Received 12 October 2014; revised 8 November 2014; accepted 20 November 2014

ABSTRACT

Cordyceps (CS) is a Chinese herb that produces various effects including immune modulation, and now CS culture product is interested in the use as a functional food. We prepared CS mycelium culture extract (Paecilomyces hepiali, CBG-CS-2), CS extract, for functional foods. This study aimed to deduce the molecular mechanism of immunomodulatory effect of CS extract in Peyer’s patches (PPs), a main gut immune site, in mice that treated with mitomycin C (MMC), an immunosuppressing antibiotics. Nutrigenomics give us invaluable molecular information about both of foods and nutrition to improve or maintain good health. Here we performed nutrigenomics using DNA microarray to investigate the effect of CS extract on gene expression altered in PPs of the immunosuppressed mice. Interestingly, CS extract protected from the MMC-mediated downregulation of 22 genes, which are associated with IgA production and other immune response in PPs. These suggested that CS extract alleviated the downregulated expression of immune related genes in the gut immune site in an immunosuppressed state. Thus CS extract appears to be practical functional food for immunodepression and/or its related hypofunction.

Keywords:

DNA Microarray, Functional Food, Gene Expression, Immunosuppression

1. Introduction

Nutrigenomics is a genomic study on the effects of foods and food constituents on the level of gene expression. It gives us high-throughput methods and tools to look for disease preventing and health promoting foods, functional foods [1] [2] . From this point of view, nutrigenomics was introduced in recent paper of this Journal [3] . DNA microarray is powerful nutrigenomic tool to investigate global gene expression events in target tissues or cells and provides invaluable information to elucidate the molecular mechanism of the effects of functional foods.

Cordyceps (Cordyceps sinensis, CS) has been used as a medicine in China for over 300 years and was featured in the book, “Ben-Cao-Bei-Yao”, which was edited by Wang Ang in 1694. CS has several effects including immunomodulation [4] - [6] . CS-derived natural products are comprised of complicated components, cordycepin derivatives, cordycepic acid, ergosterol, polysaccharides, and nucleosides [7] [8] . Cordycepin and cordycepic acid are thought to be the main active ingredients in CS; however, this is still debated [8] . Recently people have interested in CS and its culture products to use as a functional food [9] - [11] . CS mycelium culture techniques were developed to yield alternative CS products with a constant composition [12] [13] . However, there is little information regarding the molecular mechanisms underlying function of CS culture product.

In the current study, we performed nutrigenomis to elucidate the in vivo immunomodulatory function of CS mycelium culture extract (Paecilomyces hepiali, CBG-CS-2) on gut immunity. Peyer’s patches (PPs) are main gut immune site and participate in essential gut immune responses against orally invading infectious microorganisms [14] [15] and IgA immunity [16] - [18] . We examined the effect of CS extract on gene expression in PPs of an immunosuppressed mice treated with mitomycin C (MMC). MMC is an apoptosis-inducing antibiotic that inhibits DNA replication, and it is utilized for cancer therapy and organ transplantation due to its immunosuppressive action [19] - [21] . This antibiotics was also applied to induce immunodepression in animals to study the effect of herbal medicines in a previous paper [22] . For these reasons we used MMC to prepare immunosuppressed mice in this study.

Here we performed nutrigenomic study and revealed previously unknown functional mechanism of CS extract to improve downregulated expression of immune-related genes in PPs of MMC-treated mice.

2. Material and Methods

2.1. Preparation of Cordyceps Mycelium Culture Extract (Paecilomyces hepiali, CBG-CS-2)

Paecilomyces hepiali, a pure strain from the culture collection of Chebigen Inc., was initially grown on potato- dextrose agar medium slants in Petri dishes and then transferred into a seed culture medium composed of 4% Dextrose, 1% yeast extract, and 1% peptone (DYP). The seed culture was grown in a 250 mL flask containing a nutritive medium (100 mL of 2% potato-dextrose broth) at 25˚C in a shaking incubator (150 rpm for 5 - 7 days). The obtained seed culture was transferred into a stirred tank fermentor containing DYP medium with 10% (v/v) working volume, and the culture was fermented at 25˚C, 150 rpm, for 5 days. The fermented product was concentrated and extracted with a vacuum concentrator and extractor at 80˚C to 10% of the initial volume. The extracts were freeze dried and used as CS mycelium culture extract (Paecilomyces hepiali, CBG-CS-2) in this study.

The main components of the mycelium culture extract were as follows: 32% cordyceps polysaccharide, 7.3% cordycepic acid, 0.13% adenosine, and 0.001% cordycepin.

