Aluminum (Al) is the most abundant metal in nature and is widely used in daily life [1
]. The principal exposure route to Al for humans is through food, water and beverages [2
]. The mean levels of Al exposure via this route range from 0.2 to 1.5 mg/kg of body weight (bw) weekly in Europe and 1.3 mg/kg·bw/week for adults and 3.3 mg/kg·bw/week for children in China [3
]. The European Food Safety Authority (EFSA) established a health-based guidance value for 1 mg/kg·bw/week, which is thus exceeded in China [4
]. The excess Al could accumulate in most of the tissues, including brain, liver, kidneys, spleen and bone, and may cause Alzheimer’s disease, dialysis encephalopathy, microcytic anemia, and osteomalacia [5
]. The intestinal tract is the first barrier against Al exposure. Previous studies reported that the intestinal tract was the main storage organ for Al: forty percent of the ingested Al was found to accumulate in the intestinal mucosa [6
]. The intestinal barrier consists of a single layer of epithelial cells and tight junction (TJ) proteins, acting as the body’s first line of defense against harmful substances, including heavy metals [8
]. Once Al is absorbed, it can induce oxidative stress and intestinal inflammation [2
]. At the same time, the absorbed Al can damage TJ proteins and impair the intestinal barrier function and cause cell apoptosis, thereby increasing the intestinal permeability [2
Probiotics exhibit a variety of health-promoting functions [10
] and there is accumulating evidence that some probiotics have the potential to alleviate metal toxicity [11
]. Moreover, probiotics can provide benefits to the host gut through a diverse set of mechanisms that include modulation of immune responses, alleviation of oxidative stress and reduce intestinal permeability by maintenance of intestinal barrier integrity by expression and distribution of TJ proteins [13
]. TJ proteins comprise a large group of proteins, including the scaffolding proteins zonula occludens-1 (ZO-1), and the transmembrane proteins occludin and claudins, which are critical in maintaining barrier function [16
]. A number of lactobacilli have been shown to improve intestinal barrier function, partly by modulating the expression and distribution of the TJ proteins [13
Our previous studies demonstrated the protection against Al toxicity in vitro and in vivo by the probiotic strain Lactobacillus plantarum
CCFM639 was due to its enhanced Al-binding and antioxidative capabilities [17
]. It significantly inhibits Al absorption in the intestines of mice, thereby decreasing Al accumulation in tissues and alleviating tissue injury caused by this metal. A number of studies have demonstrated that probiotics can protect intestinal health [19
]. We hypothesized that L. plantarum
CCFM639 can inhibit Al absorption by protecting the intestinal integrity and modulating the inflammatory response.
2. Materials and Methods
2.1. Bacterial Strains and Culture Conditions
L. plantarum CCFM639 (CGMCC9664) was obtained from the in-house Culture Collections of Food Microbiology in Jiangnan University (CCFM, Wuxi, China) and stored in the China General Microbiological Culture Collection Center (CGMCC, Beijing, China). It was grown in MRS broth (Qing Dao Hopebio-Technology Co., Ltd., Qingdao, China) at 37 °C.
2.2. Cell Culture Experiment
HT-29 cells were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI-1640 medium, supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (PS) at 37 °C in an atmosphere of 5% CO2. The media and supplements were purchased from Gibco (New York, NY, USA).
The HT-29 cells were seeded in and cultured in different formats, including 96-well plates, 12-well plates, 6-well plates and 2-cm-diameter cover glass-bottom dishes, at a density of 105
cells/mL, and grown in an atmosphere of 5% CO2
at 37 °C. When the cells attached to the plates and formed monolayers, Al ions and the prepared L. plantarum
CCFM639 were added at final concentrations of 4 mM and 108
CFU/mL, respectively (Table 1
). The dose of Al exposure was selected based on a previous study [2
] and on the data obtained from our preliminary cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Figure S1
Medium, RPMI-1640 supplemented with fetal bovine serum (FBS) and penicillin–streptomycin (PS); Medium + Al, 4 mM Al ion in medium; Medium + 639, 1 × 108 CFU/well L. plantarum CCFM639 in medium; Medium + Al + 639, 4 mM Al ion and 1 × 108 CFU/well L. plantarum CCFM639 in medium. PW, plain water for drinking; SM, 0.2 mL skim milk; Al, Al ion at 200 mg/L in drinking water; DFP, 0.2 mL DFP (2.5 g·L−1) in plain water. SM + 639, 0.2 mL skim milk contained 1 × 109 CFU L. plantarum CCFM639 once a day administered via oral gavage. SM + DFP, 0.2 mL DFP administered once a day via oral gavage.
