of female to male patients was 2: 1.4 Although the cause of

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MS is unknown, one theory is that it is triggered by immune system destruction of

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myelin,5 and there is evidence that autoreactive T helper (Th) cells are critically

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involved in the development of MS.5-7 Experimental autoimmune encephalomyelitis

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(EAE) is a T-cell-mediated autoimmune disease displaying the key characteristics of

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human MS; it is therefore widely used as a preclinical model of MS.8 The model can

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be induced either by immunizing animals with a variety of antigens, such as myelin

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proteins, CNS homogenate and so on, or by adoptive transfer of encephalitogenic T

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cells. 9 In general, the clinical symptoms of EAE are progressive paralysis and weight

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loss. More importantly, EAE can mimic the typical neuropathological characteristics

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of MS, such as inflammatory demyelination and destruction of the BBB. The

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development of MS and EAE has been attributed to large numbers of Th1 and Th17

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cytokine-secreting CD4+ T cells. 10, 11

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MS is still incurable, but several treatments have proven effective. It is generally

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treated with immunomodulators such as glucocorticoid, intravenous immune globulin,

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interferon beta, glatiramer acetate, methotrexate, cyclophosphamide, etc.12-17 The

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calcineurin (CN) inhibitor, cyclosporine A (CsA), which has outstanding

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immunosuppressive and anti-inflammatory effects, is also a commonly used oral drug

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for MS patients in the clinic.18 Although some of these drugs are given orally, most of 3



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those used in MS are given by intravenous or subcutaneous injection, which is painful

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and inconvenient and carries a high risk of infection. Moreover, these drugs can only

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slow down the progression of the disease, and often have significant adverse effects in

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long-term

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nephrotoxicity and cardiotoxicity.19 Therefore, the development of an oral drug with

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high efficacy and low toxicity is urgently needed.

therapy

including

depression,

nausea,

anemia,

hepatotoxicity,

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It is generally recognized that increasing fruit consumption is a safe way to reduce

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the incidence of cancers, cardiovascular disease and inflammatory and autoimmune

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diseases.20-22 The mangosteen (Garcinia mangostana L.), is a tropical evergreen tree

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originating in the Sunda Islands and the Moluccas of Indonesia, which is well-known

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for its sweet, slightly acid-tasting fruit. This consists of a milky-white edible portion

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and a purple pericarp. There is a long history of use of the fruit, especially its pericarp,

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to treat diarrhea, dysentery and skin and wound infections.23 Recent studies have

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shown that extracts of mangosteen have anti-inflammatory, antitumor, antioxidant and

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antibacterial activities,24, 25 and that it can to treat diarrhea, dysentery and skin and

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wound infections, as well as ameliorating Alzheimer's disease and diabetes.26

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CN is generally used as a

target for screening potential immunosuppressive

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agents, and our laboratory has built a mature system for identifying CN inhibitors

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among natural products.27,

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mangostana L. fruit in our laboratory, inhibits CN activity, but its mechanism of

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action differs from that of CsA. In previous work we showed that it inhibits the

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proliferation of spleen T-lymphocytes induced by concanavalin A (ConA), as well as

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the mixed lymphocyte reaction, and has an excellent therapeutic effect in a variety of

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animal models, including those of delayed type hypersensitivity (DTH), graft survival

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in allogeneic skin transplantation, rheumatoid arthritis (RA) and Systemic Lupus

28

Isogarcinol (Figure 1A), extracted from Garcinia

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Erythematosus (SLE).27, 29, 30

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Isogarcinol also has anti-inflammatory activity both in vitro and vivo.29 More

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importantly, it was less hepatotoxic and nephrotoxic than CsA in experimental

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animals. Therefore, it may be a safer immunosuppressant. In this study, we explored

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its effects on inflammatory/autoimmune disorders and its possible mode of action in

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the EAE model.

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MATERIALS AND METHODS

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Mice and drug. Female C57BL/6 mice (6–8 weeks) were purchased from Beijing

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Vital River (Beijing, China). All mice were maintained under standard conditions with

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standard mouse chow and water ad libitum. All animal experimental procedures were

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authorized by the Animal Ethics Committee of Beijing Normal University (NO.

