Background
Colla corii asini (donkey-hide gelatin, E-Jiao) is a well-known traditional Chinese medicine [
1] and has been used for more than 2000 years in Asia for health-care food [
2]. Previous reports have shown that the main components of
Colla corii asini comprise amino acids, microelements and small molecular weight hydrolysates of collagen proteins [
3]. Some pharmacological properties of
Colla corii asini include sedation, anticoagulation, vasodilatation, hematopoiesis, as well as the improvement of cellular immunity and radio-protection [
3,
4]. It has been widely used for the treatment of gynecologic diseases (e.g., dysmenorrhea, menoxenia, metrorrhagia, abortion) [
5] and some chronic diseases (e.g., anxiety, insomnia, apostaxis, hemoptysis, hematuria, hemafecia) [
6]. Also,
Colla corii asini has the anti-aging effect through enhancing antioxidant activity, scavenging free radicals, and modulating aging-related gene expression in aging mice model [
7]. Furthermore,
Colla corii asini promotes anemia and optimizes hemoglobin components in pregnant women with thalassemia [
8]. Despite the increasing number of studies on the pharmacological effects of
Colla corii asini, the antibacterial effect of
Colla corii asini has not yet been investigated.
The gram-negative bacterium
Salmonella enterica serovar Typhimurium (
S. typhimurium) is the main cause of gastroenteritis in humans and animals around the world, and it occurs via bacterial gastrointestinal infections [
9]. These bacterial infections are caused by oral intake of contaminated food or water or by fecal contamination [
10,
11].
Salmonella epidemics associated with contaminated food have been shown to result from insufficient hygiene conditions.
S. typhimurium is classified as a non-typhoidal serovar, which can infect a broad range of animals and humans and can cause acute intestinal inflammation and diarrhea [
12]. The systemic spread of
S. typhimurium may cause severe disease in people with underlying infections or immunodeficiency disorders and can trigger an enormous economic loss in animal husbandry.
S. typhimurium relies on two distinct groups of genes,
Salmonella pathogenicity islands (SPI)-1 and SPI-2, which play pivotal roles in the colonization of the host [
13]. SPI-1, which encodes a type III secretion system (T3SS), secretes effectors such as SipA, SipB and SipC, which promote host invasion in the small intestinal tract via the formation of actin filaments at the bacterial entry site [
14]. In contrast,
Salmonella pathogenicity islands 2 (SPI-2) are required for proliferation inside the host cells [
15].
In this study, we investigated the antibacterial effects of Colla corii asini by using S. typhimurium in vitro and in vivo. Furthermore, our findings suggest clues that may aid in understanding the antibacterial action of Colla corii asini and determining whether Colla corii asini may be a good candidate for treating bacterial diseases.
Methods
Source of bacterial strain and culture condition
The gram-negative bacterium S. typhimurium KCTC 1926 was obtained from the Korean Research Institute of Bioscience & Biotechnology (KRIBB). For the experiment, bacteria were cultured at 37 °C with shaking in a 5 ml Luria-Bertani (LB) broth medium, until 0.5–0.6 OD value at 600 nm, pelleted at 5000 x g for 10 min, washed in PBS, and resuspended in 1 ml Dulbecco Modified Eagle Medium (DMEM) completed media for cell infection (1 × 108/ml).
Cell culture
Human colorectal cancer cells, caco-2 cells were maintained in DMEM with 1% antibiotics (penicillin-streptomycin), and 10% fetal bovine serum (FBS) at 37 °C in 10% CO
2. Cells were seeded at 1 × 10
6 cells per well in 6-well tissue culture plates containing or not coverslips and maintained as differentiated monolayers for 21–28 days, changing the media every 2–3 days [
16].
Preparation of Colla corii asini extract
Colla corii asini were purchased from the Korea Medicinal Herbs Association (Yeongcheon, Korea). The identification of Colla corii asini was confirmed by Professor KiHwan Bae of the College of Pharmacy, Chungnam National University (Daejeon, Korea), and all voucher specimens were deposited in the herbal bank in the Korea Institute of Oriental Medicine (KIOM, Korea).
To prepare the Colla corii asini ethanol extract (CEE), Colla corii asini (50 g) was grounded to powder, then suspended in 70% ethanol (300 ml) on shaking incubator (100 rpm) for 24 h at 40 °C. The solution was filtered through a nylon net filter (60 μm; Millipore Co., Denver, MA, USA), and then deposited overnight. The supernatant was lyophilized, and then the dried pellet (the yield, 1.66%) was stored at −20 °C until use. Also, to prepared the Colla corii asini water extract (CWE), Colla corii asini (50 g) were placed in 1000 mL distilled water and then extracted during 2 h of heating at 115 °C (Gyeongseo Extractor Cosmos-600, Inchon, Korea), and the solution was filtered using standard testing sieves (140 μm) (Retsch, Haan, Germany). The supernatant was lyophilized, and then the dried pellet (the yield, 38.70%) was stored at −20 °C until use.
