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International Immunopharmacology xxx (2013) xxx–xxx

INTIMP-02902; No of Pages 6

Contents lists available at SciVerse ScienceDirect

International Immunopharmacology

j ourna l homepage: www.e lsev ie r .com/ locate / in t imp

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Preventive effects of andrographolide on the development of diabetes in autoimmunediabetic NOD mice by inducing immune tolerance

Chengliang Zhang 1, Ling Gui 1, Yanjiao Xu, Tao Wu, Dong Liu ⁎Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430033, China

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⁎ Corresponding author at: Department of Pharmacy,College, Huazhong University of Science and Technology430030, China. Tel./Fax: +86 27 83663643.

E-mail address: ld_2069@yahoo.cn (D. Liu).1 These authors contributed equally to this work.

1567-5769/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.intimp.2013.05.002

Please cite this article as: Zhang C, et al, Preveby inducing immune tolerance, Int Immunop

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Article history:Received 13 November 2012Received in revised form 1 May 2013Accepted 1 May 2013Available online xxxx

Keywords:AndrographolideAutoimmune diabetesTh1Th2Th17

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RAndrographolide, an active component in traditional anti-diabetic herbal plants, is a diterpenoid lactone iso-lated from Andrographis paniculata because of its potent anti-inflammatory and hypoglycemic effects. How-ever, the effect of andrographolide on the development of diabetes in autoimmune non-obese diabetic (NOD)mice remains unknown. This study aimed to investigate the protective effects of andrographolide on the de-velopment of autoimmune diabetes and clarify the underlying mechanism. NODmice were randomly dividedinto four groups and administered with water and andrographolide at 50, 150, and 500 mg/kg body weightfor four weeks. ICR mice were also selected as the control group. Oral glucose tolerance and histopathologicalinsulitis were examined. Th1/Th2/Th17 cytokine secretion was determined by ELISA. The transcriptionalprofiles of T-bet, GATA3, and RORγt in the pancreatic lymphatic node samples derived from the NOD micewere detected by RT-PCR. After four weeks of oral supplementation, andrographolide significantly inhibitedinsulitis, delayed the onset, and suppressed the development of diabetes in 30-week-old NOD mice in a dosedependent manner. This protective status was correlated with a substantially decreased production of inter-feron (IFN)-γ and interleukin (IL)-2, increased IL-10 and transforming growth factor (TGF)-β, and a reducedIL17. Andrographolide also increased GATA3mRNA expression but decreased T-bet and RORγt mRNA expres-sions. Our results suggested that andrographolide prevented type 1 diabetes by maintaining Th1/Th2/Th17homeostasis.

© 2013 Elsevier B.V. All rights reserved.

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RR1. Introduction

Type 1 diabetes (T1D) is caused by immunologically mediatedselective destruction of insulin-producing β-cells in pancreatic isletswith consequent insulin deficiency in humans and non-obese diabetic(NOD) mouse models [1]. Although pathogenesis and autoimmuneprocesses of T1D have been determined, the clinical onset of T1Dhas not been intervened in, delayed, or prevented effectively [2]. Ingeneral, the progression of T1D is inhibited by two strategies: (1)suppressing or eliminating autoimmunity or (2) replacing β cells toreplenish insulin [3]. Methods suppressing autoimmunity against βcells, such as the use of anti-CD3, anti-CD20 monoclonal antibody,or GAD65, alter Th1/Th2 balance and destroy autoreactive immunecells [4,5]. Nevertheless, prophylactic or therapeutic drugs currentlyavailable for T1D are scarce.