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CLS-EAW-2013-015) and were executed in strict accordance with institutional

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guidelines. Isogarcinol was extracted and purified by J. Cen of our lab, and was more

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than 95% pure27, 31.

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Reagents. A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular

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Technologies, Inc. (Beijing, China). ConA, complete Freund’s adjuvant (CFA) and

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dimethyl sulfoxide (DMSO) were from Sigma–Aldrich (St. Louis, MO, USA).

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35–55

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(MEVGWYRSPFSRVVHLYRNGK), of purity > 95%, was synthetized by Scilight

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Biotechnology, LLC (Beijing, China). Mycobacterium tuberculosis H37Ra was from

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Difco Laboratories (Detroit, MI, USA), pertussis toxin (PTX) from List Biological

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Laboratories (Campbell, CA, USA), and. Percoll from GE Healthcare (Piscataway, NJ,

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USA). Flow cytometry antibodies against CD4, CD8, B220 and CD11b, and the

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mouse Th1/Th2/Th17 Cytokine Kit and Skim Milk were from BD Biosciences (San

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Jose, CA, USA), and the mouse CD4 isolation kit and MACS separation columns

residue

peptide

of

myelin

oligodendrocyte

5


glycoprotein

The

(MOG)


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were from Miltenyi (Germany). RPMI 1640, fetal bovine serum (FBS) and TRIzol

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reagent were from Life Technologies (Carlsbad, CA, USA), and oligo dT Primer,

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dNTP mixture, RNase inhibitor, Reverse Transcriptase M-MLV and SYBR Premix Ex

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Taq were from TaKaRa Bio (Kusatsu, Shiga, Japan). The bicinchonininc acid (BCA)

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protein quantitation kit was purchased from Thermo Fisher Scientific (San Jose, CA,

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USA), and Western blotting antibodies, the Stat Antibody Sampler Kit and the

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Phospho-Stat Antibody Sampler Kit were from Cell Signaling Technology (Danvers,

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MA, USA).

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EAE induction. EAE was induced as previously described.5,

8

The first

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immunization day was defined as day 0. Mice were immunized s.c. with 200 µg

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MOG35–55 and 400 µg Mycobacterium tuberculosis H37Ra emulsified with 200 µl of

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CFA. Then they were administered i.p. 200 ng PTX in PBS on days 0 and 2

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postimmunization. The clinical behavior of the mice was examined daily and scored

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on the standard scale of Kuerten et al.32

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Preparation of splenocytes, mononuclear cells and CD4+ cells. Brain, spinal

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cord and spleen were filtered through 70-µm nylon cell strainers to prepare single-cell

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suspensions. MNCs from the CNS were purified using a 37/70% Percoll gradient at

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780 g for 25 min (4 ℃). CD4+ cells were purified by negative selection MACS with a

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Miltenyi CD4 isolation kit.

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ConA/MOG35-55-induced T cell proliferation. Splenocytes (5×105 per well in

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96-well plates) derived from EAE mice stimulated with MOG35-55 (20 µg/mL), and

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normal congenic mice stimulated with ConA (5 µg/mL), were cultured with different

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concentrations of isogarcinol (2.5, 5 and 10 µM) for up to 48 h. Proliferation was

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measured with the CCK-8 assay and growth inhibition was calculated from the

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equation: viability (%) = (OD450 of isogarcinol group / OD450 of control group) ×100. 6


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Drug administration. For treatment, isogarcinol (100 mg/kg) or CsA (50 mg/kg)

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was given daily starting 11 d after immunization until 28 days postimmunization. The

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naïve and vehicle groups received the same volume of solvent (23% ddH2O, 12%

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ethanol, 60% peanuts oil and 5% Tween-80) as the treatment group. The dosages were

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based on our previous findings. 27, 29

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Histopathological Analysis. Spinal cords were obtained from animals and

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immediately fixed in 4% paraformaldehyde. After being embedded in paraffin, they

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were cut and stained with Hematoxylin and Eosin (H&E), and Luxol Fast Blue (LFB).