Amino acid content in Colla corii asini
The amino acid composition of Colla corii asini were assayed with an L-8900 amino acid analyzer (Hitachi, Japan) after hydrolysis of 20 mg sample with 0.02 N HCl at 110 °C for 24 h. The Aspartic acid (Asp), Threonine (Thr), Serine (Ser), Glutamic acid (Glu), Glycine (Gly), Alanine (Ala), Cysteine (Cys), Valine (Val), Methionine (Met), Isoleucine (Ile), Leucine (Leu), Tyrosine (Tyr), Phenylalanine (Phe), Lysine (Lys), Histidine (His), Arginine (Arg), Proline (Pro), Valine (Val), β-alanine and ethanol amine content were quantified by standard peak values.
Agar well diffusion assay
The extracts were tested for antibacterial effects by using agar diffusion on Luria agar [
17]. The solid agar was punched with 7 mm diameter holes using the steriled tips. The agar plates included 1% S. typhimurium and were then treated with 100 μl of Colla corii asini extract. The concentrations of the extracts (EtOH and water) applied were 0.25–2 mg/ml, and ampicillin (5–40 μg) was used as a positive control. The solid agar was then incubated at 37 °C for 24 h. After incubation, the clear zone associated with each concentration was measured.
Determination of the minimum inhibitory concentration and minimum bactericidal concentration
To determine the MIC (minimum inhibitory concentration) of the CEE, we detected the lowest concentration that did not demonstrate visible bacterial growth after 24 h of incubation at 37 °C. The MIC was determined by using two-fold serial dilutions of the extracts [
18]. The MBC (minimum bactericidal concentration) value was determined by sub-culturing the test dilutions on a solid medium and incubating the cultures at 37 °C for 24 h. The MBC value indicated the lowest dilution concentration at which there was no bacterial growth on the solid medium.
Analysis of protein secretion
S. typhimurium (1 × 108 cells) was incubated in 6-well plates. CEE was treated to S. typhimurium for 1 h, and then collected the DMEM media, and centrifuged, and the supernatants (2 ml) were collected. The supernatants were filtered (0.45-μm pore size), and the proteins were precipitated with 100% trichloroacetic acid (TCA) by using high-speed centrifugation (14,000×g for 30 min). The pellet was washed in cold acetone and resuspended in PBS. The solutions contained proteins and were mixed with Sodium Dodecyl Sulphate (SDS) sample buffer and boiled for 5 min. Each sample was separated by SDS-10% polyacrylamide gel electrophoresis (SDS-PAGE). The expression of SipC protein were analyzed by Western blot. Bacteria were lysed using ice-cold lysis buffer, and the protein concentration were determined by bicinchoninic acid (BCA) assay. Equal amounts of protein (30 μg/mL) were electrophoresed on 4–10% SDS-acrylamide gels and transferred to nitrocellulose membranes using an electric transfer system. Blots were incubated for overnight at 4 °C with primary antibody against anti-SipC (1:100; ABIN335178). After three times washes with TBS-T, the membranes were incubated with HRP conjugated goat anti-mouse IgG (1:2000) for 1 h at room temperature. The signals were detected with a chemiluminescence reagent (Millipore Corporation, Billerica, USA), and analyzed with the ChemiDoc Touch Imaging System (Bio-Rad, Hercules). Protein bands were quantified by densitometry using Image J
Quantitative real-time PCR
S. typhimurium (1 × 10
8 cells) was incubated in 6-well plates. CEE was treated to S. typhimurium for 1 h and mRNA was purified using TRIzol reagent and chloroform. Reverse transcription was conducted in 20 μl reactions with 1 μg of total RNA transformed into cDNA using AccuPower Cycle Script RT premix (Bioneer). The measurements of SipA, SipB and SipC mRNA and 16 s rRNA were conducted under the following conditions: 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s using a LightCycler 480 II (Roche, Rotkreuz, SWI). The mRNA level in each sample was quantified on the basis of the threshold cycle (Ct). The target genes were normalized relative to the reference gene 16S rRNA [
19].
Cell viability
Cell viability was determined by using a Cell Counting Kit-8 (CCK-8) according to the manufacturer’s instructions by utilizing Dojindo highly water-soluble tetrazolium salt (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H–tetrazolium, monosodium salt] (WST-8) was reduced by dehydrogenases in cells, to forming a yellow product (formazan) generated by the activity of dehydrogenases in cells in a manner directly proportional to the number of live cells. Briefly, Caco-2 cells (1 × 103 cells) were plated on a 96-filter plate. After CEE treatments (1.56–0.04875 mg/ml) for 24 h, 10 μl of the CCK-8 solution was added to each well of the plate and incubated for 1 h at 37 °C with 5% CO2. Absorbance was measured at 450 nm using a precision microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Selectivity index (SI)
SI was calculated dividing the IC
50 value by the MIC value for CEE [
20,
21].
