Screening Immunocompetent Lactic Acid Bacteria And Its Immunoregulatory Effect On Mice

Immunity is the body's identification of"self"and"non-self(different)",generating an immune response to eliminate"different"antigens or induce immune tolerance,thereby maintainingitsinternalenvironment.Immunosuppression(immunitydamageor immunodeficiency)refers to the reduction of the body's immune system activity and the reduction or even disappearance of the immune response in a short time or for a long period of time due to damage to the immune system.Immunosuppression can cause the body to reduce the immune response to the antigen and increase the sensitivity to the disease,which is an abnormal immune state.There are many substances on the surface of lactic acid bacteria such as peptidoglycan,cell wall teichoic acid,lipoteichoic acid,surface protein and other cell wall related polysaccharides.In addition,lactic acid bacteria can also secrete extracellular components with immunomodulatory activity,such as extracellular polysaccharides and other secreted proteins.These components are all involved in immune regulation or can directly elicit an immune response,and thus constitute a material basis for lactic acid bacteria to exert their probiotic effects.The purpose of this project is to screen lactic acid bacteria with high immunocompetence from 10 strains of lactic acid bacteria preserved in the laboratory and to study the related biological characteristics of the screened lactic acid bacteria.And by establishing an immunosuppressive mouse model,it was observed whether the lactic acid bacteria had immunoregulatory capabilities.Cell Counting Kit-8(CCK-8)method was used to observe the effects of different doses of ten lactic acid bacteria on the proliferation of mouse spleen lymphocytes and the energy metabolism of mouse peritoneal macrophages.Macrophage phagocytic neutral red method was used to evaluate the effects of different doses of ten Lactobacilli on the phagocytosis of macrophages.The results of in vitro experiments showed that ten strains of lactic acid bacteria showed a dose-effect relationship on the proliferation activity of spleen lymphocytes and on phagocytosis of macrophages and on the macrophage energy metabolism levels,among which Lactobacillus plantarum KLDS1.0318 had the highest regulatory activity on splenic lymphocytes and macrophages.The growth curve of Lactobacillus plantarum KLDS1.0318 was determined by plate counting and UV-Vis spectrophotometry,and the acid production capacity was measured by a pH meter.The plate count method was used to determine the survival rate of the strain after its action in the acidic and high bile salt environment and the survival rate was calculated.The experimental results showed that Lactobacillus plantarum KLDS 1.0318 entered the logarithmic phase after 4 h and reached a stable phase after 12 h.Its acid-producing ability is strong,and the pH drops to 4.16after 12 h.When the strains were cultured at pH 2.5 and 3.5 for 3 h,their survival rates were 91.20%and 97.50%,respectively,and they survived for 4 h in a medium with a bile salt mass concentration of 0.3 and 0.5 g/100 mL,respectively.The rates were 95.40%and 87.76%,respectively.Ninety-five specific pathogen free(SPF)BALB/c mice were habituated in the animal house for 1 week until their body weight reached(20±2)g.The mice were randomly divided into 6groups:normal control(NC),model control(MC),positive control(PC),lactic acid bacteria low-dose group(1×10~7 CFU/mouse),lactic acid bacteria medium-dose group(1×10~8 CFU/mouse),lactic acid bacteria high-dose Group(1 x 10~9 CFU/mouse).Normal control mice were injected with an equal volume of phosphate buffer saline(PBS),and the remaining mice were injected intraperitoneally with cyclophosphamide 80 mg/kg for 3 d to establish an immunosuppressive mouse model.Mice in each group were given oral administration once a day for 20 days.At the end of the experiment,each mouse was weighed and the mice were sacrificed by eyeball sampling.The thymus index and spleen index,the proliferation activity of T lymphocytes,the activity of NK cells,the phagocytic capacity of macrophages,and the macrophage energy metabolism were measured in each group.The levels of interleukin-2(IL-2),interleukin-6(IL-6),interferon-?(IFN-?),tumor necrosis factor-?(TNF-?)and immunoglobulin G(IgG)in mice were measured by enzyme-linked immunosorbent assay.Hematoxylin-Eosin(HE)staining and Alcian Blue Periodic Acid Schiff(AB-PAS)staining were used to observe the pathological structure of the small intestine.The results of animal experiments showed that Lactobacillus plantarum KLDS1.0318could act on immune organs of mice,increase the thymus index and spleen index of immunosuppressed mice,and at the same time can enhance the activity of immune cells,promote the proliferation of T lymphocytes,enhance the killing activity of NK cells to YAC-1 target cells,increase macrophage phagocytic neutral red capacity and its energy metabolism;significantly up-regulate IL-2 and IFN-?levels in spleen cells of mice,TNF-?levels in macrophages,IL-2,IL-6,IFN-?and IgG levels in serum;significantly increase the small intestine villi length,crypt depth,goblet cell number and mucin area in mice.Conclusion:(1)The effect of ten strains of lactic acid bacteria on the activity of immune cells was evaluated in vitro.The results showed that Lactobacillus plantarum KLDS1.0318 has high immunomodulatory activity.(2)Lactobacillus plantarum KLDS1.0318 grows faster and has stronger acid-producing ability and acid and bile-resistance.(3)Lactobacillus plantarum KLDS1.0318 can increase the thymus index and spleen index of immunosuppressed mice,enhance their immune cell activity,significantly increase their cytokine levels,and significantly improve the morphological structure of intestinal mucosa.In summary,the results of this study show that the screened Lactobacillus plantarum KLDS 1.0318 can be used as a probiotic Lactobacillus with potential immunomodulatory effects.