| Cadmium(Cd) is a toxic heavy metal which can be easily accumulated in humans and animals. This non-essential metal is also classified as the sixth toxic substance by Agency for Toxic Substances and Disease Registry(ATSDR, U.S.). Cd exposure causes a variety of adverse health effects in humans including hepatic, renal, reproductive, skeletal and cardiovascular dysfunctions. The soil pollution condition investigation announcement published by the government of China in 2014 indicates that Cd has become the contaminant with highest rate of exceeding allowable limit, causing great public health concerns. Chelation therapies, the most direct treatments to alleviate Cd toxicity by promoting Cd excretion, are de?cient in safety and ef?cacy. Lactic acid bacteria(LAB) are food-grade microorganisms which can be generally recognized as safe, and have been widely used in the food industry. Previous reports have demonstrated that LAB strains can bind and remove heavy metals such as cadmium in vitro, alleviate oxidative stress in human subjects, and provide protective effects on gut barrier, indicating these bacteria have potency against Cd toxicity. But to our knowledge, very few studies on the protective effects of LAB against Cd toxicity have been carried out so far. Therefore, the objective of this study was to screen LAB strains with potency on the alleviation of Cd toxicity, to investigate their biological characteristics, and to evaluate the effects of the strain against Cd toxicity in cell and animal models. In addition, the underlying protective mechanisms were studied and the application of LAB-fermented food for Cd toxicity protection was considered.Firstly, LAB strains were screened for their Cd binding ability, Cd tolerance, antioxidative capacity and acid and bile salt tolerance. Lactobacillus plantarum CCFM8610 showed the highest binding ability in the experiments with several different initial Cd concentrations. This strain also had signi?cantly better Cd tolerance than other tested strains, with the minimum inhibitory concentration of over 1000 mg/L. With the evaluation of DPPH radical scavenging, hydroxyl radical scavenging, lipid peroxidation inhibition and reducing activity, CCFM8610 exhibited relatively good antioxidative capacity. Further principal component analysis showed characteristics of CCFM8610 were the most similar to those of L. rhamnosus GG, a reference strain that has been reported to have good antioxidative ability both in vitro and in vivo. Additionally, survival rates of CCFM8610 in simulated gastric and intestinal juices were 88.72% and 92.98%, respectively. These results indicated that CCFM8610 had potential protective effects against Cd toxicity. As a reference strain, L. rhamnosus GG had good Cd tolerance, antioxidative capacity and acid and bile tolerance, but the Cd binding ability of this strain was poor. Other tested strains also had defects in one or several evaluated parameters, therefore CCFM8610 was selected for further studies.The Cd binding mechanisms of CCFM8610 were firstly investigated by observation with transmission and scanning electron microscope-energy dispersive X-ray analysis, and the occurrence of Cd binding was clearly noticed. Using the methods to separate cellular components, it was observed that most Cd was bound in the surface of cell wall and cell membrane, only 10% entered the protoplast. After blocking the surface groups of the cell, it could be found that amino- and carboxyl- groups played important roles in the binding process, but phosphate group was not closely related with the binding ability. The binding thermodynamic analysis showed that the binding process of CCFM8610 can be explained by Langmuir–Freundlich dual isotherm model(R2=0.9928), and the maximum binding ability(Qmax) of CCFM8610 was significantly higher than that of commercial probiotic strains such as L. rhamnosus GG and L. casei Shirota. The binding kinetics analysis showed that the binding process is fast and effective, which could be well explained by pseudo-second-order kinetics equation and reached 90% of the equilibrium absorption capacity at the time point of 100 min. The Weber-Morris model showed that the binding process of CCFM8610 had two stages, including the diffusion of Cd2+ from the bulk solution to the cell surface and the Cd2+ binding of the active sites in the cell surface. Additionally, protective effects of CCFM8610 against Cd toxicity were evaluated in HT-29 cell model. This strain could recover the oxidative stress induced by Cd exposure, modulate the immune response of the cell, inhibit cell apoptosis and protect cell viability, indicating CCFM8610 can alleviate Cd-induced cytotoxicity.The protective effects of CCFM8610 agianst Cd toxicity were subsequently investigated in animal models. In the acute Cd exposure experiment, compared to the mice that received Cd only, CCFM8610 treatment could effectively decrease the mortality of mice, increase the Cd levels in the feces, reduce hepatic and renal Cd accumulation, alleviate Cd-induced tissue oxidative stress, and ameliorate tissue histopathological changes. With the establishment of therapy group and prevention group, the protective effects of both living and dead CCFM8610 treatments were observed and the results showed living CCFM8610 administered after Cd exposure offered the most signi?cant protection. In the chronic Cd exposure experiment, CCFM8610 exhibited similar protective effects against Cd toxicity, and induced the metallothionein(MT) production in the tissues. Compared with the protection of L. bulgaricus CCFM8004, a commercial yogurt starter culture, CCFM8610 showed significantly better effects, indicating the protection of LAB strains against Cd toxicity is strain specific.To further investigate the underlying protective mechanism of LAB strains against Cd toxicity, mice in chronic Cd exposure experiments were then treated with three L. plantarum strains which have different biological properties, respectively. The results showed that chronic Cd exposure caused the damage of intestinal barrier, including the decreased expression of tight junctions, reduced levels of secretory immune globulin(s Ig A), increased levels of inflammatory factor and altered intestinal permeability, which exacerbated the Cd absorption in the gut. Besides the binding ability, L. plantarum strains could recover the above mentioned intestinal barrier damage, which in turn inhibited Cd absorption in the gut. The protection against intestinal barrier damage could be due to the Cd binding and antioxidative stress capacities of the strains. As CCFM8610 possessed both capacities, it exhibited better protection than that of the control L. plantarum strains. Additionally, the intestinal Cd sequestration route of CCFM8610 was bypassed by introducing Cd directly into mice via intraperitoneal injection. The results showed that besides intestinal Cd sequestration, CCFM8610 treatment offers direct protection against Cd-induced oxidative stress. Compared with the protection of L. bulgaricus CCFM8004, it could be concluded that such protection is strain specific.The evaluation of the protective effects of LAB fermented soymilk against chronic Cd toxicity in mice showed that non-fermented soymilk gave limited protection against chronic Cd toxicity. However, oral administration of L. plantarum CCFM8610 fermented soymilk was able to increase fecal Cd excretion, reduce tissue Cd burden, alleviate tissue oxidative stress, reverse changes in hepatic and renal damage biomarkers, and ameliorate tissue histopathological changes in mice. The treatment of L. bulgaricus CCFM8004 fermented soymilk provided similar protection, although the effects were less signi?cant than for CCFM8610 treatment. The conjunct effects of the strains and the soymilk may be attributed to two aspects including the increased Cd excretion ability by the synergistic action of LAB strains, soymilk and aglycones, and the increased antioxidative capacity by LAB-fermentation-induced alteration of glucoside/aglycone ratio. |
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