Gentamicin assay
S. typhimurium was used to infect Caco-2 cells (1 × 10
6 cell/well) in in 6 well plate. CEE was used to treat Caco-2 cells with/without bacterial infection (50 multiplicity of infection (MOI)) in antibiotic-free medium, non-adherent bacteria were washed three times by PBS. Gentamicin (100 μg/ml) was then added for 1 h to kill any remaining bacteria. The Caco-2 cells were washed three times with PBS and lysed with 1% Triton-X-100. Bacteria in the lysis solutions were plated on solid agar, and colony numbers were counted after O/N incubation at 37 °C [
22].
Infection of Salmonella enterica serovar Typhimurium in mice
The infection of S. typhimurium was initiated in 6 to 7-week-old male ICR mice (Samtako Inc., Gyunggi-do, Korea). All mice were divided into 4 groups, each consisting of 5 mice. Mice were administered Colla corii asini extract (100 or 200 mg/kg) via oral gavage daily. Mice were administered either 100 μl of S. typhimurium (5 × 10
7 cells) or a vehicle control (PBS) by gavage. Forty-eight hours post-infection, mice were euthanized, and tissues (small intestine) were obtained from each treatment and the control group. Intestine samples were stored using the “Swiss roll” technique for immunohistochemistry (IHC) analysis [
23]. All procedures were approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee (KIOM-IACUC) and were conducted in accordance with the US guidelines (NIH publication #83–23, revised in 1985).
Histological analysis
After tissue fixation, Swiss rolled intestinal tissues were embedded in paraffin and were sectioned into 5 μm sections using a microtome. The serial sections were subjected to immuno-fluorescence staining using fluorescence-tagged antibodies for measuring the number of S. typhimurium in the small intestine (ileum) using a confocal microscope.
Statistical analysis
For analysis of the statistical significance of differences between two groups we generally performed two-tailed Student’s t-tests. For analysis of the statistical significance of differences among more than two groups, we performed one-way ANOVAs with Dunnett’s multiple comparisons tests. All statistical analysis was performed using GraphPad Prism version 5.03 for Windows (GraphPad Software Inc., San Diego, CA, USA).
Discussion
S. typhimurium is the main cause of gastroenteritis, and it occurs via bacterial gastrointestinal infections from contaminated food, thus causing various diseases and enormous economic loss. Normally,
S. typhimurium-related diseases occur when bacteria penetrate the gastro-intestines [
24]. Despite the growing recognition of hygiene and health, salmonella infections have increased worldwide, and the underlying mechanisms remain unclear. Additionally, mutant bacteria and antibiotic resistance has increased, and new therapeutic agents without side effects are needed [
25].
Colla corii asini (E‘jiao) is prepared from the skin of
Equus asinus and is used in traditional Chinese medicine and nutritional supplements in China and Korea [
2]. We conducted experiments to determine the antibacterial effects of approximately four hundred kinds of Korean medicine on
S. typhimurium, and
Colla corii asini was the most effective. We hypothesized that
Colla corii asini had an antibacterial effect through killing
S. typhimurium as well as inhibiting the invasion of the host.
In the present work, the extracts obtained from
Colla corii asini showed strong activity against
S. typhimurium. Our data showed that CEE at a high dose (the inhibition of bacterial growth) possessed an antibacterial effect. An agar well diffusion assay and MIC/MBC values indicated that
Colla corii asini might be the new antibacterial agent (Figs.
1 and
2, Table
2).
In previous studies, human and animal models of salmonella infection have investigated many TTSS effector proteins such as SipA, SipB and SipC and have shown that they are involved in inflammatory responses and diarrhea [
26]. Salmonella invasion proteins play a pivotal role in the infection process and facilitate bacterial invasion in non-phagocytic cells in the host [
27,
28]. SipA is an important protein promoting salmonella invasion [
29]. SipB is a substrate of the SPI1 type III export system [
30] and deficient bacterial SipB do not enter eukaryotic cells [
27]. SipC is a protein with diverse functions, and its actin nucleation and bundling activities play a critical role in salmonella-induced actin cytoskeleton reorganization and bacterial invasion [
31]. Our data showed that CEE at low doses (no effect on bacterial growth) significantly decreased the expression of salmonella invasion proteins including SipA, SipB and SipC in
S. typhimurium. Additionally, the mRNA expressions of salmonella invasion proteins were suppressed in a dose-dependent manner (Fig.
3a and b). To confirm the effect of CEE on salmonella invasion, we conducted gentamicin assays. CEE did not affect Caco-2 cell viability and attenuated the bacterial invasion in the Caco-2 cell monolayer (Fig.
4a and b). Furthermore, CEE inhibited the invasion of
S. typhimurium in the small intestines (Fig.
5).