Open Access

Characterization of an Ebosin derivative produced by heterologous gene replacement in Streptomyces sp. 139

  • Yang Zhang1,
  • Junjie Shan2,
  • Yonggang Bao1,
  • Liping Bai1,
  • Rong Jiang1,
  • Lianhong Guo1,
  • Chen Yao1,
  • Ren Zhang3 and
  • Yuan Li1Email author
Microbial Cell Factories201413:103

https://doi.org/10.1186/s12934-014-0103-6

Received: 7 May 2014

Accepted: 7 July 2014

Published: 22 July 2014

Abstract

Background

Ebosin is a novel exopolysaccharide (EPS) produced by Streptomyces sp. 139 and evidenced to possess an anti-rheumatic arthritis activity in vivo. The Ebosin biosynthesis gene cluster (ste) consists of 27 ORFs and ste7 has previously been demonstrated to code for a fucosyltransferase, which plays an essential role in the formation of repeating sugar units during Ebosin production. Aiming to generate derivatives of Ebosin for better activity, we replaced ste7 with a gene encoding for a glucosyltransferase (gtf) from Streptococcus thermophilus.

Results

This alteration resulted in a novel Ebosin derivative (EPS-7 g) with its monosaccharide composition dramatically changed, especially in the proportion of glucose which increased from 1.1% (Ebosin) to 84.01% (EPS-7 g). In an ELISA analysis, EPS-7 g exhibited a higher binding activity for IL-1R, as a competitor of interleukin-1, than that of Ebosin. It also exhibited a higher inhibitory effect on the activity of IL-1β-converting enzyme and production of IL-1β in fibroblast-like synoviocytes (FLS). In addition, experiments with acute inflamed mice induced by croton oil showed a significantly higher anti-inflammatory activity of EPS-7 g compared with Ebosin.

Conclusions

The new Ebosin derivative EPS-7 g is more bioactive than Ebosin evaluated by a series of experiments. This is the first report demonstrating a modification of EPS structure via heterologous gene replacement in Streptomyces.

Keywords

Ebosin derivativeHeterologous gene replacementGene gtfGene ste7StreptomycesStreptococcus thermophilus

Background

One type of microbial polysaccharides are secreted out of the cells [1] and therefore called exopolysaccharides (EPSs), which are long-chain polysaccharides consisting of branched, repeating units of sugars or sugar derivatives [2]. In bacteria, EPS biosynthesis starts with the intracellular formation of EPS precursors and the sugar nucleotides, followed by the formation of a repeating unit on a lipid carrier which is located in the cytoplasmic membrane. The later steps involve transport of the repeating units across the membrane to the outer layer and polymerization of tens and even hundreds of such units to form the final EPSs [3]. During the process, glycosyltransferases play important roles to sequentially transfer sugars from intracellular nucleotide sugars to a lipid carrier acceptor [4].

In order to improve applications of EPSs, genetic engineering can be used in the production of desired polysaccharides targeting predefined macroscopic properties.

Based on the available genetic information, genetic modification of eps genes should lead to EPSs with a different repeating unit or with a different chain length [5]. Manipulation of genes which function in export, polymerization, and determination of chain length during EPS biosynthesis were also proved effective for altering EPS structure [6]. Heterologous production of an EPS has been successful by transfer of the complete eps gene cluster alone of a LAB strain into a non EPS-producing heterologous host, provided that the heterologous host possessed all necessary genetic information for precursor synthesis [7]. Ingeborg et al [8] described increased exopolysaccharide production in Lactoccus lactis due to manipulated overexpression of the NIZO B40 eps gene cluster, the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactoccus lactis leads to increased EPS production.

Streptomyces are a group of gram-positive bacteria that have been intensively studied for their secondary metabolites, particularly antibiotics. However, little is known of the production of EPSs in Streptomyces. Recently a novel EPS namely Ebosin was isolated from the supernatants of fermentation cultures of Streptomyces sp. 139 [9], which has remarkable anti-rheumatic arthritis activity in vivo[10]. Its biosynthesis gene cluster (ste) consisting of 27 ORFs was also identified [11]. Efforts have been made in elucidating the function of individual ste genes [12]–[15]. Insights into the biosynthesis pathways of polysaccharides are crucial for the exploitation of microorganisms to produce polysaccharides of industrial or medicinal importance. With a number of the ste genes identified, it is now possible to carry out specific manipulations for creating Ebosin derivatives which can then be screened for better bioactivities. This paper reports our endeavor to generate such derivatives by tackling the ste7 gene encoding a fucosyltransferase, which catalyzes the transfer of fucose specifically from GDP- fucose to a fucose acceptor during Ebosin biosynthesis [16]. In this study, we replaced ste7 with the glucosyltransferase gene (gtf) from Streptococcus thermophilus, which is a gram-positive bacterium and most valuably used in food industry. Stingele et al [17] identified the eps genetic locus of S. thermophilus Sfi6, revealing a 15.25-kb region containing 16 open reading frames (ORFs), within a 14.52-kb region encodes 13 genes (epsA to epsM) capable of directing EPS synthesis. The resultant EPS-7 g produced by the strain Streptomyces sp. 139 (gtf) has a very high content of glucose and exhibited remarkably higher bioactivities than that of Ebosin in both in vitro and in vivo assays.

