Selection Of Probiotics Against Helicobacter Pylori And The Study Of Antagonistic Mechanisms

Objective:1.To comparethe adhesive ability and toxic effects on GES-1 cells of Lactobacillus acidophilus,Lactobacillus salivarius,Clostridium butyricum,Bacillus licheniformis,Bifidobacterium infantis,Bifidobacterium longum and Lactobacillus bulgaricus.2.To determine the capacity of live probiotics and their cell-free supernatants to inhibit Helicobactcr pylori growth.3.To evaluate the effect of probiotics on the secretion of IL-8 in GES-1 cells induced by HP-LPS.4.To investigate the effect of probiotics on TLR₄-NF-?B signal transduction pathway.Methods:1.Gram staining and trypan-blue staining were used to detect the adhesive ability and the toxic effect on GES-1 cells of the probiotics.2.Measuring the PH values of the cell-free supernatantsof the probiotics and adjust the PH value to3,4,5,6.We performed agar plate diffusion assays to determine the capacity of live probiotics and their cell-free supernatants to inhibit Helicobactcr pylori growth.A modified phenol red method was used to evaluate the inhibition of Helicobactcr pyloriadhesion to GES-1 cells.3.The expression of TLR₄ and the transfer rate of NF-?Bp65 from the cytoplasm to the nucleus of GES-1 cells induced by HP-LPS were detected by immunohistochemical method.4.GES-1 cells,pre-treated in the presence or absence of live probiotics for 2 h,were stimulated with HP-LPS.TLR₄,NF-?B p65?the whole cell and the nuleus?,p-I?B?and I?B?were then measured by western blots.The amount of interleukin-8?IL-8?in the cell culture medium was measured by ELISA and IL-8 mRNA was measured by reverse transcription polymerase chain reaction.Results:1.Lactobacillus acidophilus,Lactobacillus salivarius,Bifidobacteriuminfantis,Bifidobacterium longum,Lactobacillus bulgaricus can adhere to GES-1 cells.The adhesion index number was in accordance withcell adhesion rateof the probiotics.The adhesion index number:Lactobacillus acidophilus>Lactobacillus bulgaricus>Bifidobacterium infantis>Bifidobacteriumlongum>Lactobacillus Salivarius>Bacillus licheniformis>Clostridium butyricum.2.The probiotics inhibit the viability of GES-1cellin a time-dependent and dose-dependent manner.When MOI =1:100,there was no toxic effect to GES-1 cells within 4h.When MOI=1:1000,there was no toxic effect to GES-1 cells in 2h,GES-1 cells began to die after2h.3.Live cells andcell-free supernatants with PH value<2of Lactobacillus acidophilus, Lactobacillus salivarius,Bifidobacterium infantis,Bifidobacterium longum,Bacillus licheniformis,Lactobacillus bulgaricus and Clostridium butyricum showed anti-H.pylori activity.However,only the cell-free supernatantsof Bacillus licheniformis displayed anti-H.pylori activity.The active substance of the cell-free supernatants against Helicobacter pylori is mainly organic acid except Bacillus licheniformis. 4.Pretreatment of Lactobacillus acidophilus,Lactobacillus salivarius,Bifidobacterium infantis,Bifidobacterium longum and Lactobacillus bulgaricus for 2h,the adhesive ability of Helicobacter pylori on GES-1 cells was inhibited,Lactobacillus acidophilus had the maxium inhibitory ability?P<0.05?.However,the post-treament did not have the same effect.5.Pretreatment with live Lactobacillusbulgaricus cells attenuated the expression of TLR4,p-I?B?,increased cytoplasmic I?B?,prevented the activation of NF-?B,and consequently blocked IL-8 production.Conclusion:Lactobacillus acidophilus and Lactobacillus bulgaricus are effective in reducing a Helicobacter pylori load and Lactobacillusbulgaricus can reduce gastric inflammation through a TLR₄-I?B?/NF-?B pathway.Food containing Lactobacillus acidophilus and Lactobacillus bulgaricus may be considered as adjuvant therapy for gastric diseases caused by Helicobacter pylori and be a promising preventive food against Helicobacter pylori infection.