2.3. Animals and Experimental Design
Forty adult C57BL6 mice (6-week-old males, 18–25 g) were bought from the Shanghai Laboratory Animal Center (Shanghai, China). The mice were housed in a room with constant temperature and humidity and a 12 h light-dark cycle was maintained. The standard commercial mouse food and deionized water were given ad libitum. All of the protocols were approved by the Ethics Committee of Jiangnan University (Wuxi, China, JN No. 20150312-0717-10). The care procedures for the animals were implemented following the European Community guidelines (directive 2010/63/EU).
The mice were randomly divided into control, Al only, Al plus CCFM639 and Al plus deferiprone (DFP) groups (Table 1
). An oral dose of Al ions (200 mg/L) in drinking water was used for establishing the Al exposure model [21
]. The probiotic strain was given via gavage at a dose of 1 × 109
CFU in 0.2 mL of skim milk once daily. The skim milk was used as a protectant to avoid the loss of viability during lyophilization. The DFP was administered in 0.2 mL at a concentration of 2.5 g/L via oral gavage as a positive control. At the end of the experiment, each mouse was anesthetized by ether and then sacrificed.
2.4. Measurement of Cytotoxicity
The cell viability was measured by MTT assay according to the procedure previously described [23
]. The assay kit was obtained from the Institute of Beyotime Biotechnology (Jiangsu, China). The result of the cell viability was expressed as the ratio to the level in the control group. Apoptotic rate was measured with an Annexin V-FITC/PI kit (Institute of Beyotime Biotechnology, Jiangsu, China), and the FITC levels were then detected by flow cytometry (FACSCalibur, Becton Dickinson, San Diego, CA, USA) [23
2.5. Measurement of Tight Junction Proteins in HT-29 Cell Line
The morphologies of the TJ proteins were evaluated by immunofluorescence staining as previously described [24
]. Firstly, HT-29 cells were fixed, permeabilized and blocked. They were then incubated with the primary antibodies at 1:50 dilution, including anti-ZO-1, anti-claudin-1 and anti-occludin (Life Technologies, Rockford, AL, USA), overnight at 4 °C. The cells were then incubated with secondary antibodies, Alexa Fluor 488-conjugated goat anti-rabbit at 1:50 dilution (Life Technologies, Rockford, AL, USA), for 1 h at room temperature. Finally, the cells were exposed to the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Images of the stained cells were acquired using confocal laser scanning microscopy (TCS SP8, Leica, Mannheim, Germany).
2.6. Measurement of Tight Junction Proteins in Mice Intestinal Tissues
The small intestine and colon sections were prepared by blocking with 5% bovine serum albumin (BSA) (Sigma, Dorset, UK) and 0.5% Triton X-100 (Sigma, Dorset, UK) in PBS. The tissues were incubated with primary antibodies, anti-occludin (Santa, San Fransisco, CA, USA, sc-271842) and anti-claudin-1 (Santa, San Fransisco, CA, USA, sc-166338) and anti-ZO-1 (Abcam, Cambridge, UK, ab59720). They were then incubated with secondary antibodies, goat anti-mouse Alexa 488 (Abcam, Cambridge, UK, ab150113) or goat anti-rabbit Alexa 488 (Abcam, Cambridge, UK, ab150077) for 1 h at room temperature. Nuclear staining and imaging were performed as described above.
