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

https://doi.org/10.1016/j.intimp.2013.05.002 Get rights and content

Highlights

  • Andrographolide significantly suppressed the development of diabetes in NOD mice.

  • Andrographolide regulated Th1/Th2/Th17 cytokine secretion.

  • Andrographolide modulated mRNA expression of T-bet, GATA3, and RORγt.

  • Andrographolide prevented type 1 diabetes by maintaining Th1/Th2/Th17 homeostasis.

Abstract

Andrographolide, an active component in traditional anti-diabetic herbal plants, is a diterpenoid lactone isolated from Andrographis paniculata because of its potent anti-inflammatory and hypoglycemic effects. However, 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 development of autoimmune diabetes and clarify the underlying mechanism. NOD mice were randomly divided into four groups and administered with water and andrographolide at 50, 100, and 150 mg/kg body weight for four weeks. ICR mice were also selected as the control group. Oral glucose tolerance and histopathological insulitis were examined. Th1/Th2/Th17 cytokine secretion was determined by ELISA. The transcriptional profiles of T-bet, GATA3, and RORγt in the pancreatic lymphatic node samples derived from the NOD mice were detected by RT-PCR. After four weeks of oral supplementation, andrographolide significantly inhibited insulitis, delayed the onset, and suppressed the development of diabetes in 30-week-old NOD mice in a dose dependent manner. This protective status was correlated with a substantially decreased production of interferon (IFN)-γ and interleukin (IL)-2, increased IL-10 and transforming growth factor (TGF)-β, and a reduced IL-17. Andrographolide also increased GATA3 mRNA expression but decreased T-bet and RORγt mRNA expressions. Our results suggested that andrographolide prevented type 1 diabetes by maintaining Th1/Th2/Th17 homeostasis.

Introduction

Type 1 diabetes (T1D) is caused by immunologically mediated selective destruction of insulin-producing β-cells in pancreatic islets with consequent insulin deficiency in humans and non-obese diabetic (NOD) mouse models [1]. Although pathogenesis and autoimmune processes of T1D have been determined, the clinical onset of T1D has not been intervened in, delayed, or prevented effectively [2]. In general, the progression of T1D is inhibited by two strategies: (1) suppressing or eliminating autoimmunity or (2) replacing β cells to replenish insulin [3]. Methods suppressing autoimmunity against β cells, such as the use of anti-CD3, anti-CD20 monoclonal antibody, or GAD65, alter Th1/Th2 balance and destroy autoreactive immune cells [4], [5]. Nevertheless, prophylactic or therapeutic drugs currently available for T1D are scarce.

Andrographolide (Fig. 1) is a diterpenoid lactone isolated from Andrographis paniculata, a traditional Chinese medicine that has been extensively used to treat infection, inflammation, cold, fever, and diarrhea in China [6], [7]. Andrographolide is also considered a pharmacologically immunological, antibacterial, antiviral, anti-inflammatory, antithrombotic, and hepatoprotective substance [8], [9], [10] and functions in glycemic control. Zhang et al. [11] demonstrated that andrographolide-lipoic acid conjugate is hypoglycemic and β-cell protective in alloxan-treated mice (a T1D model) because this conjugate has antioxidant and NF-κB inhibitory activities. In addition, oral treatment of andrographolide can lower plasma glucose concentrations dose dependently in streptozotocin-induced diabetic rats [12]. Moreover, andrographolide elicits immunomodulatory effects on the immune system. Iruretagoyena et al. [13] reported that andrographolide significantly alleviates experimental autoimmune encephalomyelitis by inhibiting T cell and antibody responses to myelin antigens. This study aimed to determine whether or not andrographolide controls the prevention of diabetes development in autoimmune NOD mice and whether or not such an effect originates from an immune regulatory activity.

Section snippets

Reagents

Andrographolide (purity > 98%; Hubei Jianyuan Chemical Co., Ltd., Wuhan, China) was used to prepare a 50 mM stock solution by dissolving in dimethyl sulfoxide. The resulting mixture was serially diluted in PBS immediately prior to experiments.

Experimental animals

Female NOD mice (NOD/LtJ) and female ICR mice (as age-matched control group) aged seven weeks were purchased from Beijing HFK Bio-Technology Co., Ltd. (Beijing, China). The experimental mice were maintained at the Experimental Animal Center of Tongji Medical

Delayed onset and decreased incidence of diabetes in NOD mice by andrographolide treatment

We initially examined the cumulative incidence of diabetes in NOD mice treated with or without andrographolide for 30 weeks. T1D spontaneously developed in approximately 70% of the non-treated female NOD mice, and this finding is consistent with that in a previous study [14]. By contrast, intragastrical (i.g.) injection of andrographolide prevented 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

Discussion

The loss of immune tolerance to β cells causes autoimmune-mediated destruction of insulin-producing β cells in T1D NOD mice and humans [15]. Few therapies have successfully restored euglycemia and self-tolerance to islets in overtly diabetic NOD mice [16], [17]. Agents inducing immune tolerance have been developed to combat T1D. For example, some agents inhibit pro-inflammatory responses that indirectly but powerfully influence the activity of Ag-activated T cells to various protective (Treg)

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    These authors contributed equally to this work.

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