Results

Construction of the heterologous gene replacement strain Streptomyces sp. 139 (gtf)

The replacement of ste7 gene by the gtf gene of S. thermophilus (Figure 1A) was evidenced by Southern hybridization using a 1.03-kb DNA (F1) as probe upstream of ste7 (Figure 1B). The hybridization signals appeared with the expected sizes of 4.0 kb for Streptomyces sp. 139 (gtf) and 3.1 kb for Streptomyces sp. 139 (ste7 -). This result indicated that the colonies with thior Ams phenotypes had integrated the gtf-thio r cassette into the kanamycin resistance gene, therefore, confirmed the establishment of heterologous gene replacement strain Streptomyces sp. 139 (gtf).
Figure 1

The diagram of gene replacement and Southern blot analysis. (A) Diagram of gene replacement of ste7 with gtf originated from Streptococcus thermophilus through a double crossover via homologous recombination. Gray box indicates the location of Kmr gene in Streptomyces sp. 139 (ste7 - ). Restriction maps of Streptomyces sp. 139 (ste7 - ) and the gene replacement strain Streptomyces sp. 139 (gtf) show the predicted fragment sizes upon Bam HI digestion. (B) Southern blot autoradiograph of Streptomyces sp. 139 (ste7 - ) and Streptomyces sp. 139 (gtf). 1. Chromosome DNA of Streptomyces sp. 139 (ste7 - ) digested with Bam HI; 2. chromosome DNA of Streptomyces sp. 139 (gtf) digested with Bam HI.

Sugar composition of EPSs

GC analysis of Ebosin, EPS-7 m produced by the knock mutant Streptomyces sp. 139 (ste7 -) and EPS-7 g by Streptomyces sp. 139 (gtf) (Figure 2A, B, C) showed that these EPSs consisted of the same monosaccharides: glucose, mannose, arabinose, galactose, fucose, xylose and rhamnose, of which the most striking was an increase of glucose proportion from 1.1% (Ebosin) to 84.01% (EPS-7 g). While, some proportional changes of other sugars also occurred in comparison with Ebosin (Figure 2D). Using a method based on uronic acid carbazole reaction, galacturonic acid was also found in EPS-7 m and EPS-7 g.
Figure 2

GC chromatograms of sugar analysis of Ebosin (A), EPS-7 m (B) and EPS-7 g (C). 1. rhamnose, 2. fucose, 3. arabinose, 4. xylose, 5. mannose, 6. glucose, 7. galactose. (D). Monosaccharide compositions of Ebosin, EPS-7 m and EPS-7 g. Galacturonic acid was analyzed using a method based on uronic acid carbazole reaction (Bitter et al., 1962).

The competitive binding activity of Ebosin derivates with IL-1 for IL-1R

Using an ELISA assay, the competitive binding activities of Ebosin, EPS-7 m and EPS-7 g with IL-1 for IL-1R were determined.

For EPS-7 m they were 6.1% and nil (P < 0.05) at dosages of 0.64 ng/μL and 0.128 ng/μL respectively, which were remarkably lower than those of Ebosin (24.2%, 13.7%) at the same dosages. Higher binding activities of EPS-7 g were detected to be 27.1% and 24.3% (P < 0.05) respectively compared with that of Ebosin at same concentrations (Figure 3).
Figure 3

Binding activities of Ebosin, EPS-7 m and EPS-7 g to IL-1R determined by an ELISA assay.