Andrographolide (Fig. 1) is a diterpenoid lactone isolated fromAndrographis paniculata, a traditional Chinese medicine that has been ex-tensively used to treat infection, inflammation, cold, fever, and diarrhea in

Tongji Hospital, Tongji Medical, 1095 Jiefang Avenue, Wuhan

rights reserved.

ntive effects of andrographolharmacol (2013), http://dx.d

China [6,7]. Andrographolide is also considered a pharmacologically im-munological, antibacterial, antiviral, anti-inflammatory, antithrombotic,and hepatoprotective substance [8–10] and functions in glycemic control.Zhang et al. [11] demonstrated that andrographolide-lipoic acid conjugateis hypoglycemic and β-cell protective in alloxan-treated mice (a T1Dmodel) because this conjugate has antioxidant and NF-κB inhibitory ac-tivities. In addition, oral treatment of andrographolide can lower plasmaglucose concentrations dose dependently in streptozotocin-induceddiabetic rats [12]. Moreover, andrographolide elicits immunomodulatoryeffects on the immune system. Iruretagoyena et al. [13] reported thatandrographolide significantly alleviates experimental autoimmune en-cephalomyelitis by inhibiting T cell and antibody responses tomyelin an-tigens. This study aimed to determine whether or not andrographolidecontrols the prevention of diabetes development in autoimmune NODmice andwhether or not such an effect originates from an immune regu-latory activity.

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2. Materials and methods

2.1. Reagents

Andrographolide (purity > 98%; Hubei Jianyuan Chemical Co., Ltd.,Wuhan, China)was used to prepare a 50 mMstock solution by dissolving

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Fig. 1. Chemical structure of andrographolide.

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in dimethyl sulfoxide. The resulting mixture was serially diluted in PBSimmediately prior to experiments.

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2.2. Experimental animals

Female NOD mice (NOD/LtJ) and female ICR mice (as age-matchedcontrol group) aged seven weeks were purchased from Beijing HFKBio-Technology Co., Ltd. (Beijing, China). The experimental mice weremaintained at the Experimental Animal Center of Tongji MedicalCollege (Huazhong University of Science and Technology, China)under specific pathogen-free conditions. The mice were housed instainless steel cages and kept at a controlled temperature (25 ± 2 °C)and ambient humidity (50% to 75%). Light was maintained following a12 h dark-light cycle. All of the mice were continuously provided withchow diet and tap water throughout the experiment. The experimentswere carried out according to the National Institutes of Health Guidefor the Care and Use of Laboratory Animals approved by the AnimalEthics Committee of Tongji Medical College, Huazhong University ofScience and Technology.

Fig. 2. Effect of andrographolide on the prevention of diabetes in NODmice. (A) Cumulativediabetic incidence from 8 to 30 weeks of age, which wasmonitored in the female NODmicetreated with vehicle (control) and andrographolide (50, 100, and 150 mg/kg) from 8to 12 weeks. (B) OGTT was performed at the age of 12 weeks. The mice orally received2.5 g/kg glucose after 12 h of fasting, and the blood samples were collected from the caudalvein at t = 0, 30, 60, 90 and 120 min to determine the glucose concentrations. Bars refer tomeans ± SDwith eightmice per group. IC: age-matched control (ICRmice). CO: control. AL:low-dose andrographolide (50 mg/kg bw). AM:medium-dose andrographolide (100 mg/kgbw). AH: high-dose andrographolide (150 mg/kg bw).

UNCO2.3. In vivo experimental design

Eight-week-old NOD/LtJ were randomly divided into four groups:(1) control group (CO, 0.3 ml of deionized water); (2) low-doseandrographolide group [AL, 50 mg/kg body weight (bw)]; (3) medium-dose andrographolide group (AM, 100 mg/kg bw); and (4) high-doseandrographolide group (AH, 150 mg/kg bw). Female ICR mice (IC,0.3 ml of deionized water) were selected as the age-matched group.During the study period, the grouped mice were orally administeredwith additional 0.3 ml of deionized water or andrographolide solutiondirectly into the stomach by using a gavage needle continuously forfour weeks. Eight 12-week-old mice per group were randomly selectedand executed to perform an oral glucose tolerance test (OGTT). Histo-pathological insulitis was evaluated by grading each islet in the pancre-atic sections under double-blinded conditions. The degree of insulitiswas graded as follows: normal islet, score 1; perivascular/periductalinfiltration, score 2; peri-insulitis, score 3; mild insulitis (b25% of theislet infiltrated), score 4; and severe insulitis (>25% of the islet infil-trated), score 5. The remaining 12 mice per group were fed until theywere 30 weeks old to assess the incidence of diabetes.