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Inflammation and demyelination were assessed according to Wei et al and Li et al.33,

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Magnetic resonance imaging (MRI). MRI experiments were performed on a

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3.0-T scanner (Prisma, Siemens Healtcare, ER, GER). Mice were anesthetized with 1%

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nembutal and scanned for T1-weighted images (TR/TE = 433.2/11, FOV = 17 × 24,

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matrix acquisition = 256 × 389, slice thickness = 0.5 mm). Gd-DTPA-enhanced T1

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images, with scan sequences the same as for T1, were made after i.p. injection of 0.5

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mmol/kg gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).

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Drug toxicity in EAE mice. To compare the toxicity of isogarcinol and CsA, blood

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samples of treated EAE mice were collected to measure biochemical parameters and

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carry out routine blood test.

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Cell population analyses and cytokine measurements. Splenocytes and MNCs

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from the CNS were prepared as described above and incubated with

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fluorochrome-conjugated antibodies to CD4, CD8, B220, and CD11b. Culture

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supernatants were collected from 20 µg/mL MOG-stimulated splenocytes after 48 h to

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measure concentrations of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A and IL-10 with a

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Th1/Th2/Th17 Cytokine Kit by flow cytometry. 7



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Real-time PCR for transcription factors related to Th cell differentiation. Total

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RNA was extracted from CD4+ T cells and MNCs with TRIzol reagent. cDNA

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synthesis and relative quantitative real-time PCR were performed according to the

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manufacturer’s instructions. The mRNA levels of T-bet, GATA3, RORγt and Foxp3

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genes were analyzed with an Applied Biosystems 7500. The sequences of the primers

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used were as follows: β-actin, AGAGGGAAATCGTGCGTGAC (forward) and

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CAATAGTGATGACCTGGCCGT (reverse); T-bet, GTTCAACCAGCACCAGACA-

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GAG (forward) and TGGTCCACCAAGACCACATC (reverse); GATA3, GGATGT-

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AAGTCGAGGCCCAAG

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(reverse); RORγt, TCTGCAAGACTCATCGACAAGG (forward) and CACATGTT-

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GGCTGCACAGG (reverse); Foxp3, TGCCTTCAGACGAGACTTGGA (forward)

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and GGCATTGGGTTCTTGTCAGAG (reverse).

(forward)

and

ATTGCAAAGGTAGTGCCCGGTA

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Western blot analysis of the Jak/STAT signaling pathway. Protein samples were

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obtained by centrifugation after 24 h stimulation with MOG35-55 (20 µg/mL). A BCA

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kit was used to measure protein concentrations. The samples were electrophoresed on

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SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked with

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5% skim milk powder in TBST (TBS + 1‰ Tween-20) and blotted with primary and

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secondary

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electrochemiluminescence (ECL) kit.

antibodies.

Specific

bands

were

visualized

with

an

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Statistics. Data are expressed as means ± SEM. Student’s t-tests were used to

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analyze differences between paired samples. Other differences were assessed by

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one-way ANOVA. p values < 0.05 were deemed statistically significant.

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RESULTS

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Isogarcinol inhibits the proliferation of Encephalitogenic T Cells and

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efficiently ameliorates the Clinical Symptoms of EAE Mice. Splenocytes from 8


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EAE mice stimulated with MOG35-55 (20 µg/mL) or from normal congenic mice

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stimulated with ConA (5 µg/mL) were cultured in the presence of different

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concentrations of isogarcinol, and their proliferation was measured using the CCK-8

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assay after 48 h. The concentration of isogarcinol used was based on our previous

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study of ConA-stimulated T cells proliferation.27 As shown in Figure 1B, isogarcinol

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inhibited the proliferation of encephalitogenic T cells induced by MOG35-55 at a low

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concentration (2.5 µM), whereas a concentration of up to 10 µM was needed to inhibit

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ConA-induced nonspecific proliferation. This result suggested that encephalitogenic T

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cells were very sensitive to isogarcinol treatment. Therefore, we examined the effect

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of isogarcinol on the development of an EAE model. C57BL/6 mice were immunized

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as described in MATERIALS AND METHODS, and divided into 3 groups of vehicle,

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isogarcinol and CsA mice. EAE developed in 100% of the mice in all 3 groups, with,

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as expected, very similar onset times. Isogarcinol (100 mg/kg) or CsA (50 mg/kg)

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treatments were started from day 11 postimmunization, when the disease generally

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develops in mice, and was continued daily thereafter. As shown in Figure 2A,

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isogarcinol significantly decreased disease severity, as did CsA. The body weights of

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the isogarcinol/ CsA- treated mice were unaffected at the peak of the disease (Figure

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2B).