2.2. Animals and Administration of Cordyceps Mycelium Culture Extract and Mitomycin C (MMC)

All procedures were carried out according to the Institutional Animal Care and Committee Guide of Intelligence and Technology Lab. Male BALB/c mice (9-week-old; Japan SLC, Inc., Hamamatsu, Japan) were used for this study. Four to five mice per cage were housed under a specific pathogen-free condition (controlled temperature of 20˚C - 26˚C and humidity of 40% - 60%) and fed standard laboratory food (FR-2; Funabashi Firm, Inc., Funabashi, Japan) ad libitum.

CS mycelium culture extract (Paecilomyces hepiali, CBG-CS-2) was prepared by Chebigen Inc. (Jeonju, S. Korea). The CS mycelium extract solution was dissolved in sterile water, and it was orally administered to mice for 28 days at 120 or 400 mg/kg/day. Mitomycin C (MMC; Kyowa Hakko Kirin Co., Ltd, Tokyo, Japan) solution (3 mg/kg) was dissolved in saline and intraperitoneally administered to mice at day 21, day 24, and day 27. The mice were divided into 6 groups: 1. control group (Cont; n = 9), 2. CS group with 120 mg/kg (CS120; n = 9), 3. CS group with 400 mg/kg (CS400; n = 9), 4. MMC group without CS (MMC; n = 9), 5. MMC group with 120 mg/kg CS (MMC + CS120; n = 9), and 6. MMC group with 400 mg/kg CS (MMC + CS400; n = 9). At 28 days, all mice were anesthetized with isoflurane for blood collection, followed by sacrifice to harvest PPs.

2.3. RNA Preparation

RNA was prepared as described previously [23] . Briefly, total RNA was extracted from the harvested PPs using Isogen (Nippon gene, Tokyo, Japan) and DNase-treated in the aqueous phase using the RNase-Free DNase Set (Qiagen, Valencia, CA). The extracted RNA was further purified using an RNeasy MinElute Cleanup Kit (Qiagen), and the quantity and purity were evaluated photometrically at 260 nm, 280 nm and 320 nm using an Ultrospec 2000 (Pharmacia Biotech/GE Healthcare, Uppsala, Sweden).

2.4. DNA Microarray Analysis

Two-hundred nanograms of total RNA pooled from each group was used to generate cDNA and Cy3-labeled cRNA using the Low Input Quick-Amp Labeling Kit (Agilent Technologies, Santa Clara, CA), according to the manufacturer’s instructions. The labeled cRNA was photometrically examined to determine the quantity and dye-incorporation ratio using an Ultrospec 2000, and it was hybridized to a Mouse Gene Expression 4 × 44K v2 Microarray (Agilent Technologies). The array was scanned with GenePix 6000B (Molecular Devices, Sunnyvale, CA), and the obtained image was processed using GenePix Pro 6.0 Software (Molecular Devices). Features were manually examined, and poor quality spot were flagged and filtered out.

Signal data for the features (spots of array) were then imported to GeneSpring 12.6 (Agilent Technologies, Inc.), and further analysis was performed using this software. Signal data from the arrays was normalized using the 75th percentile method [24] , and baseline transformation was performed using the median of the control samples. Quality control was performed to filter out signal data with a standard error over 0.2.

To analyze differentially expressed genes, the fold change (FC) vs. control samples was calculated, and genes with a FC greater than 1.5 were extracted for the CS-administered and MMC-injected groups. To further analyze the alleviative effects of CS on MMC treatment, genes shifted toward control levels in MMC + CS groups in comparison with MMC groups were extracted with the criteria set as 25% upregulation or downregulation induced by MMC treatment. Gene Ontology analysis was performed on the extracted genes using software of GeneSpring 12.6, and gene sets with a corrected p-value < 0.1 were selected as significant gene groups.

2.5. Assay of Plasma IgG

Plasma samples were prepared from collected blood by centrifugation, and the plasma level of total IgG was determined using a sandwich ELISA with a capture antibody (goat anti-mouse IgG (ab97031, Abcam com., Cambridge, MA, USA)), a detection antibody (goat anti-mouse IgG-HRP (ab98717, Abcam)) and a substrate (TMB (3,3',5,5'-Tetramethylbenzidine, T8665, Sigma-Aldrich Corporation, Saint Louis, MO, USA)). The optical density at 450 nm, with a reference at 630 nm, was measured using a Well Reader SK601 (Seikagaku Corporation, Tokyo, Japan).

3. Results

3.1. Preliminary Results on Mitomycin C (MMC)-Treatment

Plasma IgG levels markedly decreased from 18.3 ± 1.9 mg/mL of normal to 10.3 ± 2.9 mg/mL of MMC-treated mice (data not shown). The wet weight of spleen also markedly decreased from 89.4 ± 1.7 mg of normal to 53.6 ± 3.2 mg of MMC-treated mice (data not shown). These indicated that some immunosuppressive state occurred in MMC-administrated mice.

