Elsevier

Fitoterapia

Volume 100, January 2015, Pages 88-94
Fitoterapia

Oridonin: A small molecule inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) isolated from traditional Chinese medicine

https://doi.org/10.1016/j.fitote.2014.11.001Get rights and content

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel regulating the transepithelial transport of electrolyte and water. In the recent years, CFTR chloride channel becomes the new molecular target of treating secretory diarrhea. The objective of this study is to find out a novel CFTR inhibitor from traditional Chinese medicine (TCM) and study on its pharmacological activity. About 34,000 factions of TCM extracts were screened by high throughput screening (HTS) in this research. We found that Rabdosia rubescens show a potent inhibition on CFTR. Under the bio-active analysis guidance, an ent-kaurane diterpenoidoridonin (PubChem CID: 34378) was isolated from R. rubescens. A series of intensive studies showed that oridonin remarkably reduced iodide influx in wt-CFTR and ΔF508-CFTR FRT epithelial cells in a dose-dependent and irreversible way. Oridonin sharply blocked FSK-stimulated short-circuit current in both rats and mice intestine in vitro. In mouse closed-loop model, oridonin reduced cholera toxin-induced fluid secretion significantly over 6 hours in vivo. Thus we concluded that oridonin is a new inhibitor of CFTR Cl channel. It will be a good leading compound for developing the new drug of cholera toxin-induced secretory diarrhea.

Introduction

Cholera is a great threat to human health. Cholera outbreaks worldwide and leads to several global devastating pandemics in human history [1]. The World Health Organization (WHO) said that 58 countries reported about 590,000 cholera cases in 2011. Cholera toxin (CT) binds to the apical surface of enterocyte and causes an irreversible increase in intracellular cyclic adenosine monophosphate (cAMP) level [2]. Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-dependent epithelial chloride channel, and it provides a pathway for Cl movement across epithelia and regulates the rate of Cl flow [3]. The increasing cAMP over-stimulates CFTR chloride channel and leads to a ceaseless Cl secretion. The irreversible severe chloride ion loss disturbs physiological mechanism of intestinal ion transport, which causes fulminant water loss in intestine. If left untreated, the patient could die of dehydration within a single day.

Hence, CFTR inhibitors serve as targets for therapy of cholera toxin-induced secretory diarrhea [4], [5]. CFTR inhibitors declined electrolytes and water loss by closing CFTR chloride channel, which is the final way of intestinal fluid secretion [6]. The inhibitors of CFTR, such as CFTRinh-172 [7], Lysophosphatidic acid (LPA) [8], Stevioside [9] and GlyH-101 [10], significantly decreased CT-induced intestinal fluid accumulation in mouse closed-loop model [11].

With the booming of traditional Chinese medicine (TCM) in the globe, it is thought as an important resource of discovering bioactive leading compounds and new drugs [12], [13], [14]. In this study, we utilize the high-throughput screening (HTS) technique to screen the potential inhibitors of CFTR in traditional Chinese medicine. HTS monitors the real-time transmembrane rates of halide by the fluorescent intensity. It is swift and efficient that HTS screened inhibitors from thousands of fractions of Chinese herbal extracts.

R. rubescens is a common herbal plant used for treating stomach ache, sore throat and cough in traditional Chinese medicine. The major activity of R. rubescens ingredient is Oridonin (C20H28O6, PubChem CID 34378) — an ent-kaurane diterpenoid compound [15]. Oridonin has various pharmacological functions and physiological activities such as treating tumor and leukemia, restraining bacterial growth, scavenging active oxygen free radicals and eliminating inflammation. [16], [17], [18], [19], [20] Oridonin has been used to manufacture drugs and health products.

In this study, we try to identify a novel natural CFTR inhibitor from traditional Chinese medicine by high through-put screening, and discuss its efficacy by a series of in vitro and in vivo experiments.

Section snippets

Compounds

FSK, GST, IBMX, F-12 Coon's medium, indomethacin, amiloride and cholera toxin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Solvents (HPLC grade) were purchased from Honeywell (New Jersey, USA). CFTRinh-172 and GlyH-101 were synthesized as reported previously [7], [10], and the purity were verified by analytical HPLC. Compounds were stored in DMSO. For protecting the cells and the tissues, DMSO was less than 0.1% in operating fluid.

Cells

Human wt-CFTR and the halide-sensing green

Results

In this study, we aim to find out a new natural inhibitor of CFTR from traditional Chinese medicine (TCM). The schematic of High-throughput Screening (HTS) procedure was shown in Fig. 1A. For screening the inhibitors of CFTR, we extracted 423 Chinese herbal plants with Soxhlet extraction. Every herbal extract had been separated into 80 factions by chemical polarity. On initial study, 423 kinds of traditional Chinese herb — nearly 34,000 factions had been screened. Among them, only R. rubescens

Discussion

The inhibitors of CFTR have been a new strategy of treating cholera toxin-induced secretory diarrhea for the past few years [30], [31]. However, it is generally known that CFTR inhibitor is rare in nature. Many international pharmaceutical companies screen leading compounds in traditional Chinese medicine [32], [33]. In this study, we used high throughput screening to screen small molecular inhibitor of CFTR from extracts of TCM. Among 34,000 factions of TCM extracts, only eight successive

Competing interests

The authors have declared that no competing interests exist.

Acknowledgments

This work was supported by the National Natural Science Fund (No. 81473265; 30973577) and the Special Fund for Doctorate Disciplines Construction in Universities (20112136110002).

References (34)

  • T. Ma et al.

    Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion

    J Clin Invest

    (2002)
  • C. Li et al.

    Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhoea through CFTR-dependent protein interactions

    J Exp Med

    (2005)
  • P. Pariwat et al.

    A natural plant-derived dihydroisosteviol prevents cholera toxin-induced intestinal fluid secretion

    J Pharmacol Exp Ther

    (2008)
  • M. Chatchai et al.

    Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure–activity analysis, and in vivo efficacy

    Physiology

    (2004)
  • Weiqiang Zhang et al.

    Recent advances and new perspectives in targeting CFTR for therapy of cystic fibrosis and enterotoxin-induced secretory diarrheas

    Future Med Chem

    (2012)
  • X.T. Li et al.

    Comparative study on the sensitivities of seven human cancer cell lines to rubescensine A

    Acta Pharm Sin

    (1985)
  • T. Ikezoe et al.

    Oridonin induces growth inhibition and apoptosis of a variety of human cancer cells

    Int J Oncol

    (2003)
  • Cited by (0)

    View full text