Sinomenine suppresses collagen-induced arthritis by reciprocal modulation of regulatory T cells and Th17 cells in gut-associated lymphoid tissues

https://doi.org/10.1016/j.molimm.2015.01.014Get rights and content

Highlights

  • Sinomenine, administered orally, markedly attenuates the clinical signs of CIA.

  • Sinomenine regulates the balance of Th17/Treg cells in intestinal lymph node.

  • Sinomenine induces the migration of Treg cells from gut to joint.

Abstract

Sinomenine (SIN) has long been used as a therapeutic agent of rheumatoid arthritis (RA) in China. However, the discrepancy between low oral bioavailability and higher minimal effective concentration made its action mode mysterious. The present study aimed to gain insight into the mechanisms by which SIN suppressed collagen-induced arthritis (CIA) in rats in view of Th17 and regulatory T (Treg) cell balance. SIN was orally administered, and the clinical symptoms of CIA rats were monitored; inflammatory cytokines levels in serum were measured by ELISA; pharmacokinetic studies were performed in normal and CIA rats; Th17 and Treg cell frequencies were analyzed by flow cytometry. The data showed that SIN treatment resulted in a dramatic decrease of arthritis scores and paw volume of CIA rats, which was accompanied by down-regulation of IL-17A and up-regulation of IL-10 in rat serum. The frequency of Treg cells was increased and the frequency of Th17 cells was decreased in the gut lymphoid tissues of SIN-treated rats. Immunohistochemistry assay demonstrated that more α4β7-positive cells were detained in joint tissues after SIN treatment. Moreover, the anti-arthritis efficacy of SIN disappeared when it was given by intraperitoneal injection, further confirming the action of SIN was gut-dependent. In conclusion, SIN exerts anti-RA action probably through modulating the frequencies of Treg cells and Th17 cells in intestinal lymph nodes and yielding a trafficking of lymphocytes (especially Treg cells) from gut to joint.

Introduction

Sinomenine (SIN) is an alkaloid constituent occurring in Sinomenium acutum Rehd. et Wils. and other medicinal plants. In China, it has been used for the treatment of rheumatoid arthritis (RA) for several decades. The satisfactory therapeutic efficacy and fewer side effects of SIN in patients have been confirmed in open clinical trials (Huang et al., 2007, Shi et al., 1985). When orally administered at the doses of 100 and 300 mg/kg, SIN could attenuate adjuvant-induced arthritis (AIA) in rat and collagen-induced arthritis (CIA) in mouse through suppressing the production of autoantibody and modulating Th1/Th2 response (Feng et al., 2007, Mu et al., 2013). In vitro mechanistic studies showed that SIN could suppress the activation of T lymphocytes, induce the apoptosis of macrophage, reduce antigen-induced activation of basophils, and hinder migration of synoviocytes at concentrations over 0.5 mM (He et al., 2005, Huang et al., 2008, Liu et al., 1994, Ou et al., 2011). Overall, the minimal effective concentration of SIN against immunity or inflammation effector cells is quite high. In contrast, pharmacokinetic studies manifested that the oral bioavailability of SIN was low. When it was orally administered at a dose of 90 mg/kg in rats, the Cmax was about 13.9 μg/mL (≈42 μM) (Liu et al., 2005), which was obviously lower than the in vitro minimal effective concentration as mentioned above. How SIN exerts anti-arthritic effects needs to be clarified.

RA is a chronic autoimmune disease that primarily targets the synovium, cartilage, and bone of multiple joints. As the concept of an imbalance between pro-inflammatory and anti-inflammatory forces exists for several decades, RA is formerly considered as a disorder driven by T helper 1 (Th1) cells through producing inflammatory cytokines, such as interferon-γ (IFN-γ). Recently, research interest has expanded to the balance between IL-17-producing T cell (Th17) and IL-10-producing regulatory T cell (Treg). Rebuilding the balance of Th17/Treg cells through either boosting the number of Treg cells or reducing the number of Th17 cells is likely to be one of the most effective strategies for RA intervention (Kohm et al., 2002, Park et al., 2014a). Interestingly, a large number of compounds from medicinal plants have manifested beneficial regulatory effects on the Th17/Treg balance, such as epigallocatechin-3-gallate (EGCG) (Wang et al., 2012), grape seed proanthocyanidin extract (GSPE) (Park et al., 2011), halofuginone (Park et al., 2014b), and curcumin (Park et al., 2013).

Considering the possible residence of SIN in intestines after oral administration and the central roles of Th17/Treg cell imbalance in RA pathogenesis, the present study explores the anti-arthritis mechanism of SIN with a focus on the regulation of Th17/Treg balance, especially in the intestinal tract.

Section snippets

Animals

Specific pathogen-free Wistar female rats (weighed 130–150 g) were obtained from B&K Universal Group Limited (Shanghai, China). They were maintained at a constant temperature and humidity, with a 12-h light–dark cycle. Water and a nutritionally adequate diet were provided ad libitum. All rats were observed daily for general health and clinical signs of disease. At the end of the study, rats were euthanized by CO2 asphyxiation, followed by exsanguination, and tissues were collected postmortem.

SIN ameliorated the clinical symptoms of rat CIA

Rats were orally administered with SIN for 14 days after the second week of the first immunization of collagen. Compared with rats in model group, the rats received SIN (120 mg/kg) showed significantly lower arthritic scores (4.38 ± 0.47 versus 8.25 ± 0.73; P < 0.01) and paw swelling (1.07 ± 0.14 mL versus 1.99 ± 0.08 mL; P < 0.01). LEF (2 mg/kg) also yielded a significant decrease in arthritic index scores and paw volumes (Fig. 1A–C).

Histological analysis of the inflamed hind paws showed a massive area with

Discussion

Orally administered SIN could remarkably inhibit the development and progression of CIA in rats, which was well consistent with previous reports (Feng et al., 2007, Zhou et al., 2008). However, lower plasma concentration of SIN in normal rats is hard to support its explicit efficacy. Therefore, we compared its pharmacokinetic parameters between normal and CIA rats, and found that the absorption of SIN was actually increased in arthritic rats. In spite of this, the Cmax value in CIA rats was

Conflicts of interest

The authors have declared that no competing interests exist.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81373426) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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    These authors equally contributed to this paper.

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