Tectorigenin inhibits the in vitro proliferation and enhances miR-338* expression of pulmonary fibroblasts in rats with idiopathic pulmonary fibrosis

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Abstract

Tectorigenin is one of the main components in rhizomes of Iris tectorum, which is traditionally used to treat disorders such as hepatic cirrhosis caused by fibrosis. Idiopathic pulmonary fibrosis (IPF), one of the most common interstitial lung diseases, is caused by accumulation of fibroblasts in lungs.

Aim of the study

In this work we sought to examine the effects of tectorigenin on pulmonary fibroblasts in the IPF animal model and investigated the molecular mechanism (microRNA regulation) of tectorigenin treatment.

Materials and methods

A well-known animal disease model of pulmonary fibrosis in rat was established by intratracheally instilling of bleomycin. In vitro cultured pulmonary fibroblasts in bleomycin-treated rats and in controls were treated with or without tectorigenin. Comparative analyses of cell proliferation, apoptosis and cell cycle of pulmonary fibroblasts in bleomycin-treated rats and in controls were performed. Expression of miR-338* and its candidate gene LPA1 related to IPF of tectorigenin-treated pulmonary fibroblasts in bleomycin-treated rats were further investigated.

Results

Tectorigenin significantly inhibited the proliferation of pulmonary fibroblasts in bleomycin-treated rats but not in controls. However, no altered cell cycle and apoptosis of pulmonary fibroblasts in bleomycin-treated rats and in controls was observed after tectorigenin treatment. Tectorigenin remarkably enhanced miR-338* expression of pulmonary fibroblasts in bleomycin-treated rats and downregulated LPA1 in the protein level.

Conclusions

Tectorigenin inhibits the proliferation of pulmonary fibroblasts in vitro and enhances miR-338* expression, which might in turn downregulate LPA1. This indicates a potential inhibitory role of tectorigenin on the pathogenesis of IPF.

Graphical abstract

Tectorigenin inhibits the in vitro proliferation (A) and enhances miR-338* expression (B) of pulmonary fibroblasts in rats with idiopathic pulmonary fibrosis (IPF). In addition, the IPF-related LPA1, which was predicted to be one of the candidate targets of miR-338* was downregulated (C) in tectorigenin-challenged pulmonary fibroblasts in IPF animal model.

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Introduction

The rhizome of Iris tectorum Maxim (Iridaceae) is well known as a Chinese traditional medicine for the treatment of inflammatory diseases (Fang et al., 2008). For example, it is used to treat abdominal distension and hepatic cirrhosis (Song et al., 2001), the latter of which is due to the accumulation of extracellular matrix caused by activation of fibroblasts (Guyot et al., 2006). As one of the main components in rhizomes of I. tectorum, tectorigenin is a type of O-methylated isoflavone. It is pharmacologically characterized to inhibit inflammatory responses in the murine macrophage RAW264.7 (Pan et al., 2008, Conforti et al., 2009). Moreover, it has been shown to inhibit the proliferation of prostate cancer cells (Morrissey et al., 2004, Thelen et al., 2005). Increasing evidence show that tectorigenin could inhibit cell cycle at G2/M phase as demonstrated in the Chinese hamster lung fibroblast (V79-4), and promote apoptosis (Lee et al., 2001, Morrissey et al., 2004, Kang et al., 2005, Fang et al., 2008).

Idiopathic pulmonary fibrosis (IPF), one of the most common interstitial lung diseases, is defined as a chronic progressive fibrosing and eventually fatal interstitial pneumonia with an unknown etiology, characterized by a histological pattern of usual interstitial pneumonia (UIP) (American Thoracic Society, 2002). Of note, fibroblasts are recruited to the lung tissue and proliferated during the progress of pulmonary fibrosis (King et al., 2001, Scotton and Chambers, 2007). Even though the clinical state of IPF lasts several years, its prognosis is very poor and the survival time from diagnosis is only about 3–5 years (American Thoracic Society, 2000, Collard et al., 2003, Flaherty et al., 2003, Latsi et al., 2003).

