Studies on the cell-immunosuppressive mechanism of Oridonin from Isodon serra

https://doi.org/10.1016/j.intimp.2007.03.001Get rights and content

Abstract

Distinct effects of Oridonin and Lasiodonin, which were extracted from Isodon serra was compared by the ratio of IC50 versus EC50, the therapeutic index. After choosing the more effective one, Oridonin, its immunosuppressive effect and mechanism were investigated using BALB/c mouse splenic lymphocytes both in vitro and in vivo. When murine splenic lymphocytes was incubated with Oridonin, the novel extract effectively suppress the overproduction of the cell stimulated by Concanavalin A in a dose and time-dependent manner. This inhibitive activity was mainly due to interfering DNA replication in G1 stages and regulating cell cycle and minorly due to decreasing the CD4+/CD8+ lymphocytes level, according to Flow cytometry analyses (FCAS) results. Xylene-induced mouse tumescence model result suggested that Oridonin depressed the murine ear-swelling extent and the level of Interleukin-2 in the blood serum of experimental animals. The exciting results of enzyme-linked immunosorbent assay (ELISA) indicated that Oridonin could inhibit the secretion of Interleukin-2, Interferon-γ, Interleukin-12p40 and Tumor necrosis factor-α in murine splenic lymphocytes. Moreover, the results of reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that the inhibition was through decreasing the expression level of these cytokines mRNA. Consequently, the results of our research showed that Oridonin suppressed overproduction of murine splenic lymphocytes through interference of DNA replication, regulation of cell cycle and inhibition of cytokine secretion both at protein and mRNA level.

Introduction

An increasing number of people are adopting alternative systems of medicine owing to the irreversible effects of modern drugs and therapies. Use of medicinal plant products for treatment of various acute and chronic diseases is gaining increasing importance around the globe [1]. Isodon serra (MAXIM.) HARA, commonly called Xi Huangcao in Chinese, is one of the perennial herbs. Traditionally, it is used in the treatment of arthritis, enteritis, jaundice, hepatitis, lepromatous leprosy, ascariasis and acute cholecystitis [2], [3], [4]. Our previous studies [5], [6] on I. serra extracts have led to the extractions of some novel ent-Kaurene Diterpenoids, which showed different molecular components and distinct immunosuppressive effect on T lymphocytes. In this study, we obtained another extract, Oridonin which has a similar chemical structure to Lasiodonin. So, we first compared the pharmacological effect on them. After the comparison, we then aimed to investigate detailed and systemic immunosuppressive mechanisms on the more effective extract both in vitro and in vivo.

As we known, lymphocytes play an important role on the immune system. T lymphocytes have two subclasses, CD4+ T cells and CD8+ T cells. CD4+ T cells assist and induce T cells and B cells into action. On the contrary, CD8+ T cells obstruct the active B cells producing antibody [7]. The ratio of CD4+/CD8+ could partly reflect an illness. It was reported that patients with autoimmune disease had high CD4+/CD8+ ratio in their blood. It meant that unbalance T lymphocyte subclasses could damage the immune system and cause diseases.

Cytokines are a series of bioactive polypeptides and glycoproteins produced by immunocytes and unimmunocytes. Generally, cytokines include lymphokines, monocytokines, and cytokines produced by other cells [8]. IL-2 is mostly produced by mature T lymphocytes and could increase the proliferation of lymphocytes and activate the secretion of lymphokines. It was reported that IL-2 level is increased in blood serum, synovial fluid and lymphocytes serum of arthrosis autoimmune disease patients [9], [10]. IFN-γ could increase the identification and submission to autoantigen for inducing T cells. IL-12p40 and TNF-α secretion is important for the initiation of T cell-mediated immune responses [11]. Studies on most immunosuppressants lie on the inspection of these relative cytokines.

Recently, our studies on Oridonin from I. serra [12], [13], [14] have found that it could effectively suppress the overproduction of BALB/c mouse splenic lymphocytes. However, the inhibition mechanism is not reported. So the aim of our study is to figure out the immunosuppressive mechanism of Oridonin both in vitro and in vivo.

Section snippets

Reagents

Oridonin, one ent-Kaurene Diterpenoids of I. serra, was extracted and purified by the school of Pharmacy, Shanghai Jiao-Tong University, China [4]. The chemical structure was shown in Fig. 1. The purity was over 98%. Oridonin was prepared in growth media with 0.5% DMSO added to improve solubility. The concentration of DMSO was low enough not to damage the activity of immune cells.

Multi-glycoside from Tripterygium wilfordii Hook f. (GTW) was used for various immune and inflammatory diseases in

Cytotoxicity and inhibitive activity evaluation of Oridonin

Lymphocytes with and without ConA were treated with different doses of Oridonin (3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml) and measured for 48 h by MTT method. Fig. 2.1 showed that Oridonin had a cytotoxicity on normal lymphocyte, while GTW had a slight inhibitive effect. Half inhibitive concentration (IC50) was calculated from the result, which was 8.5 μg/ml.

Oridonin showed a dose-dependent inhibition on Con A-induced lymphocyte proliferation (Fig. 2.2). The viability rate of

Discussion

Cell proliferation was clearly accommodated within a chronic disease state such as rheumatoid arthritis (RA), where there was T cell infiltration of synovium and marked cytokine production[8], because stimulated T cells were able to support osteoclast formation. Our previous studies [6] had elucidated that Enmei, one of I. serra extracts, had the dose-dependent inhibitive effect on ConA-induced lymphocytes over-proliferation. In this study, we tried to examine the immunosuppressive mechanism of

Acknowledgements

This work has been supported by the National Nature Science Foundation of China— Project for Young Scientists Fund (No.30600598) and the Project of Shanghai Science Committee for Young Scientists (No. 06QA14043). The author would also like to thank some fellow members namely Xiaodong Zhang, Shuihua Zhang, Liyun Shen, Ting Li and Kejia Sun for completing the research.

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