The mRNA expressions of the TJ proteins in intestinal tissues were evaluated by real-time quantitative PCR (RT-qPCR). Approximately 0.1 g of small intestine and colon fragments were isolated quickly and preserved in liquid nitrogen immediately. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s operating instructions, and the extracted RNA samples were analyzed by NanoDrop and via gel electrophoresis. The RNA was then transcribed to cDNA using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Tokyo, Japan). Gene expression was investigated using a RT-qPCR system (Bio-Rad, Hercules, CA, USA) and the sequences of the RT-qPCR primers used were as follows [25
]: claudin-1 forward (F): 5′-GATGTGGATGGCTGTCATTG-3′ and reverse (R): 5′-CCTGGCCAAATTCATACCTG-3′; occludin F: 5′-CACACTTGCTTGGGACAGAG-3′ and R: 5′-TAGCCATAGCCTCCATAGCC-3′; ZO-1 F: 5′-CTTCTCTTGCTGGCCCTAAAC-3′ and R: 5′-TGGCTTCACTTGAGGTTTCTG-3′; and β-actin F: 5′-GGCTGTATTCCCCTCCATCG-3′ and R: 5′-CCAGTTGGTAACAATGCCATGT-3′.
2.7. Measurement of Intestinal Permeability of Mice
The intestinal permeability of the mice was analyzed by endotoxin assay as described in a previous study [26
]. After sacrifice, the endotoxin levels in the serum were detected using an ELISA kit (Abcam, Cambridge, UK).
2.8. Measurement of Oxidative-Stress-Related Parameters in HT-29 Cell Cultures and Serums
The activities of superoxide dismutase (SOD) and catalase (CAT) and the level of malondialdehyde (MDA) were used to evaluate oxidative stress [17
]. The level of MDA and activities of SOD and CAT in HT-29 cells and serums of mice were determined by assay kits (Nanjing Jiancheng Bioengineering, Jiangsu, China). The level of reactive oxygen species (ROS) in HT-29 cells was measured by S0033 assay kit (Institute of Beyotime Biotechnology, Jiangsu, China) following manufacturer’s instructions.
2.9. Measurement of Pro-Inflammatory Cytokines in HT-29 Cell Cultures and Intestinal Tissues
The HT-29 cells were centrifuged at 15,000× g
for 15 min at 4 °C to obtain the supernatant. The small intestine and colon samples were homogenized with 1 mL of radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Jiangsu, China) and then centrifuged under the same conditions to obtain the supernatant [26
]. The levels of TNF-α, IL-1β and IL-6 in the supernatant were detected using an ELISA kit (Abcam, Cambridge, UK) following the manufacturer's instructions.
2.10. Statistical Analysis
The data were expressed as the mean ± standard error of the mean (SEM). The data were statistically analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Differences between the data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Values of p < 0.05 were considered to be statistically significant.
A number of studies have documented the adverse effects of Al on the brain, liver and bone [28
]. Recently, more attention has been paid to the harmful effects of Al on the intestine, including injury of intestinal barrier, decrease of tight junction (TJ) protein expression and stimulation of intestinal inflammation [2
]. This study demonstrated that chronic Al exposure can cause the disruption of TJ proteins, increase intestinal permeability and induce oxidative stress and inflammatory response. Moreover, we observed that L. plantarum
CCFM639 treatment could increase Al excretion [17
] and protect the intestinal barrier, which acts as the body’s first line of defense against oral Al exposure. Meanwhile, L. plantarum
CCFM639 has the ability to alleviate oxidative stress and inflammatory response. Therefore, we hypothesized that L. plantarum
CCFM639 can reduce Al absorption and protect intestinal health possibly via initial intestinal Al sequestration, protection of TJ proteins, alleviation of oxidative stress and inflammatory response.
Moreover, Lactobacilli need to survive exposure to gastric acid and bile before reaching the intestines and this passage can reduce the numbers of viable bacteria. A large number of studies show that oral administration of probiotics should be more than 108
CFU per day, which can permit viable bacteria levels to be more than 106
CFU in human or animal intestines that can then provide the beneficial effects [30
]. Therefore, a dose of 109
CFU was selected in the present study.