The usual therapy is corticosteroid treatment in combination with immunosuppressant and cytotoxic drugs. So far, no effective treatment has been developed (Flaherty et al., 2001, Davies et al., 2003, Richeldi et al., 2003). A bleomycin (BLM)-induced animal model of pulmonary fibrosis has similar features to human conditions and is widely used for studying its pathogenesis and treatment strategies. Cultured pulmonary fibroblasts from such animal models have been widely used for studying the pathogenesis and potential treatment strategies for pulmonary fibrosis (Thrall et al., 1979, Thrall et al., 1982, Usuki and Fukuda, 1995, Chua et al., 2005). In vitro cultured pulmonary fibroblasts in animal models are equally popular (Konigshoff et al., 2007). In this study, we hypothesized that tectorigenin could regulate the function of pulmonary fibroblasts in the IPF model, which in turn affects the development of IPF.

MicroRNAs (miRNAs) are endogenous small non-coding 21–24 nucleotide regulatory RNAs that play a role in post-transcriptional regulation of mRNAs by binding their 3′ untranslated region (3′UTR) (Flynt and Lai, 2008). Increasing evidence show that differentially expressed endogenous miRNAs contribute to the regulation of fibroblast proliferation and differentiation (Nimmo and Slack, 2009). A recent study by Lal et al. (2009) reported that antagonizing miR-24 enhanced fibroblast proliferation while overexpression increased the G1 compartment. Overexpression of miR-150 and miR-194 inhibits the proliferation, activation and extracellular matrix (ECM) production of hepatic stellate cell (HSC) which is the major cell type responsible for the progression of liver fibrosis (Venugopal et al., 2009). MiR-1 and miR-133 are demonstrated to have distinct roles in modulating skeletal muscle proliferation in cultured myoblasts (Chen et al., 2006). These findings support the need for a better understanding of miRNAs expression and their effect on cell function.

Lysophosphatidic acid receptor 1 (LPA1), also known as EDG2, has been identified to be involved in such biological responses as tumor cell invasion, cell differentiation and apoptosis (van Corven et al., 1989, Mills and Moolenaar, 2003). Contos et al. (2000) had shown that cell viability, proliferation and migration decreased in embryonic fibroblasts from LPA1-knockout mice in response to LPA. Moreover, Tager et al. (2008) reported that LPA1-deficient mice were markedly protected from pulmonary fibrosis induced by bleomycin. Therefore, LPA1 plays a critical role in the pathogenesis of IPF.

To explore if tectorigenin regulated the function of pulmonary fibroblast, we established the BLM-induced pulmonary fibrosis model in rat, from which pulmonary fibroblasts were cultured in vitro. In this study, the cell proliferation, apoptosis and cell cycle of pulmonary fibroblasts after tectorigenin treatment were examined. Expression of miR-338* and the IPF-related key gene LPA1, which is one of the candidate targets of miR-338*, was also investigated.

Section snippets

Reagents

Bleomycin, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Alkaline hydrolysis kit was purchased from Jiancheng Bioengineering (Nanjing, China). RPMI 1640 medium, non-essential amino acids and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA). High capacity

Evaluation of IPF model in rat

To evaluate the BLM-induced disease model, we firstly analyzed the lung index. Compared to controls, the lung index of BLM-treated rats increased significantly (Fig. 1A, p < 0.01). Moreover, the hydroxyproline content in BLM-treated rats was significantly higher than in controls (Fig. 1B, p < 0.05).

Histological analysis indicated that treatment with bleomycin resulted in enhanced pulmonary alveolus inflammation, as compared to controls. Additionally, broadened alveolar septa and increased

Current therapies for IPF

To date, corticosteroids remain a commonly used therapy for treating IPF (Davies et al., 2003, Walter et al., 2006, Kim and Meyer, 2008). However, in older studies in which the definition of IPF was less specific, only a small proportion of patients after treatment with corticosteroid were reported to show physiologic or radiographic improvement. Moreover, when high doses of corticosteroids are used for treating patients with IPF, significant and often irreversible toxicity is consistently

Conclusion

Tectorigenin inhibits the in vitro proliferation of pulmonary fibroblasts and enhances miR-338* expression, which might in turn downregulate LPA1. This indicates a potential inhibitory role of tectorigenin on the pathogenesis of IPF.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant nos. 30800546 & 30972535), the Doctoral Foundation of Education Ministry of China (grant nos. 20070284015 & 200802841008), and the International Science and Technology Cooperation Project of Jiangsu, China (grant no. BZ2008055).

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