| Objective: The study was aimed to research the effect of Lactobacillus casei on the immunomodulatory function and intestinal barrier function of 7,12-dimethylbenz(a)anthracene-induced breast cancer in rats.The effect of Lactobacillus casei on the immunomodulatory function of rats was evaluated by the changes of the cytokine in serum,natural killer(NK)cell activity and T lymphocyte subsets distribution in peripheral blood.The effect of Lactobacillus casei on the intestinal barrier function was assessed by the improvement of the intestinal flora and the expression level of intestinal tight junction(TJ)proteins in rats.Methods: 1.The inhibitory and immunoregulatory effects of Lactobacillus casei on 7,12-dimethylbenz(a)anthracene(DMBA)-induced breast cancer in rats.(1)Animal grouping and model building: After one week adaptation with general meals,sixty female Spraque-Dawley(SD)rats were randomly divided into control group(C),model group(M),low dose of Lactobacillus casei group(L1)and high dose of Lactobacillus casei group(L2)based on body weight.The rats were subcutaneously injected with 100mg/kg DMBA to establish breast cancer rat model.Four and eight m L/(kg.d)Lactobacillus casei were administered to L1 and L2 by gavage,and five m L/(kg.d)soybean oil was administered to control and model groups by gavage,respectively,1 time/day,6 times/week for 16 weeks in total.As soon as the experimental rats were sacrificed,the blood,the thymus,spleen and the small intestine were all collected from the dead rats.(2)The incidence rate of breast cancer,the latency of breast cancer,the average tumor weight,tumor suppression rate and organ index were calculated.(3)The activity of NK cells and the percentages of T lymphocytes were quantified in peripheral blood of rats by flow cytometry.(4)The levels of interleukin 4(IL-4),IL-6,IL-10,IL-12,interferon γ(IFN-γ)and tumor necrosis factor α(TNF-α)in serum were detected.2.The effects of Lactobacillus casei on the intestinal flora and the intestinal barrier function of DMBA-induced breast cancer in rats.(1)In addition,the level of four major bacterial populations of the enteric flora was detected by real-time PCR(q PCR).(2)Hematoxylin-eosin(HE)staining was used to observe the pathological changes of jejunum tissue in rats.(3)The levels of D-lactate(DLA)in serum in different groups were detected.(4)The expression levels of tight junction(TJ)proteins,occludin and ZO-1,were measured using western blotting.Results:1.The inhibitory and immunoregulatory effects of Lactobacillus casei on DMBA-induced breast cancer in rats.(1)No tumor was detected in group C,whereas the tumors were formed in remaining three groups.Compared with the model group,the L2 group have the extended latency of tumors and the tumor incidences and tumor weights were both reduced(P<0.05).The tumor inhibition rate in the L2 group reached to 41.2%.Compared with the model group,the thymus index in the group treated with high-dose of Lactobacillus casei was increased significantly(P<0.05).(2)Compared with the model group,the percentage of TCRαβ+ CD161a+ NK cells and the CD3+CD8+ T cells in L2 groups were both increased significantly,while the proportion of CD3+ Foxp3+ T cells was decreased significantly(P<0.05).(3)ELISA results showed that,compared with the control group,in the model group,the concentration of IL-4 was decreased,while the concentrations of IL-6,IL-12 and TNF-α were increased(P<0.05).In the group treated with high-dose of Lactobacillus casei,the concentration of IL-4 and IL-10 were significantly higher than those in the model group,while the concentrations of IL-6 and IL-12 were significantly decreased respectively(P<0.05).In L1 and L2 groups,TNF-α concentrations were decreased significantly compared with the model group(P<0.05).2.The effects of Lactobacillus casei on the intestinal flora and intestinal barrier function of DMBA-induced breast cancer in rats.(1)Compared with the control group,the copy numbers of Bifidobacterium,Lactobacillus and Enterococcus faecalis were all decreased in the model group of rats.Notably the copy number of Bifidobacterium and Enterococcus faecalis were significantly decreased(P<0.05).In the group treated with Lactobacillus casei,Bifidobacterium was increased significantly(P<0.05),while in the intervention group,Escherichia coli was decreased compared with the model group.(2)Pathological observation of jejunum tissue in rats by HE staining showed that,compared with model group,in low and high dose of Lactobacillus casei,the morphology of the villi and the crypt was clear and complete,the structure was clear,and the damage of the villi was lighter.But compared with the control group,the small intestinal mucosal thickness was decreased,the arrangement of villi were more messy,the tip of villi showed the phenomenon of edema in L1 and L2 groups.(3)The D-LA level in group model was increased,compared with that in group control(P<0.05).Interestingly,D-LA level in group L2 was significantly lower than that in group model(P<0.05).(4)Western blotting showed that the expression levels of occludin and ZO-1 was decreased significantly in group model,while the expression of occludin and ZO-1 were significantly higher in group L2 than group model(P<0.05).Conclusions: 1.Lactobacillus casei displayed the inhibitory effect on breast cancer rat.2.After Lactobacillus casei supplementation,the levels of inflammation-associated cytokines were improved,the distribution of CD3+CD4+,CD3+CD8+ T cells,NK cells and regulatory T cells were changed,and the immunoregulatory function were enhanced.3.Lactobacillus casei can improve the distribution of intestinal flora,reduce the concentration of D-LA in serum,increase the expression of intestinal TJ protein occludin and ZO-1,and improve the intestinal barrier function. |
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