Screening Of Anti-HP Lactobacillus Acidophilus And Anti-CagA～+HP Specific Binding Peptides And Its Effects On C57BL/6 Mice With HP Infection

PARTⅠEstablishment of HP infection models and analysis of histopathology in different inbred strains of miceObjective Establish different HP strains infected models in different inbred strains of mice. Observe HP colonization and histopathology in mice. Methods SPF C57BL/6 and BALB/C mice were inoculated with either 0.5 ml of HP SS1 or HP NCTC 11637 (1×109CUF/ml) four times interval 48 h. Prior to inoculation, all animals were given 0.5ml 0.1M sodium bicarbonate. Control groups were inoculated with PBS alone. At 4, 8, 12 weeks after the last inoculation, mice were sacrificed and their stomaches were removed for rapid urease test , bacterial culture and pathological examination. Plasme HP -IgG- antibody was tested by ELISA. Results All C57BL/6 mice inoculated with HP SS1 were infected with HP at 4, 8, 12 weeks after the last inoculation. The bacteria inoculated mainly in the antrum and transitional zones between the antrum and body, and its the highest level of colonization was about 105 CFU/g. At the same time lymphocytes and monocytes infiltration could be seen in submucosa of stamoch. However, C57BL/6 mice inoculated with HP NCTC 11637 and BALB/C mice inoculated with HP SS1 or HP NCTC 11637 were few infected with HP, and showed low level of colonization and slight infiltration of lymphocytes and monocytes. Conclusion The susceptibility and pathogenicity of HP infection were variable between differences inbred strains of mice inoculated by different HP strains. Both host background genes and HP strains are important factor in HP infection. C57BL/6 mice inoculated with HP SS1 showed the highest level of both susceptibility to colonization and persistent infection, indicating that this model is a reliable model for human infection.PARTⅡScreening of anti-HP Lactobacillus acidophilus and analysis of inhibitory effects on HP in vitro Objective To screen the strains of Lactobacillus acidophilus which have obvious antagonistic activity against Helicobacter pylori in vitro. Methods Lactobacillus acidophilus were isolated from the feces of 120 health persons by anaerobic culture and were identified by culture properties, biological characteristics and biochemical reactions. In vitro studies we investigated the inhibitory effects of Lactobacillus acidophilus on HP SS1, NCTC 11637 and clinical isolate by well diffusion assay and liquid culture assay. Results 62 suspected stains of Lactobacillus acidophilus were isolated from the feces of healthy persons. Among them 16 strains of Lactobacillus acidophilus had more inhibitory activity on HP than 10g/L lactic acid and were called L1~L16. And their inhibitory activity was independent of lactic acid level. 2 strains of Lactobacillus acidophilus (L4 and L6) had highest inhibitory activity on HP in well diffusion assay . The 2 strains of Lactobacillus acidophilus (L4 and L6) and their culture supernatants significantly decreased the viability and urease activity of HP in liquid culture assay. Conclusion The 2 strains of anti-HP Lactobacillus acidophilus L4 and L6 and their spent culture supernatant significantly decreased the viability and urease activity of HP in vitro.PARTⅢAnalysis of preventive and therapeutic effects of anti-HP Lactobacillus acidophilus in HP infection C57BL/6 miceObjective To investigated the prevention and treatment of Helicobacter pylori infection by anti-HP Lactobacillus acidophilus that have significantly inhibited HP in vitro in C57BL/6 murine model. Methods SPF C57BL/6 mice were inoculated with HP SS1 , in the prevented group, inoculated with anti-HP Lactobacillus acidophilus at 1 week before inoculation HP , the treated group inoculated with anti-HP Lactobacillus acidophilus were managed at 4 weeks after the last inoculated HP, MRS culture solution or Staphylococci were inoculated, respectively as positive and negative controls. HP -IgG- antibody, urease levels of gastric mucosa , HP culture, HP colonization and histopathology was assessed at different times. Results At 4, 8, 12 weeks after the last inoculated HP, the rate of HP infection in the prevented group was 0%, respectively. At 4, 8, 12 weeks after treated with anti-HP Lactobacillus acidophilus , the rate of HP infection in the treated group as same as control groups. However, a significant reduction in the levels of HP -IgG Antibody, urease and HP colonization in treated groups compared with control groups. This reduction was accompanied by a significant decline in the associated chronic gastric mucosal inflammation observed at each time point throughout the observation period. Conclusion Preinoculated with anti-HP Lactobacillus acidophilus can prevented HP infection in C57BL/6 mice. Treatment with anti-HP Lactobacillus acidophilus in HP infected C57BL/6 mouse, could significantly decrease chronic gastric mucosal inflammation, and reduce the levels of HP -IgG antibody, urease and HP colonization. The therapeutic effects could be significantly improved by prolong the time of therapy.PARTⅣScreening of anti-CagA+ HP specific binding peptides and analysis of anti-HP effect in vitro and in vivo Objective Specific HP Cag A antigen as target for screen the anti-Cag A+ HP specific binding peptides from 12 mer phage-display peptide library, and analysis of anti-HP effect in vitro and in vivo. Methods Using recombinate HP Cag A antigen as target, screened 12 mer phage-display peptide library, identified the positive clones by phage ELISA and competive ELISA, and investigated the inhibition of HP in vitro and in vivo. Results After 3 rounds of screening, 19 of 24 clones were identified as positive clones and all the 19 phage clones can bind with specific HP Cag A antigen. The anti-CagA+ HP specific binding peptides significantly decreased the viability and urease activity of HP in liquid culture assay. Preinoculation with highe level anti-CagA+ HP specific binding peptides could prevent HP infection in C57BL/6 mice. A significant reduction in the levels of HP -IgG antibody, urease, HP colonization and chronic gastric mucosal inflammation in HP infected C57BL/6 mice treated with anti-CagA+ HP specific binding peptides . Conclusion The anti-CagA+ HP specific binding peptides which screened with HP Cag A antigen as target can bind to HP Cag A antigen specific, and can inhibit CagA+ HP in vitro and in vivo.