Influence Of The Microflora In Stomach To H.pylori Field Planting And Efficacy Of CMS005 Treatment To H.pylori Infection

ObjectiveThe antagonistic action on H. pylori and tolerance to the environment simulating acid and bile of a Bacillus bifidus strain which named CMS005 was carried.MethodsA plate well diffusion assay was employed to examine the antagonistic activity of the Bacillus bifidus strain against Helicobacter pylori cultures. The pH value of MRS liquid medium was modulated to 2.0 to simulate gastric acid environment, bile was added to MRS liquid medium to simulate duodenal bile environment and MRS liquid medium with pH modulated to 6.8 and without bile salt was control. the Bacillus bifidus strain was quantificatd and was counted at the time of every 30min after inoculating respectively. Then the difference between experimental and control group was compared by statistics.Results1. The Bacillus bifidus strain CMS005 had obvious antagonistic activity against Helicobacter pylori in vitro. the minimum antagonistic activity concentration was 1×10~7CFU/ml, and there was no difference between CMS005 with concentration of 1×10~8CFU/ml and ampicillin with concentration of 0.25mg/ml (P＞0.05).2. The Bacillus bifidus strain CMS005 can grow well in MRS of pH 2.5 and above, but not in MRS of pH 2.0 and lower. There is no significant difference between the viable bacteria change of CMS005 in MRS containing bile and the change in MRS control(P＞0.05).ConclusionThe Bacillus bifidus strain CMS005 had obvious antagonistic activity against Helicobacter pylori in vitro,and was confirmed to be acid-tolerant and bile-tolerant. ObjectiveSPF BALB/c Mice with lessen bacteria in stomach was selected to establish a new Helicobacter pylori infection model. At first local flora in stomach was ruined by feeding antibiotics and balance of micro-ecosystem was interrupted which made it easy for Helicobacter pylori to colonizate. Then Helicobacter pylori was feeded to Mice to establish the model which needs less time and is more stable. Achievement ratio between this method and available method which was carried by feeding Helicobacter pylori dierectly were compared. Flora in Mice Stomach was compared before and after the proceed, and means of micro-ecosystem balance in the establishment was discussed to supply evidence for the hypothesis which Helicobacter pylori was conditional pathogenic bacteria.MethodsA total of 60 SPF grade BALB/c mice were randomly divided into 6 groups. The first and the second group of mice were pretreated with antibiotics and inoculated with H. pylori. The third and the fourth group were only inoculated with H. pylori, The fifth and the sixth group were inoculated with sodium chloride solution. The first, the third and the fifth groups were sacrificed 4 weeks postinoculation (p. i.), and the second, the fourth and the sixth groups were sacrificed 8 weeks postinoculation (p. i.). The infection of H. pylori in gastric mucosa of the mice was analyzed by rapid urease test, Giemsa staining and culture. The changes of microflora in stomach and duodenum were confirmed by Gram staining and anaerobic culture. The histopathological changes in the gastric mucosa of mice were assessed in hematoxylin-eosin stained sections.Results1. Assessment of H. pylori infection in miceH. pylori colonization rate was 100% in the stomach of the first and the second group, and the colonized bacterial number increased accompanied with time(8w vs 4w, P＜0.05). H. pylori colonization rate was 60% in the stomach of the third and 50% of the fourth group. There was no difference between the third and the fourth group in the quantitatation of colonized bacterial number. (P＞0.05). No bacteria were found in the two control groups inoculated with H. pylori(P＜0.05).2. Assessment of microflora in stomachThe bacterial numbers in stomach of the first and the second group of mice was 10~4～10~5CFU/g.The bacterial numbers in stomach of the third and the fourth group of mice was10~5～10~6CFU/g. The bacterial numbers in stomach of the fifth and the sixth group of mice was10~5～10~6CFU/g.The bacterial numbers in stomach of the first and the second group were significantly decreased comparise to the third and the fourth and the fifth and the sixth group(P＜0.05). There is no significant difference between the third and the fourth group and the fifth and the sixth group in bacterial numbers in stomach.3. Assessment of Histological examinationThe chronic inflammatory response in stomach of the first group was more serious than the second group, and they were all more serious than the third and the fourth group. There is no significant difference between the third and the fourth group in the chronic inflammatory response.ConclusionThe gastritis model in SPF grade BALB/c mice challenged with H.pylori SS1 wassuccessfully established