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Isogarcinol Treatment Alleviates Inflammation and Demyelination in the

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Brain and Spinal Cord. Inflammatory infiltration and demyelination in the CNS, and

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BBB lesions, are typical characteristics of MS and EAE.1, 3 Histological studies of

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spinal cords were carried out on day 18, and the results are shown in Figure 2C.

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Compared with healthy mice, infiltration of a large number of inflammatory cells and

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large areas of demyelination can be seen in H&E and LFB staining of the

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vehicle-treated mice. Infiltration and demyelination were greatly reduced in the mice 9



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treated with isogarcinol or CsA (Figure 2D and 2E). At the same time, the number of

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CNS MNCs was clearly reduced by isogarcinol (Figure 2F). Since the integrity of the

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BBB can be evaluated by imaging, we used MRI to check for intracranial signal

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changes in the mice on day 18 post-immunization. A number of sporadic hyperintense

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lesions were observed in the vehicle-treated mice after injection with Gd-DTPA,

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indicating damage to the BBB. In contrast, no abnormal signals appeared in the

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isogarcinol or CsA-treated mice (Figure 3). These results show that isogarcinol can

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prevent intracranial lesions in MOG35-55-treated mice.

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Isogarcinol has no significant Toxicity in the EAE Model.

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To evaluate any toxic side effects of isogarcinol in the EAE model, biochemical

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and hematological parameters were examined. Blood samples of the mice were

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collected on day 28. As shown in Figures 4 and 5, treatment with 100 mg/kg

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isogarcinol did not cause any major hematopoietic or biochemical changes in the mice,

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whereas treatment with 50 mg/kg CsA led to a reduced red blood cell count (P =

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0.0048) and increased alkaline phosphatase (ALP, P = 0.0055) and total bilirubin

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(TBIL, P = 0.0024) levels. CsA also raised platelet numbers and creatinine (CRE)

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levels compared with the isogarcinol group, but the effects were not statistically

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significant.

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Isogarcinol reduces activation of the CD4+ and CD11b+ Cell Populations. There

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is some evidence that the tissue damage in MS and EAE is correlated with certain

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immune responses. 8, 35 We therefore isolated MNCs from the spleens and CNS of the

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mice, and used flow cytometry to analyze the percentage expression of surface CD4,

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CD8, B220, and CD11b, representing Th cells, cytotoxic T cells, B cells and

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infiltrated macrophages/microglia, respectively. The percentages of CD4+ (spleen, P =

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0.0011; CNS, P = 0.0002) and CD11b+ (spleen, P = 0.0152; CNS, P = 0.0073) cells 10


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were significantly reduced by isogarcinol in both spleen and CNS. However, the

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percentages of CD8+ and B220 cells were not altered (Figure 6).

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Isogarcinol reduces numbers of Th1 and Th17 by inhibiting the Jak/STAT

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Signaling Pathway. Since isogarcinol inhibited the activation of CD4+ T cells, we

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investigated the subset of Th cells affected by it. Because the differentiation of Th

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cells is promoted by the Jak/STAT pathway, we hypothesized that isogarcinol affects

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the STAT protein family. Protein samples were obtained from isogarcinol- and

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vehicle-treated mice as described in MATERIALS AND METHODS. As shown in

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Figure 7A and B, the phosphorylation levels of STAT1 and STAT3, which are closely

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related to the differentiation of Th1 and Th17 cells, were markedly reduced in

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isogarcinol-treated mice, whereas STAT6 and STAT5, whose levels are related to Th2

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and Treg cell differentiation, were not affected. We then used CD4+ cells, obtained