Effect of Ebosin derivates on the enzymatic activity for IL-1β-converting enzyme (ICE)

At concentrations of 0.64 ng/μL and 0.128 ng/μL, Ebosin suppressed the enzyme with inhibition ratios 35.5% and 25.0% respectively, compared with 15.5% (P < 0.05), 14.3% for EPS-7 m and 47.6%, 35.7% (P < 0.05) for EPS-7 g respectively (Figure 4A). The results demonstrated a higher activity of EPS-7 g to inhibit the IL-1β-converting enzyme.
Figure 4

Suppression of ICE enzymatic activity (A) and inhibited production of IL-1β in FLS (B) by Ebosin, EPS-7 m and EPS-7 g. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus FLS without stimulating with LPS (control 1).

Inhibiting effect of Ebosin derivates on production of IL-1β in FLS cells

To assess the effects of Ebosin, EPS-7 m and EPS-7 g on production of IL-1β in FLS (fibroblast-like synoviocytes), cell cultures were carried out and stimulated with LPS at 37°C for 72 h before analyzed by ELISA. The results indicated (Figure 4B) that Ebosin, EPS-7 m and EPS-7 g at dosage of 3.2 ng/μL reduced the IL-1β production 52.03% (P < 0.01), 26.17%, 58.70% (P < 0.01) respectively and 45.57% (P < 0.5), 15.39%, 52.34% (P < 0.01) at dosage of 0.64 ng/μL separately. With dosage of 0.128 ng/μL, the IL-1β production were suppressed 31.61%, 10.16% and 47.82% (P < 0.01) individually.

Effect of Ebosin and EPS-7 g on the inflammatory activity in the acute inflamed mice induced by croton oil

To evaluate the effect of Ebosin and EPS-7 g on the acute inflamed mice induced by croton oil, the mice were randomly divided into three groups described as above. Ebosin and EPS-7 g (100 mg/kg) were orally administered to each mouse of the respective experimental groups for 1 h before induction by croton oil. Compared with the acute inflamed mice groups untreated (control), the inhibition ratios of EPS-7 g and Ebosin for mice ear edema were 48.61% (P < 0.05) and 21.60% respectively (Figure 5), indicating that EPS-7 g surpasses Ebosin by over 100% on suppressing the inflammatory activity in the acute inflamed mice.
Figure 5

Effect of EPSs on inflammatory activity in the acute inflamed mice induced by croton oil.

Discussion

Exopolysaccharides include a range of diverse polymers that play vital roles in variety of biological processes. In addition, EPSs have also significant industrial applications, including their use as biothickeners in foods [4]. Notably, EPSs produced by lactic acid bacteria contribute significantly to the structure and viscosity of fermented milk products [18]. The health value of these macromolecules has also emerged in recent years; many reports indicate that they can confer health benefits on consumers arising from their immunogenic and cholesterol-lowering properties [19],[20]. EPSs produced by Trichoderma erinaceum DG-312 was shown to have a strong anti-inflammatory activity in inflamed mice [21]. Enterobacter cloacae was also found to produce EPSs with anti-diabetic activity [22].

Metabolic engineering has enabled generation of “designer” polysaccharides in lactic acid bacteria (LAB), which mostly involved manipulations of glycosyltransferases [2]. Introducing new or existing glycosyltransferases into LAB [4] or gene shuffling with glycosyltransferases are effective means for controlling EPS structure [5]. Masja et al [23] reported that heterologous production of the pneumococcal serotype 14 polysaccharide in Lactococcus lactis resulted in the recombinant product secreted into culture medium, which simplified downstream processing. This was achieved by coexpressing the pneumococcal gene cluster cpsFGHIJKL 14 with the lactococcal regulatory and priming glucosyltransferase-encoding genes (epsABCD B40) specific for B40 polysaccharide. Knoshaug et al [24] reported evidence for a novel gene organization expressing EPS in Lactococcus lactis subsp. cremoris Ropy352 and showed the specificities of polymerization and export enzymes blocking the function of just one glycosyltransferase abolished the production of ropy EPS. Heterologous expression of glycosyltransferases has been found to result in different sugars added at strategic positions in generating EPSs with new properties [5].

