Anti-Helicobacter Pylori Effect And Mechanism Of Patchouli Alcohol

ObjectiveMinimal inhibitory concentration(MIC),bactericidal,anti-motile,anti-adhesive,morphological broken and gene expression restrain capacity of patchouli alcohol(PA)against Helicobacter pylori(H.pylori)were all examined in vitro.In vivo experiment,C57BL/6 mice H.pylori SS1 infected model was established and the therapeutic effect was defined.MethodH.pylori was cultured and authenticated by using gram stain,rapid urease test(RUT)and 16s DNA sequencing method.Agar dilution method was recruited to define the MIC of PA and bactericidal ability was also evaluated at the series concentration from 2 to 5 times MIC value.Besides,the possible influence of H.pylori motile ability was observed by means of swarm agar assay.In the coculture experiment,FITC-labeled bacteria was employed to explore the anti-adhesion effect of PA in GES-1 cell line.For the gene expression,Q-PCR assay was adopted to test the effect on AlpA,AlpB,BabA,FlaA and FlaB gene expression.Also,the flagella formation and ultrastructural change of H.pylori was studied based on scanning electron microscopy and transmission electron microscopy,respectively.In vivo experiment,C57BL/6 mice were infected with H.pylori SS1 to conduct the therapeutic effect of PA.Briefly,second passage H.pylori SS1 strain was adjusted to Mc=1 and inoculated in brain-heart infusion(BHI)supply with 10%fetal calf serum at radio of 1:10.Bacteria grow to early-to-mid logarithmic phase with 120 rpm shake for 16-18 h.Mice were fasted for 12 h and then administrated orally with 0.25ml H.pylori suspension by using gauge needle in three times at 2 days intervals over 5 days.The blank control was administrated with BHI equally.Two week later,PCR,RUT and bacteria culture experiments were adopt to validate the H.pylori infection status.Next,the infected mice were randomly divided into 10 groups including control,model,triple therapy(omeprazole+ metronidazole +clarithromycin),PA 1.25,2.5,5,10,20,40,80 and 160 mg/kg groups.With 14 days orally administration of PA,mice were sacrificed and stomachs were removed.RUT and boric acid methylene blue technology were employed to verify the effect.ResultIn pH=5.3,7 and 9,MIC values of PA against H.pylori standard strains NCTC11637 were 25,25 and 50 ?g/ml,against NCTC26695 were 50,25 and 25 ?g/ml,and against SS1 were 25,25 and 75 ?g/ml;MIC values of PA against H.pylori clinical strains Hp1869,Hp1870,Hp1874 and Hp1875 were 50 ?g/ml;MIC values of PA against H.pylori clinical strains Hp1873,Hp1876 and AH-258 were 25 ?g/ml;MIC values of PA against H.pylori clinical strains Hp1872 and Hp1877 were 20 ?g/ml;MIC values of PA against H.pylori clinical strains Hp1868 and Hp1871 were 12.5 ?g/ml;MIC value of PA against H.pylori standard strain SS1 was 25 ?g/ml under the pH=5.3.And the bactericidal experiment reviewed that 2 or more times MIC value of PA was all presented a strong killing ability in neutral or weak acid condition.Besides those,5 times MIC of PA could kill all the bacteria in just 15 min in neutral environment.Meanwhile,anti-H.pylori motile and adhesive ability were both showed under the concentration of 5,10 and 20 ?g/ml of PA.According to the result of transmission electric microscopy,more coccoid bacteria and ultrastructure damage,including plasmolysis,vesicle formation and necrosis,were observed under the effect of PA at 25 and 50 ?g/ml in 1 or 2 hours.In addition,PA could restrain the gene expression of adhesion relative gene(AlpA and AlpB)and motility relative gene(FlaA and FlaB)at 5,10,20 ?g/ml in 1 hour.The result of rapid urease test,PCR and bacterial culture revealed that the H.pylori infected model was established successfully by administrating orally with early-to-mid logarithmic phase H.pylori in three times at 2 days intervals over 5 days.Besides,PA(5mg/kg)could reduce efficaciously infected rate of H.pylori based on the result of mice challenge study.ConclusionThe strong bacteriostat and bactericidal capacity of PA are exemplifiled.Also,anti-motile,anti-adhesive and ultrastruture damage ability are all observed.The PA anti-motile ability mechanism is related to flagella structure gene(FlaA and FlaB)expression restraint.And the anti-adhesive mechanism may be associated with down-regulation of adhesive gene(AlpA and AlpB)expression.Meanwhile,bacterial clearing ability of PA is presented in vivo and the optimal dose is 5 mg/kg.