Effect Of Bacillus On The Antioxidative Capability Of Caco-2 Cells And Mice

The indispensable oxidation in organisms is the main source of the free radical. The major approach to decrease oxidative stress is to keep the homeostasis of free radical. As a high-quality and safe probiotic, Bacillus has been developed into various forms of additive used in animal production. But few researches on their antioxidative function has been done and reported. Based on their antioxidative activities in vitro, three strains of Bacillus were selected to study their antioxidative effects on Caco-2 cells and mice under oxidative stress. The main results were as follows:(1) The antioxidative capability of twelve Bacillus strains were studied and compared, including the capacity of scavenging DPPH and hydroxyl radical, the reduction activity and antioxidant enzymes activities. The results indicated that both supernatants and cell-free extracts of all these twelve Bacillus strains showed antioxidative capacities. For the supernatants, the DPPH scavenging rate ranging from 8% to 48%, and the reduction activity, GSH-Px activity, SOD activity, and no hydroxyl radical scavenging rate were detected. For the cell-free extracts, the DPPH scavenging rate ranged from 29% to 48%, all 12 strains had the reduction activities, GSH-Px and SOD activities, and the hydroxyl radical scavenging rate of 11 strains ranged from 3% to 16%. Therefore, two strains with higher antioxidative capacity (B1 and B3), and one strain with relative lower antioxidative performance (B10) were selected for further study.(2) H2O2 was added into the cell culture medium at final concentrations of 0,50,100 and 200μmol/L, respectively. After being cultured for 12h,24h,48h, the oxidative injuries of Caco-2 cells were determined. Results showed that compared with the control,50μmol/L of H2O2 didn't cause the damage of cells significantly.The recoverable oxidative damage to cells was detected when 100μmol/L H2O2 was added, with the cell survival rate being decreased at 12h,but increased at 48h significantly.200μmol/L H2O2 could damage cells seriously, which caused lower cell survival rate, LDH activity and free radical scavenging rate. Thus oxidative stress of Caco-2 cells could be induced when they were cultured for 12h with addition of 100μmol/L H2O2.(3) The effects of antioxidant TBHQ and Bacillus (B1, B3 or B10) on the antioxidative activity of Caco-2 cells under oxidative stress were determined. Results showed that addition of Bacillus (B1, B3 or B10) increased T-AOC significantly (P＜0.01), and decreased MDA content (P>0.05) in supernatant of Caco-2 cells cultured for 12 hours. In addition to the enhanced anti-superoxide anion rate, SOD and CAT activities, the supernatant of Caco-2 co-cultured with B10 also showed the increased the hydroxyl scavenging rate and POD activity. The increased T-AOC for the group addition with B1 or B3 was mainly due to the increased anti-superoxide anion capacity, SOD and CAT activities in supernatant and elevated POD and SOD activities in the cell lysate. After being cultured for 48 hours, cells co-cultured with Bacillus showed the increased T-AOC (P＜0.01) and antioxidant enzymes activities, and the lower MDA content and LDH activities (P＜0.01) in supernatants. In addition, the antioxidative activities of three Bacillus strains were higher than TBHQ.(4) 75 ICR male mice with similar initial weight (35 g), were randomly divided into 5 groups, with 3 replicates each group and 5 mice per replicate. Group A was fed with basal diet while Group B was fed with high fat diet. Group C-E were fed with high fat diet supplemented with 0.01%TBHQ,0.1% B1 and 0.1% B10 (108CFU/g), respectively. The test lasted for 30 days. At the end of the trail,6 mice were randomly selected from each group to collect blood samples, livers and small intestines for further analysis. The results showed that the daily gain of groups supplemented with Bacillus (groupD and E) were lower than that of the control (group B), decreased by 57.89% (P＜0.01) and 42.20% (P＜0.05) respectively, but had no significant differences with the control (group A). It indicated that Bacillus could alleviate the obesity of mice caused by high fat diet. Addition of Bacillus in high fat diet could decrease the levels of TG (P<0.01) and TC (P＜0.01) in liver, and increase level of HDL-C in serum. In addition, two Bacillus strains could decrease the level of glucose, GOT and GPT activities in serum. It could be concluded that Bacillus could decrease the liver injuries of mice caused by high fat diet.Addition of Bacillus in high fat diet could increase the antioxidative activities in intestinal mucosa, liver and serum of mice. The increased level of GSH and CAT activity in liver, T-AOC in intestinal mucosa, and the lower level of MDA and XOD activity for the group addition with B1 were detected, while the increased GSH-Px activity in liver, SOD activity and anti-superoxide anion capacity in intestinal mucosa and the lower level of MDA in liver were observed for the group addition with B10. The two Bacillus strains could increase the T-AOC, the level of GSH and GSH-Px activity in serum of mice. Compared to the control group, the level of 8-OHdG in liver of Group D and E decreased significantly (P＜0.01), but that in intestinal mucosa had no significant differences.Above all, Bacillus can not only secrete antioxidative substances and antioxidant enzymes by themselves, but also improve the antioxidative functions of cells and animals under oxidative stress conditions, so as to prevent oxidative damage. Meanwhile, Bacillus may also prevent obesity through reducing levels of triglyceride and cholesterol in liver.