Please cite this article as: Zhang C, et al, Preventive effects of andrographolby inducing immune tolerance, Int Immunopharmacol (2013), http://dx.d

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2.4. OGTT

To determine the ability of metabolizing glucose, eight mice ineach group were subjected to OGTT during the experimental period.The mice were fasted for 14 h before oral glucose challenge andthen orally administered with 2.5 g/kg bw glucose [D-(+)-glucoseanhydrous; MP Biomedicals Inc., Eschwege, Germany] suspended inapproximately 0.1 ml to 0.2 ml of deionized water. The whole bloodof each experimental mouse was drawn from the tail vein at 0, 30,60, 90, and 120 min to determine the glucose levels. Area under theglucose-time curve (AUC) was calculated.

2.5. Serum collection for cytokine determination

The experimental mice were sacrificed after four weeks ofandrographolide treatment. The mice were fasted for 12 h, weighed,anesthetized, and immediately bled using retro-orbital venous plexus

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puncture to collect the blood into a 1.5 ml vial. The blood was allowedto stand for 2 h at room temperature and then centrifuged at12,000 ×g for 15 min at 4 °C to separate the serum. The sera werecollected and stored at −20 °C for analysis. The concentrations ofcytokines, including IL-2, IL-10, IFN-γ, TGF-β, and IL-17 in the serumwere determined by quantitative sandwich ELISA with commerciallyavailable ELISA kits. The concentration values were expressed as pg/ml.

2.6. RT-PCR assay

Eight NOD mice per group were sacrificed after four weeks ofandrographolide treatment. RNA was isolated from a pancreaticlymph node (PLN) by using TRIzol according to the manufacturer'sprotocol (Invitrogen Life Technologies) and converted to first-strandcDNA templates by using a cDNA synthesis kit (AmershamBiosciences).These cDNA templates were used to perform PCR with T-bet primers(5′-GTGACCCAGATGATTGTGCT-3′ and 5′-GGTTGGGTAGGAGAGGAGAG-3′), RORγt primers (5′-ACCTCCACTGCCAGCTGTGTGCTGTC-3′and 5′-TCATTTCTGCACTTCTGCATGTAGACTGTCCC-3′), GATA3 primers(5′-CCTTAAAACTCTTGGCGTCC-3′ and5′-AGACACATGTCATCCCTGAG-3′),and G3PDH primers (5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′). These primers were designed using Primer Pre-mier 6.12. PCR products were resolved in DNA gels and visualized withethidium bromide. Densities of the resulting bands were measured

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Fig. 3. Representative images of pancreatic islets at 200× magnification from 12-week-ol(B) model control group; (C) low-dose group; (D) medium-dose group; and (E) high-dosefor insulitis. *P b 0.05, **P b 0.01 compared with the control mice. Bars refer to means ± S

Please cite this article as: Zhang C, et al, Preventive effects of andrographolby inducing immune tolerance, Int Immunopharmacol (2013), http://dx.d

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using a scanning densitometer. AU values were obtained from the ratioof the signal of each band to that of the G3PDH control.

2.7. Statistical analysis

Results were expressed as means ± SD. Data were analyzedstatistically by one-way ANOVA. If justified, statistical probability(P b 0.05) was determined by conducting Duncan's multiple rangetests in SPSS 14.0. Differences were considered statistically significantif P b 0.05.

3. Results

3.1. Delayed onset and decreased incidence of diabetes in NOD mice byandrographolide treatment

We initially examined the cumulative incidence of diabetes in NODmice treated with or without andrographolide for 30 weeks. T1D spon-taneously developed in approximately 70% of the non-treated femaleNOD mice, and this finding is consistent with that in a previous study[14]. By contrast, intragastrical (i.g.) injection of andrographolideprevented the development of diabetes in NOD mice aged 30 weeks(Fig. 2A). The incidences of diabetes were 1 of 12 (~8%), 2 of 12(~17%), and 5 of 12 (~42%) for the high-, medium- and low-dose

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d NOD mice treated with andrographolide for four weeks. (A) normal control group;group. (F) Pancreatic sections from NOD mice in different groups were scored blindlyD with eight mice per group.