in our study. The dysbactefia caused by antibiotics helped colonization of H. pylori and occurrence of gastritis. It suggested that pathogenicity and infection of H. pylori is relevant to disturbance of microflora and some commensal bacteria in the stomach could probably inhibit H. pylori. ObjectiveEvaluate the efficacy of CMSOO5 combine with PPI therapy to Helicobacter pylori-related gastritis in SPF grade BALB/c mice, and compare its efficacy to the efficacy of available trigeminy therapy. microflora in the stomach was analysised before and after the therapy to supply more evidence for the hypothesis which Helicobacter pylori was conditional pathogenic bacteria.MethodsA total of 50 SPF grade BALB/c mice were randomly divided into 5 groups. The first to the fourth group of mice were infected with Helicobacter pylori to establish the gastritis model and the fifth group was normal.The first group was treated with CMS005, The second group was treated with CMS005 and PPI, The third group was treated with available trigeminy therapy, and the fourth group of mice was treated with sodium chloride solution. the mice were sacrificed 2 weeks post-treatment. The infection of H. pylori in gastric mucosa of the mice was analyzed by rapid urease test, Giemsa staining and culture. The changes of microflora in stomach and duodenum were confirmed by Gram staining and anaerobic culture. The histopathological changes in the gastric mucosa of mice were assessed in hematoxylin-eosin stained sections.Results1. Eradication of Helicobacter pyloriThe eradication of Helicobacter pylori of The first and the second group was 60% and 70% respectively, the third group was 80%. There is no significant difference between the first and the second group and the third group in eradication of Helicobacter pylori.2. Assessment of microflora in stomachThe bacterial numbers in stomach of the first group of mice was 10~4～10~5CFU/g. The bacterial numbers in stomach of the second group of mice was 10~4～10~5CFU/g. The bacterial numbers in stomach of the third and the fourth group of mice was 10~5～10~6CFU/g. The bacterial numbers in stomach of the fifth group of mice was 10~5～10~6CFU/g.The bacterial numbers in stomach of the third and the fourth group were significantly decreased comparise to the fifth group(P＜0.05). The bacterial numbers in stomach of the first group and the second group were significantly increased comparise to the fifth group(P＜0.05). The 7bacterial numbers in stomach of the second group were significantly increased comparise to the first group(P＜0.05).3. Assessment of Histological examination The chronic inflammatory response in stomach of the fouth group was more serious than the first and second and third group(P＜0.05). There is no significant difference between the first and second and third group in the chronic inflammatory response (P＞0.05).ConclusionThe therapy of CMS005 combine with PPI to Helicobacter pylori-related gastritis in SPF grade BALB/c mice was effective. There is no significant difference between CMS005 combine with PPI and available trigeminy therapy.the therapy of CMS005 combine with PPI can reconstruction the microecological in stomach. ObjectiveComparise the change of TLR4,IL-1~β,TNF-a, IL-12 in gene and protein level of mice gastric mucosa before and after treatment of CMS005. analysis the correlation of TLR4 with IL-1β,TNF-a and IL-12 to approach the role of TLR4 in Helicobacter pylori pathogenic mechanism and CMS005 in Helicobacter pylori-related gastritis.MethodsRT-PCR, Western blotting and immunohistochemical method were used to detect the content of TLR4, IL-1β,TNF-a,IL-12 in gene and protein level of mice gastric mucosa.Results1. In gene and protein level, there was no TLR4 in the normal group, TLR4 in stomach of the first and the second and the third group were significantly decreased comparise to the fourth group(P＜0.05).2. In gene and protein level, IL-1βin stomach of the first and the second and the third group were significantly decreased comparise to the fourth group(P＜0.05), but were significantly increased comparise to the normal group(P＜0.05). 3. In gene and protein level,there was no IL-12 in the normal group, IL-12 in stomach of the first and the second and the third group were significantly decreased comparise to the fourth group(P＜0.05).4. In gene and protein level,there was no TNF-a in the normal group, TNF-a in stomach of the first and the second and the third group were significantly decreased comparise to the fourth group(P＜0.05).5. TLR4 was positive correlation to IL-1βand TNF-a,IL-12.ConclusionTLR4 participated in the pathogenic mechanism of Helicobacter pylori, and was positive correlation to downstream inflammatory factors such as IL-1βand TNF-a,IL-12. The treatment mechanism of CMS005 to Helicobacter pylori-related gastritis was concerned with TLR4.