Ebosin is a novel EPS with anti-rheumatic arthritis activity. It has been shown that Ebosin is an inhibitor of IL-1β-converting enzyme (ICE), a key enzyme in synthesis of IL-1β [25]. More recently, evidence has also been obtained demonstrating that the anti-inflammatory effect of Ebosin on rat collagen-induced arthritis is through suppressing production of interleukin-1β, interleukin-6 and tumor necrosis factor α at both transcriptional and posttranslational levels [10]. This EPS is therefore of medicinal value so improvement for better property is warranted. In this study, a glucosyltransferase gtf gene from S. thermophiles was expressed in replacement of the ste7 gene which encodes a fucosyltransferase in the Ebosin-producing strain Streptomyces sp. 139. This brought about dramatic changes in the property of EPS produced. Because of the complexity of the biosynthesis pathway of Ebosin and involvement of a large number of genes, once ste7 was replaced with the heterologous gene gtf, dramatic effect was seen not only on the increased incorporation of glucose, but also on the overall sugar profile. The reason for these changes, which are apparently not a simple proportional change of composition with increased glucose, may lie in the primary structure. The resultant EPS-7 g turns out to be more bioactive compared with Ebosin evaluated by its ICE inhibitory activity, suppression of interleukin-1β production in fibroblast-like synoviocytes (FLS), competitive binding to IL-1R against IL-1 and suppression of the acute inflammatory activity in the acute inflamed mice induced by croton oil. According to these results, it looks like that increasing proportion of glucose effects on bioactivity of EPS-7 g. A good understanding of the correlation between structure and bioactivity of Ebosin is important because it will provide a foundation for a strategy aimed at producing functionally more valuable polysaccharides. Continuing efforts in the elucidation of Ebosin biosynthesis pathway will enable more rational designs for genetic manipulation to generate highly effective derivatives.

Conclusions

EPS-7 g, a novel Ebosin derivate, was produced by heterologous gene replacement in Streptomyces sp. 139, which was more bioactive compared with Ebosin evaluated by a series of experiments. To our knowledge, this is the first report on manipulation of EPS structure by introducing heterologous glucosyltransferases into Streptomyces.

Methods

Bacterial strains and culture conditions

Streptomyces sp. 139 was isolated from a soil sample in China and kept in the China General Microbiology Culture Collection Center (No. 0405) (Table 1). Streptomyces sp. 139 (ste7 -) was generated previously [16] and kept in our laboratory. These strains were cultured at 28°C with shaking (250 rpm) in either TSB medium supplemented with 5 mM MgCI2 and 0.5% glycine or fermentation medium (1% glucose, 2% starch, 2% soybean extract, 0.2% tryptone, 0.2% beef extract, 0.4% yeast extract, 0.05% K2HPO4, 0.3% CaCO3, pH 7.3). Streptococcus thermophilus was obtained from China Industrial Microbiology Culture Collection Center (CICC 20370) (Table 1)and cultured in LB medium at 40°C.
Table 1

Bacterial strains and plasmids used in this study

Strain or plasmid

Description

Reference

Strains

  

E.coli DH5α

supE44 Δ lacU169 (φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relAI

[27]

E. coli DH5a (pUC-gtf)

E. coli DH5a clone with gtf

This study

E. coli ET12567

Methylation-deficient E. coli dam-dcm-hsdM

[29]

Streptomyces sp. 139

Ebosin producing strain

Lab stock

Streptomyces sp. 139 (ste7 - )

The gene ste7 disruption mutant

This study

Streptomyces sp. 139 (gtf)

Ams, thior, the heterologous gene replacement strain of Streptomyces sp.139

This study

Streptococcus thermophilus

Wild type

CICC 20370

Plasmids

  

pUC18

Ampr, E.coli general cloning vector with multiple cloning site

[27]

pUC-gtf

Ampr, pUC18 derived plasmid carrying gtf gene

This study

pKC7

Kmr, Amr, pKC1139 derived plasmid carrying F1, F2

This study

pKC7-gtf

pKC7 carrying gtf gene

This study

pEGM-T-tsr

pEGM-T carrying thio r gene

Lab stock

Animal

Kunmin mice (male, 18-20 g, Certificate No. SCXK 2005-0013) and Wistar rats (male, 180 ± 20 g, Certificate No.: SCXK 2005-0013) were purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences, Beijing. All rats were housed under standard laboratory conditions with the approval of the Institute of Experimental Animals and Use Committee of Chinese Academy of Medical Sciences.

Cell culture of FLS (fibroblast-like synoviocytes)

Synovial tissues obtained from the knee joints of sacrificed CIA (collagen- induced arthritis) rats [26] on day 30 after immunization were minced and digested with type II collagenase (0.4%, Gibco) at 37°C in a humidified 5% CO2 incubator for 2 h in Dulbecco’s modified Eagle’s medium (DMEM), thoroughly washed and then cultured in DMEM supplemented with fetal bovine serum (15%, Gibco). At confluence, adherent cells were trypsinized (0.25%, Hyclone) at 37°C for 0.5 h, filtered and extensively washed again. The adherent cells were cultured in DMEM containing fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 incubator for 24 h. FLS from passage 3-4 were seeded at 1 × 106/mL in 24-well plates (Nunc) or at 1 × 105/mL in 6-well plates in DMEM and cultivated at 37°C for 24 h.