3.2. Nutrigenomic Analysis

MMC treatment altered gene expression in PPs. Pretreatment with 120 or 400 mg/kg CS extract protected against MMC-induced alterations in gene expression. Table 1 and Table 2 list 84 MMC-mediated downregulated genes that were protected by CS extract, as determined by ontology analysis. Interestingly, 22 genes involved in the immune system (Aicda, Ms4a1, Bcl6, Pik3cd, Slamf1, Vav1, Syk, Bcl11a, Cxcr4, Gcsam, Scimp, Lyn, Btk, Igh-Vj558, Nuggc, Endou, Tnfrsf13c, Klhl6, Sh2b2, H2Ob, Map4k2, and Nos2) represent 26% of the total CS-alleviated genes (Table 1). Other protected genes include 22 genes (26%) associated with signal transduction or stimulus response and 11 genes (13%) associated with localization and DNA replication or transcription (Table 2).

Table 3 and Table 4 list 55 MMC-mediated upregulated genes that are alleviated by the CS extract. Four

To analyze the alleviative effects of CS extract on MMC treatment, genes shifted toward control levels in MMC + CS groups in comparison with MMC groups were extracted with the criteria set as 25% upregulation or downregulation induced by MMC treatment.

To analyze the alleviative effects of CS extract on MMC treatment, genes shifted toward control levels in MMC+CS groups in comparison with MMC groups were extracted with the criteria set as 25% upregulation or downregulation induced by MMC treatment.

To analyze the alleviative effects of CS extract on MMC treatment, genes shifted toward control levels in MMC + CS groups in comparison with MMC groups were extracted with the criteria set as 25% upregulation or downregulation induced by MMC treatment.

To analyze the alleviative effects of CS extract on MMC treatment, genes shifted toward control levels in MMC + CS groups in comparison with MMC groups were extracted with the criteria set as 25% upregulation or downregulation induced by MMC treatment.

genes (Igkv9-129, Sectm1b, Tnfsf15, Oas3) were categorized in the immune system, which represents only 7 % of the total ameliorated genes.

4. Discussion

Cordyceps (CS) is a traditional Chinese herb that displays numerous effects, including immune modulation [25] [26] . Now people have concern to develop its culture products as a functional food [9] - [11] . However, little is known about functional mechanism of CS culture products. Both of natural CS and its culture product are orally used, and the gut immune system would be its first site of action. Peyer’s patches (PPs) are essential gut lymphoid tissues associated with immune response against orally invading infectious microorganisms [17] [18] and NK cells, which induce target cell apoptosis through the perforin/granzyme pathway [27] [28] . Thus, we examined the molecular events that occurred in PPs of mice after treatment with CS, immunosuppressive antibiotic, mitomycin C (MMC), or both of CS and MMC.

In the current study, we performed nutrigenomic study using DNA microarray to deduce the mechanism of action of CS mycelium culture extract (Paecilomyces hepiali, CBG-CS-2) in mouse intestinal immune tissue (PPs). CS modulates immune responses [29] [30] , and as a result, its culture product is expected to be an effective functional food for immune-related diseases. This study confirmed the immune effect of the CS extract using functional genomics and DNA microarray analyses of PPs from MMC-treated mice. MMC is a potent immunosuppressive antibiotic, and it downregulated or upregulated the expression of various genes. Impressively, pretreatment with the CS extract protected against MMC-elicited downregulation of expression of 22 genes participating in the immune system, which represented 26% of the 84 total CS extract-alleviated genes (Table 1). PPs are central sites for intestinal immunity because they produce IgA [16] -[18] . Among the immune related genes expressed in PPs, Aicda, Tnfrsf13c, and Nos2 participate in B cell differentiation into IgA(+) B cells [31] , activating B cell for IgA class switching [32] , and regulating IgA class switch recombination of plasma cells [33] , respectively. Slamf1 is involved in NK, T, and B cell development [34] . Cxcr4 modulates lymphocyte trafficking into PPs and lymph nodes [35] , and Sh2b2/APS [36] and Klh16 [37] regulate linkage to the B cell antigen receptor. H2-Ob is selectively expressed in B cells, and it downregulates presentation of exogenous antigen to T cells [38] . CS extract also alleviated MMC-mediated upregulation of expression of four immune related genes (Table 3).

5. Conclusion

In conclusion, these results indicate that CS extract has an action to moderate the gut immunosuppressive state by modulating the expression of immune response genes in PPs. These suggest that CS extract is a possible functional food to be expected to prevent or improve malfunction in immune response or related immunity.

Conflict of Interests

The authors declare that they have no competing interests.

Acknowledgements

This study was financially supported by a contract with the Ministry of Agriculture, Food and Rural Affairs (MAFRA)/Korea National Food Cluster (FOODPOLIS to S-W C).

References

NOTES

^*Corresponding author.