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andrographolide-treated mice, indicating that andrographolide evi-dently inhibited the development of autoimmune diabetes in NODmice in a dose dependent manner.

To examine the effect of andrographolide on blood glucose re-sponses to oral glucose challenge, the experimental mice aged12 weeks were subjected to OGTT. Andrographolide-treated NODmice had significantly lower blood glucose levels at 30 and 60 min,indicating that the treated mice exhibited a higher glucose tolerancethan the untreated ones (Fig. 2B).

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3.2. Inhibition of insulitis by andrographolide

T1D is characterized by lymphocyte infiltration and corresponding in-flammation. Histological analysis of the islets at the onset of overt hyper-glycemia revealed that most of the islets were atrophic with few β cells,leukocytes invaded the islets, and β cells were partially degranulated. Bycontrast, the islet histology of the andrographolide-treated NOD micedid not reveal evident destruction of β cells or marked reduction ofinsulitis (Fig. 3A–E). The insulitis scores were significantly lower inandrographolide-treated NOD mice than those in age-matched controls(Fig. 3F).

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3.3. Inducing Th2-biased cytokine response by andrographolide treatment

We determined the plasma autoreactive Th1/immunosuppressiveTh2/Th17 cytokine levels by ELISA to understand the mechanism of theinhibited development of T1D. Andrographolide decreased the levels ofsera IL-2 and IFN-γ (Fig. 4), which benefited the therapeutic preventionof T1D. By contrast, andrographolide (100 and 150 mg/kg, i.g.) increasedthe levels of anti-inflammatory cytokine IL-10 and TGF-β. The serumIL-17 level of the andrographolide-treated NOD mice was lower. Theseresults suggested that andrographolide regulated Th cell differentiationand cytokine production in vivo.

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Fig. 4. Andrographolide treatment induced a Th2-biased cytokine response. Concentrationswith 50, 100, and 150 mg/kg andrographolide for four weeks from 8 to 12 weeks old or from⁎P b 0.05, ⁎⁎P b 0.01 compared with the control mice. Bars refer to means ± SD with eigh

Please cite this article as: Zhang C, et al, Preventive effects of andrographolby inducing immune tolerance, Int Immunopharmacol (2013), http://dx.d

3.4. Altered balance between immunity and inflammation in the PLN ofNOD mice by andrographolide treatment

We compared the transcriptional profiles of PLN samples obtainedfrom theNODmicewith euglycemia after fourweeks of andrographolidetreatment by RT-PCR (Fig. 5). The expressions of Th1-specific T-bet andTh17-specific retinoic acid-related orphan receptor γt (RORγt) tran-scription factors were remarkably decreased in the andrographolide-treated new-onset NOD mice compared with those in the untreatedones. GATA3 expression in the andrographolide-treated mice was in-creased compared with that in the chronic diabetic new-onset NODmice. Thus, andrographolide initially tilted the balance betweenpro-inflammatory cytokines and anti-inflammatory cytokines andthat between T cell Th1/Th17 effector (downregulated) and immune-suppressive Th2 (upregulated).

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O4. Discussion

The loss of immune tolerance to β cells causes autoimmune-mediated destruction of insulin-producing β cells in T1D NOD miceand humans [15]. Few therapies have successfully restored euglycemiaand self-tolerance to islets in overtly diabetic NODmice [16,17]. Agentsinducing immune tolerance have been developed to combat T1D. Forexample, some agents inhibit pro-inflammatory responses that indi-rectly but powerfully influence the activity of Ag-activated T cells to var-ious protective (Treg) or cytopathic phenotypes (Th1, Th2, Th17) andpro-inflammatory cytokines directly damaging β cells.

Andrographolide, an anti-inflammatory herbal medicine, has beenspotlighted for its therapeutic potential in treating T1D [11,18]. The pro-spective immunomodulatory activity of andrographolide has also beenconsidered. Wang et al. [19] found that andrographolide-induced in-nate and adaptive immune responses are attributed to the regulatedmacrophage phenotypic polarization and Ag-specific antibody produc-tion. Andrographolide also inhibits the activation of nuclear factor of

of IL-2, IL-10, IL-17, IFN-γ, and TGF-β in the serum from 12-week-old NOD mice treatedthe control animals. IL-2, IL-10, IFN-γ, and TGF-β in the serum were detected by ELISA.