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from spleen and CNS, to detect the expression of key transcription factors regulated

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by the STAT family. T-bet for Th1 cells and RORγt for Th17 cells were

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down-regulated in the isogarcinol-treated mice, whereas GATA3 representing Th2

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cells and Foxp3 representing Treg cells were unaltered (Figure 7C). To confirm the

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effect of isogarcinol on Th1 and Th17 cells, we measured cytokines related to Th cells

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in the supernatants of splenocytes stimulated ex vivo with 20 µg/mL MOG35-55 for 48

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h. In contrast to the vehicle-treated group, isogarcinol significantly reduced the

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production of both the Th1-related proinflammatory cytokines IFN-γ (P = 0.0137)

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and TNF-α (P = 0.0373) and the Th17-related cytokine IL-17A (P = 0.0365). In

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addition, it had a small effect effect on IL-6, which is also a Th17-related cytokine

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(Figure 7D, left). Isogarcinol had no effect on cytokines IL-4, related to Th2 cells, and

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IL-2 and IL-10, related to Treg cells (Figure 7D, right). These results together indicate

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that isogarcinol selectively inhibits the Jak/STAT signaling pathway and the 11



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production of proinflammatory cytokines (e.g., TNF-α and IL-6) and so reduces the

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symptoms of EAE mice.

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DISCUSSION

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MS is the commonest autoimmune disorder of the CNS, and its frequency increases

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year by year.36 Isogarcinol is a new immunosuppressant with dramatic effects on

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many immune-related diseases, such as acute inflammation, autoimmune diseases,

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etc.27, 29, 30 Therefore, we wished to explore whether it is effective against MS, and, if

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so, to understand its mechanism of action. The EAE murine model has been used to

268

test novel agents for treating MS, and the MOG35-55 peptide-induced C57BL/6 mouse

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is popular in EAE studies.37 Since female MS patients far outnumber males, we

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immunized female C57BL/6 mice with the MOG35-55 peptide to generate the EAE

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disease model. To determine the therapeutic properties of isogarcinol in EAE, we first

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examined the inhibitory effect of isogarcinol on the in vitro proliferation of

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encephalitogenic T cells induced by MOG35-55, before carrying out in vivo studies. We

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found that 2.5 µM isogarcinol markedly inhibited proliferation induced by MOG35-55,

275

but not by ConA. This result indicated that isogarcinol might be effective in EAE.

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MS is characterized by physical and mental clinical signs, including perivascular

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infiltration of T lymphocytes, demyelination of the white matter of the CNS and

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multifocal points, and destruction of the blood brain barrier, as seen by histopathology

279

and MRI.37, 38 In our experiments we used clinical scores, H&E staining, LFB staining

280

and MRI to evaluate the EAE model. We found that the number of infiltrating cells in

281

the model group was significantly higher than in the controls, and there was extensive

282

demyelination. Several foci were evident on the MRI scans of the model mice,

283

reflecting the acute inflammation and damage to the BBB; the meninges were also

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enhanced in Gd-DTPA-enhanced T1 images, due to the damaged BBB, all these 12


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effects resembled those in MS patients.39-41 Thus, together with clinical scores, the

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data demonstrated that we had successfully established an EAE model. We then used

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100 mg/kg isogarcinol to treat the sick mice, and found it greatly improved clinical

288

scores and the pathological changes in the brains and spinal cords of the EAE mice.

289

CD4+ Th cells exert their function by regulating other immune cells of the adaptive

290

immune system, and they differentiate into at least four major subsets, i.e., Th1, Th2,

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Th17, and regulatory T (Treg) cells in response to various factors induced by the

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Jak/STAT signaling pathway.42 CD4+ T cells play a critical role in the pathogenesis of

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MS and EAE, and the main reason for the encephalomyelitis of EAE mice is that Th1

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and Th17 cells cause activation of the microglia and macrophage infiltration.43, 44 The

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differentiation of Th1 cells, which secrete IFN-γ and TNF-α, depends on IFN-γ

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activation of STAT1 to increase expression of the transcription factor T-bet. Also

