Study On The Mechanisms Of Berberine Against Persisters Of Helicobacter Pylori

Helicobacter pylori(H.pylori)infection is the most important cause of gastrointestinal diseases.In addition to antibiotic resistance,a major reason for the failure of H.pylori eradication is the formation of persisters.In the face of the dual difficulties both in the variation of H.pylori resistance and in the phenotypic variation of persisters,it is of great significance to actively seek new anti-H.pylori drugs,especially for the treatment of its persisters.Berberine is an effective component extracted from Rhizoma Coptidis,with a broad spectrum antibacterial effect.It is of practical significance to study on the effects and the mechanisms of berberine against H.pylori persisters.Objects:To explore the effects and mechanisms of berberine against H.pylori J99 persisters.Methods:1.MIC(Minimal Inhibitory Concentration)and MBC(Minimal Bactericidal Concentration)of both amoxicillin and berberine on H.pylori were determined by liquid dilution method.2.The methods of drug exposure test,liquid culture and microscopic counting were applied to determined and compared the antibacterial effects of both berberine(10~200 ?g/ml)and amoxicillin(1~6 ?g/ml)on H.pylori cultured in logarithmic phase and stationary phase.And then the antibacterial properties of different drugs were analyzed.3.The total RNA of H.pylori cultured in stationary phase and then exposed to berberine(25 ?g/ml)for three hours were extracted by Trizol method.4.RNA-Seq(RNA-sequencing)was applied to obtain the differential genes.We applied GO functional enrichment analysis and KEGG pathway analysis to explore the possible molecular mechanisms and pathways that berberine against H.pylori persisters.Results:1.The range of MIC and MBC of berberine on H.pylori were 20.00~40.00 ?g/ml and 40.00~80.00 ?g/ml respectively,and that of amoxicillin on H.pylori were 0.05~0.10 ?g/ml and 0.10~0.20 ?g/ml,respectively.2.Berberine had stronger antibacterial effects on H.pylori in stationary phase than that in logarithmic phase.The effects displayed a concentration dependence within the concentration range tested that it could kill H.pylori in higher concentration and inhibit it in lower concentration.Amoxicillin had stronger antibacterial effects on H.pylori in logarithmic phase than that in stationary phase,and its antibacterial effects did not show dose-response relationship in the concentration range tested.3.RNA-Seq results showed there were 829 significantly difference(q<0.05)genes including 507 up-regulated genes and 322 down-regulated genes after berberine had effected on H.pylori for 3 hours.4.The results of GO functional enrichment analysis showed that the down-regulated genes were mainly enriched in respiration and energy metabolism,ribosome structure and function,material transport and intracellular homeostasis;and the up-regulated genes were mainly enriched in methylation modification of nucleic acid and repair of DNA damage.5.The results of KEGG pathway analysis showed that the down-regulated signal pathways mainly involved ribosome,oxidative phosphorylation,tricarboxylic acid cycle,two-component system,pyruvate metabolism and butanoate metabolism,and the up-regulated signal pathways mainly involved base excision repair,homologous recombination,folate biosynthesis,nucleotide excision repair,sulfur relay system,lipopolysaccharide biosynthesis,peptidoglycan biosynthesis.Conclusion:1.Berberine has the ability to kill persisters of H.pylori.2.The effects of berberine on H.pylori persisters by the possible ways include energy metabolism,biosynthesis,DNA damage repair and signal transduction.