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Fig. 5. RT-PCR-based analysis of andrographolide effects on gene expression with PLNs.Andrographolide-treated NOD mice compared with chronic diabetic mice. Gene tran-scription was analyzed according to the absolute quantification method. GAPDH wasused as an endogenous control to normalize RNA levels. IC: age-matched control (ICRmice). CO: control. AL: low-dose andrographolide (50 mg/kg bw). AM: medium-doseandrographolide (100 mg/kg bw). AH: high-dose andrographolide (150 mg/kg bw).⁎P b 0.05, ⁎⁎P b 0.01 compared with the control mice. Bars refer to means ± SD witheight mice per group.

5C. Zhang et al. / International Immunopharmacology xxx (2013) xxx–xxx

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Ractivated T cells and the phosphorylation of extracellular signal regulat-ed kinase (ERK) 1 and ERK5 in T cells [20].

In this study, new-onset overtly diabetic mice were treatedwith a short term of andrographolide. Our results indicated thatandrographolide decreased the serum glucose levels dose dependentlyafter the four-week oral supplementation. This supplementation didnot significantly affect bw, feed intake, and feed efficiency (data notshown), suggesting that the administered doses of andrographolidewere nontoxic in vivo and effectively mitigated hyperglycemia in theexperimental NOD mice. Although the mice were supplemented withandrographolide at age 8 to 12 weeks, they were not prone to diabe-tes at 26 weeks old, demonstrating that the protective effect ofandrographolide was therapeutically persistent.

Our results suggested that andrographolide was an effective im-munomodulatory prophylactic against the development of diabetesin NOD mice. After eight-week-old NOD mice were treated withandrographolide for four weeks, the inflammatory response in theislets was significantly reduced during diabetes progression bydownregulating Th1-type cytokine (IFN-γ and IL-2) and Th17-typecytokine (IL-17) production and by upregulating Th2-type cytokine(IL-10 and TGF-β) production in the serum.

Andrographolide also protected the islets from apoptosis, indicat-ing that euglycemia and immune tolerance to β cells were restored inthe andrographolide-treated NOD mice. Andrographolide therapy

Please cite this article as: Zhang C, et al, Preventive effects of andrographolby inducing immune tolerance, Int Immunopharmacol (2013), http://dx.d

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reduced destructive autoimmunity by modifying various effector(Th1, Th2, and Th17) phenotypes. Afterward, the islet-invasive formof insulitis was replaced with circumferential insulitis commonly as-sociated with tolerance to islets. Thus, the andrographolide-treatedNOD mice had larger islets of β cells than those at T1D onset. Thesharply decreased pro-inflammatory type, lineage-specific T-bet, andRORγt transcript as well as the increased anti-inflammatory typeGATA3 transcript within PLN revealed that the andrographolide-induced inflammatory changes rapidly varied the fundamental natureof T cell-dependent autoimmunity in the NOD model.

Among the key transcription genes, GATA3 is a specific gene thatmodulates Th2 cell generation, T-bet is a specific transcription factorof Th1 cells, and RORγt is a specific transcription factor of Th17 cells[21–23]. These transcription genes are closely related with the differen-tiation of naive CD4 T cells to Th1, Th2, and Th17 cells [24]. CytopathicTh17 phenotype can be destabilized with consequent acquisition ofanti-inflammatory properties by changing the inflammatory activity[17]. Regulating Th1, Th2, and Th17 transcription factors and anti-inflammatory performance of andrographolide treatment are condu-cive to balancing pro-inflammatory and anti-inflammatory cytokines,thereby preventing the development of diabetes in NOD mice.

These results indicated that the effect of andrographolide on T1Dprevention could be attributed to the modulated Th1/Th2/Th17balance, which may prevent β cell death and inhibit T cell infiltrationinto pancreatic islets.

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