297

IL-17-secreting Th17 cells differentiate in response to IL-6 phosphorylation of STAT3

298

and further induce the transcription factor, RORγt. The Th1-secreted cytokines IFN-γ

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and TNF-α, and Th17-secreted cytokine IL-17, are markedly increased in both MS

300

and EAE.38, 45 In this study, isogarcinol decreased the percentage of CD4+ Th cells,

301

and down-regulated the concentrations of secreted Th1 and Th17. These effects were

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all dependent on inhibition of the Jak/STAT signaling pathway: isogarcinol inhibited

303

the phosphorylation of STAT1 thus reducing T-bet expression and reducing the

304

differentiation of Th1 cells and interfering with cytokine secretion. Similarly, it

305

reduced the expression of RORγt by inhibiting the phosphorylation of STAT3, so

306

reducing the number of Th17 cells and blocking cytokine secretion. In addition, there

307

is ample evidence that levels of intracellular ROS can be modified by Th17

308

differentiation. Tse et al. and Ghoreschi et al. reported that the phenotype of Th17 was

309

skewed in T cells from ROS-deficient mice, and these mice were more susceptible to 13



310

EAE.46, 47 Since we recently showed that isogarcinol increased ROS in A549 and

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SMMC-7721 (data unpublished), and others have shown that isogarcinol increased

312

ROS in HL-60 cells,48 this suggests that it may reduce Th1 and Th17 cells by

313

increasing ROS levels.

314

Macrophages as antigen-presenting cells (APCs) have the capacity to activate

315

immune responses and mediate inflammatory reactions. Activation of macrophages,

316

which damages CNS structures and leads to progressive paralysis, also plays an

317

important role in MS and EAE.49 Therefore, we explored the effect of isogarcinol on

318

macrophages by measuring the proportion of CD11b+ cells and the concentration of

319

pro-inflammatory cytokines in the EAE model. The results showed that isogarcinol

320

impaired the functioning of macrophages, and downregulated levels of TNF-α, IL-6

321

and IL-17. It is worth mentioning that in previous work we found that isogarcinol

322

suppressed the inflammatory response induced by LPS in murine macrophage RAW

323

264.7, and had anti-inflammatory activity in mouse RA and SLE models29, 30. Hence

324

we believe that isogarcinol reduces the paralysis, inflammatory cell infiltration and

325

intracranial inflammation of EAE mice by weakening macrophage function.

326

Moreover, as TNF-α and IL-17 are also products of Th1 and Th17 cells, it is likely

327

that isogarcinol regulates Th1 and Th17 cells in EAE mice at least in part via its

328

anti-inflammatory effect.

329

CsA, the inhibitor of CN, is still the most popular immunosuppressant and is

330

commonly used in MS patients.18, 50 We showed above that 50 mg/kg CsA had the

331

same effect as 100 mg/kg oral isogarcinol in improving the clinical signs of EAE mice.

332

However, the EAE mice of the CsA-treated group had reduced numbers of red blood

333

cells and increased ALP and TBIL, reflecting hematological and hepatic toxicity. In

334

our previous study we showed CsA caused extensive death of mouse spleen cells and 14


Page 14 of 35

Page 15 of 35


335

had a greater effect on biochemical indicators of liver and kidney function than

336

isogarcinol.27 Furthermore, in those experiments, the CsA group mice were less active

337

and displayed more fur ruffling than the other mice. Overall isogarcinol has less toxic

338

side effects, which is an obvious advantage in long-term treatment.

339

To sum up, we have shown that isogarcinol probably alleviates the clinical signs of

340

EAE by suppressing Th1//Th17 responses by inhibiting the Jak/STAT signaling

341

pathway and by its anti-inflammatory activity. Thus isogarcinol could be an effective

342

drug against MS with low toxicity. The properties of isogarcinol remind us that we

343

should pay more attention to seeking compounds from fruit and agricultural products

344

that may have immunosuppressive, anti-inflammatory and other functions.