DNA isolation and southern blot analysis

Isolation of E. coli plasmid DNA, S. thermophilus genomic DNA and standard recombinant DNA techniques were performed as described by Sambrook and Russell [27]. Streptomyces plasmid and genomic DNA was isolated as mentioned by Kieser et al [28]. For Southern blot analysis, the DIG high prime DNA labeling and detection starter kit II obtained from Roche (USA) was used following the instructions of manufacturer.

Cloning of the gene gtf of Streptococcus thermophilus

The genomic DNA isolated from 48-hour cultured S.thermophilus was used as template. The gtf gene was amplified by PCR using primers P1 and P2 (Table 2) in the following conditions: an initial denaturation at 94°C for 10 min; 30 cycles of 1 min at 94°C, 1 min at 47°C and 3 min at 72°C; and finally 10 min at 72°C. The amplified DNA fragment was cloned into plasmid pUC18 digested with Eco RI and Sal I to construct pUC-gtf . The correct nucleotide sequence of gtf gene fragment cloned in pUC-gtf was verified by sequencing using an ABI PRISM 377XL DNA Sequencer (Applied Biosystems). Then the recombinant plasmid pUC-gtf was transformed into the strain E. coli DH5α to produce the strain E. coli DH5α (pUC-gtf). This plasmid pUC-gtf was isolated according the protocol mentioned by Sambrook and Russell [27].
Table 2

Oligonucleotide primers used in this study

Primer

Sequence 5′ – 3′

P1

5′-GCGAATTC TCTAGAATGGCGTGGCTAATTAAATG-3′

P2

5′-GCGTCGAGTTAATCGCTTTCAATA-3′

P3

5′-GCGTCGACAGGCGAATACTTCATATG-3′

P4

5′-GCAAGCTT TCTAGATGATCATCACTGACGAAT-3′

Construction of the strain Streptomyces sp. 139 (gtf)

With the plasmid pEGM-T-tsr (unpublished data) as template, the thiostrepton resistant gene (thio r ) was amplified by PCR using primers P3 and P4 (Table 2) in the following conditions: an initial denaturation at 94°C for 5 min; 30 cycles of 1 min at 94°C, 0.5 min at 58°C and 2 min at 72°C; and finally 10 min at 72°C. The amplified 1.06 kb gene fragment (thio r) was inserted into plasmid pUC-gtf digested with Sal I- Hin dIII , which was then digested by Xba I to isolate the fragment of thior-gtf. During construction of the ste7 gene disrupted strain Streptomyces sp. 139 (ste7 - ) in previous study [16], the gene ste7 disruption vector pKC7 containing a 1.03-kb F1 fragment upstream of the gene ste7 and a F2 0.899-kb fragment downstream of the gene ste7 was created. The fragment of thior-gtf was introduced into the ste7 disruption vector pKC7 at Xba I site to construct the gene replacement plasmid pKC7-gtf. Propagated in the methylation-deficient E. coli 12567 [29], pKC7-gtf was isolated and introduced into Streptomyces sp. 139 (ste7 - ) by polyethylene glycol (PEG)-mediated protoplast transformation [28]. Incubated at 28°C for 16 to 20 h, the plates were overlaid with soft R2YE (0.7% agar) containing apramycin (40 μg/mL). Plasmid pKC7-gtf bears a temperature-sensitive Streptomyces replication origin [20] that is unable to replicate at temperatures above 34°C. Therefore, the transformants were first incubated at 28°C for 2 days until pinpoint size colonies became visible and then shifted to 37°C for further incubation. Strains resulted from a double crossover via homologous recombination grew out of the original pinpoint-size colonies in several days. The heterologous gene replacement strain Streptomyces sp. 139 (gtf) was selected by both apramycin sensitivity (Ams, 40 μg/mL) and thiostrepton resistance (thior, 50 μg/mL).

Five thior Ams colonies were selected randomly and the isolated genomic DNA originated from Streptomyces sp. 139 (gtf) and Streptomyces sp. 139 (ste7 -) [16] were digested with Bam HI individually before DNA blot analysis.