Our previous study found that a probiotic L. plantarum
CCFM639 could reduce Al absorption in the intestines of mice due to its excellent Al-binding ability [17
]. The decrease of Al absorption in the intestines in turn protects the intestine from Al-induced injures. It is believed that intestinal absorption of Al includes paracellular and transcellular routes. The former is through TJ proteins by passive processes, and the latter is through the enterocytes involving passive and active transport processes [1
]. The results of in vitro therapy assay in this study showed that L. plantarum
CCFM639 can significantly maintain the vitality of cells and reverse the disruption of TJ proteins even 24 h after Al treatment (the strain had no direct contact with the aluminum). These results indicated that L. plantarum
CCFM639 can protect against Al toxicity, not only via the Al-binding route but also by other protective routes (Figure 7
). Increasing evidence indicates that some probiotics can improve gut barrier dysfunction caused by toxic substances [36
]. Lactobacilli are one of the well-known groups of beneficial members of the intestinal microbiota and very commonly are used in probiotic foods [39
]. Previous studies have demonstrated that Lactobacilli could maintain a normal mucosal barrier via modulation of TJ proteins [15
]. Al exposure damage TJ proteins located between pairs of epithelial cells, which may cause intercellular leakage and promote Al movement across the intestinal barrier [2
]. Moreover, Al induces cell damage and apoptosis, which may lead to leakage in the epithelial layer, causing a greater amount of Al to enter the systemic circulation [42
]. Therefore, maintaining the integrity of the gut barrier plays a very important role in inhibiting intestinal Al absorption. The results showed that L. plantarum
CCFM639 can significantly increase TJ proteins levels in vivo. Therefore, modulation of TJ proteins may be one route by which L. plantarum
CCFM639 protects barrier function (Figure 7
The mechanisms of Al toxicity also include oxidative stress and inflammatory response [28
]. Al exposure causes oxidative stress with a decrease in SOD and CAT activities and a higher level of MDA [17
]. It also stimulates production of pro-inflammatory cytokines [2
], including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. Moreover, pro-inflammatory cytokines can activate the NF-kB pathway, which has been identified to be critical to oxidative stress, and then generate reactive oxygen species (ROS) [44
]. At the same time, the production of ROS will aggravate the inflammatory process [11
]. Oxidative stress may result in loss of tight junction integrity and increased paracellular permeability [45
]. There is increasing evidence indicating that probiotics have beneficial effects on intestinal inflammation [10
]. They also cause an inhibition of the NF-kB pathway, which thereby alleviates oxidative stress [47
]. In present study, L. plantarum
CCFM639 was not in direct contact with Al in the therapy assay, but it still significantly inhibited the generation of ROS and pro-inflammatory cytokines, reduced the MDA levels and increased the activities of SOD and CAT, indicating that L. plantarum
CCFM639 has a direct protective effect against Al-induced oxidative stress and inflammation. These two properties of this strain may not only enable it to protect against Al-induced oxidative stress and inflammation in the intestine, but also against chain effects triggered by oxidative stress and inflammation.
Our research also showed that, although both L. plantarum
CCFM639 and DFP have good and similar Al sequestration ability in the intestine, the former has a better protective effect of decreasing Al absorption and maintaining gut health than the latter. The better protective effect of L. plantarum
CCFM639 is due to its enhanced antioxidative and anti-inflammatory activities, allowing it to further protect gut health against Al-induced oxidative stress and inflammation, but DFP only increases Al excretion and thereby decreases intestinal exposure to Al. L. plantarum
CCFM639 thus has a more comprehensive protective effect than DFP on Al induced gut injuries. Moreover, Lactobacilli are extensively used in the fermented foods and are generally regarded as safe [49
]. Therefore, it is possible to develop this strain for its application as functional probiotic and a starter culture in fermented foods, including fermented soymilk, yogurt or it can be supplemented in fruit and vegetable juices to elevate the nutritional value, and increase shelf life with additional benefits of providing protection against Al toxicity.