345 346

ABBREVIATIONS USED

347

APCs, anti-presenting cells; ALP, alkaline phosphatase; ALT, glutamic-pyruvic

348

transaminase; AST, Glutamic-oxalacetic transaminase; BBB, blood brain barrier;

349

BCA, bicinchonininc acid; BUN, urea nitrogen; CCK-8, Cell Counting Kit-8; CFA,

350

complete Freund’s adjuvant; CN, calcineurin; CNS, central nervous system; ConA,

351

concanavalin A; CRE, creatinine; CsA, cyclosporine A; DMSO, dimethyl sulfoxide;

352

DTH,

353

encephalomyelitis; Gd-DTPA, gadolinium-diethylenetriaminepentaacetic acid; H&E,

354

Hematoxylin and Eosin; HGB, hemoglobin; IFN-γ, interferon gamma; IL, interleukin;

355

LFB, Luxol Fast Blue; MNCs, mononuclear cells; MOG, myelin oligodendrocyte

356

glycoprotein; MS, multiple sclerosis; MRI, magnetic resonance imaging; NFκB,

357

nuclear factor kappa B; PLT, platelet; PTX, pertussis toxin; RA, rheumatoid arthritis;

358

SLE, systemic lupus erythematosus; STAT, signal transducers and activators of

359

transcription; TBIL, total bilirubin; Th, T helper; Jak, janus kinase; TNF-α, tumor

delayed

type

hypersensitivity;

EAE,

experimental

15


autoimmune


360

necrosis factor alpha; RBC, red blood cell; ROS, reactive oxygen species; WBC,

361

white blood cell.

362 363 364 365

Funding

366

This work was supported by the National Natural Science Foundation of China, the

367

National Important Novel Medicine Research Project, the Fundamental Research

368

Funds for the Central Universities, and the special fund of the co-construction project

369

of the Beijing Education Committee.

370

Notes

371

The authors declare no competing financial interests.

372

16


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FIGURE CAPTIONS

540 541

Figure 1. Isogarcinol. (A) Chemical structure. (B) Splenocytes from EAE mice

542

stimulated with MOG35-55 (20 µg/mL), and from normal congenic mice stimulated

543

with ConA (5 µg/mL), were cultured in the presence of different concentrations of

544

isogarcinol, and their proliferation was measured by the CCK-8 assay after 48 h (n =

545

3). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01.

546 547

Figure 2. Isogarcinol ameliorates the clinical signs of EAE induced as described in

548

MATERIALS and METHODS. (A) Mean clinical scores of EAE mice given vehicle,

549

isogarcinol (100 mg/kg) or CsA (50 mg/kg) by oral gavage daily, starting from 11 d

550

postimmunization. (B) Mean body weights of normal, vehicle-, isogarcinol- and

551

CsA-treated EAE mice on day 18 postimmunization (n = 8). (C) H&E and Luxol Fast

552

Blue (LFB) staining of spinal cords from normal mice and EAE mice treated with

553

vehicle, isogarcinol or CsA on day 18 postimmunization. (D) Quantification of CNS

554

infiltrates (n = 4). (E) Pathology scores for demyelination (n = 4). (F) The absolute

555

number of MNCs in the CNS was counted in normal, vehicle- and isogarcinol-treated

556

EAE mice on day 18 postimmunization (n = 4). All values are expressed as mean ±

557

SEM. ***P < 0.001; ###P < 0.001.

558 559

Figure 3. Isogarcinol reduces intracranial lesions and meningeal enhancement in EAE.

560

In vivo 3.0 T MRI scanned brains of normal mice or EAE mice treated with vehicle,

561

isogarcinol or CsA on day 18 postimmunization. (A-D) T1-weighted image. (E-G)

562

T1-weighted image obtained 10 min after injection of Gd-DTPA enhancer. Arrows

563

indicate focal lesions in the subcortex and enhancements in the meninges (F). 25



564 565

Figure 4. Effect of isogarcinol and CsA treatments on hematopoietic function in EAE

566

mice. White blood cell counts (A), red blood cell counts (B), hemoglobin

567

concentrations (C) and platelet counts (D) were obtained with an automated

568

hematologic analyzer after the last day of treatment (n = 8). Results are expressed as

569

means ± SEM. *P

Amelioration of Experimental Autoimmune Encephalomyelitis by

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