Isolation of EPSs

The strains of Streptomyces sp. 139, Streptomyces sp. 139 (ste7 -) and Streptomyces sp. 139 (gtf) were cultured at 28°C for 96 h respectively. Following the protocol as described before [9], Ebosin, EPS-7 m and EPS-7 g were isolated from the supernatants of fermentation cultures of respective strains.

Monosaccharide analysis of EPSs

Sugar composition analysis was performed as described by Sun et al [30]. The purified polysaccharide samples (10 mg) was dissolved in 3 ml of 2 M TFA to hydrolyze at 115°C for 3 h, then drying under a stream of nitrogen. Subsequently, the residue was treated with 200 μL of 0.5 M Na2CO3 at 30°C for 45 min and added 1.5 mL of 4% NaBH4 at room temperature for 2 h. After that, the sample was neutralized with 25% acetic acid and loaded onto a cation exchange resin column eluted with H2O, then discharged the excess NaBH4 with methanol. The sample residue was dried with P2O5 in vacuum at 85°C for 2 h and acetylated with 2.5 mL of acetic anhydride in 0.5 mL of pyridine at 100°C for 30 min. After cooling, the excess reagent was discharged under a stream of nitrogen and the sample was analyzed by Gas Chromatography (GC, HP5890, HEWLETT, Houston, TX, USA). Galacturonic acid was analyzed using a method based on uronic acid carbazole reaction [31].

Assay for IL-1R binding activity

The enzyme-linked immune specific assay (ELISA) method reported previously was used to analyze the binding activity for IL-1R of isolated EPSs [15].

Assay of the enzymatic activity for IL-1β-converting enzyme (ICE)

We found recently that Ebosin is an inhibitor of IL-1β-converting enzyme [32], a key enzyme in synthesis of IL-1β. To assess the inhibitory effects of EPS-7 m [16] and EPS-7 g on ICE enzymatic activity, the assay was conducted according to the protocol reported by koizumi et al [33]. The enzymatic reactions were performed in 100 μL reaction solution consisting of 20 mM HEPES (pH 7.5), 0.1 mM NaCl, 5 mM dithiothreitol, 10% sucrose, 250 μM substrate N-acetyl-Tyr-Val-Ala-Asp- p-nitroanilide, 2.0 IU of IL-1β-converting enzyme (ICE), 0.64 ng/μL and 0.128 ng/μL of Ebosin, EPS-7 m and EPS-7 g respectively at 37°C for 45 min.

Enzyme-linked immunosorbent assays of IL-1β in FLS cells

FLS was seeded at 1 × 106/mL in 24-well plates and cultivated at 37°C for 24 h. Ebosin (3.2-0.128 ng/μL) was added individually to each well and cultivated at 37°C for 1 h, then FLS were stimulated with LPS (5 μg/mL per well) at 37°C for 72 h. IL-1β in the supernatants were analyzed with ELISA kits (Applygen). The cultivated FLS cells un-stimulated with LPS were as contro1, while the cultivated FLS cells stimulated with LPS but not treated by Ebosin were as control 2.

Treatment of the acute inflamed mice induced by croton oil

The mice were randomly divided into three groups: acute inflammatory mice [34], Ebosin + acute inflammatory mice and EPS-7 g + acute inflammatory mice (n = 8 in each group). Ebosin and EPS-7 g (100 mg/kg) were orally administered to each mouse in the treatment groups. After 1 h, 50 μL of 2% croton oil (in absolute ethyl alcohol) was smeared on the left auricle of each mouse in three groups for 4 h. All the mice were then sacrificed and ears were excised. The degree of ear edema was measured by weighing deference between the right and left ear.

Statistical analysis

Data were presented as the mean ± SD from at least 3 independent experiments. The significance of differences between groups was evaluated by Student’s t-test. p values less than 0.05 were considered significant.

Abbreviations

EPSs: 

Exopolysaccharides

FLS: 

Fibroblast-like synoviocytes

LAB: 

Lactic acid bacteria

gtf

Glucosyltransferase gene

ORFs: 

Open reading frames

Declarations

Acknowledgements

This research was supported by a grant from the Natural Science Foundation of China (NSFC 30530830). It has been supported by the Fundamental Research Funds for the Central Universities in China (2012 N09) and a grant from the National Key Project of New Drug Study of China (2012ZX09301002-001-023-02).

Authors’ Affiliations

(1)
Key laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College
(2)
Institute of Pharmacology and Toxicology
(3)
School of Biological Sciences, University of Wollongong

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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