Research On The Pharmacokinetic Property Of Patchouli Alcohol,and Its Effect And Mechanism On Anti-intracellular H.pylori

ObjectiveGC-MS was used to identify the pharmacokinetics property of patchouli alcohol(PA)polymer suspension in rat,which was prepared by using poloxamer 407.Then,the invasive ability of Helicobacter pylori(H.pylori)standard and clinical strains was compared,and the possible mechanism was explored.Besides,the experiment tries to illustrate the effect and mechanism of PA on anti-intracellular H.pylori.Also,the post antibiotic effect,the influence of PA on H.pylori drug resistance and intestinal flora was investigated.Method? The pharmacokinetics study of PA:GC-MS method was established to analyze the content of PA in rat serum,and the detection lower limit,extract recovery,precision,accuracy,and specificity of the method were explored.SD rats were orally gavaged with PA polymer suspension prepared by poloxamer 407,and the blood collections were carried out in different time point.The content of PA in serum was detected.Finally,drawing drug-time curve,and calculating pharmacokinetic parameters based on the result.?The study on effects and mechanisms of H.pylori invasion:Gentamycin protection and fluorescence assay were adopted in order to compare the invasive abilities difference between H.pylori standard strains(NCTC 11637,NCTC26695,SS1 and ICDC111001)and clinical strains(Hp1868,Hp1869,Hp1870,Hp1872).Cytochalasin B,2-apb,SKF-96365,Nifedipine and GdC13 were utilized to investigate the possible mechanism of H.pylori entry cell.The expressions of cytoskeleton movement-related protein(ROCK1,ROCK2,MYPT1,P-MYPT1,MLC and P-MLC)were analyzed by using western blot,after the GES-1 and MKN45 cell treatment with H.pylori.?The study of PA anti-intracellular H.pylori:The effects of PA on anti-intracellular H.pylori were investigated in GES-1 and MKN45 cell via gentamycin protection and fluorescence assay,also,the activities of LDH in cell and culture media were detected.Influence of PA on MKN45 cell lamellipodia and filopodia were observed,besides,the possible restored on cell transepithelial resistance alteration induced by H.pylori were explored.Mouse gastric epithelial cell was isolated,and the intracellular calcium was stained by Fura-2.Then,the effects of PA on the increment of intracellular calcium caused by H.pylori were investigated.Vina software predicted the possible binding between PA and calmodulin,and the surface plasmon resonance method was used to verify the result.Also,the effects of PA on cytoskeleton movement-related protein expression were analyzed.GES-1 was transfection by mRFP-LC3-GFP adenovirus,and further used to investigate the effect of PA on autophagy induced by H.pylori.In in vivo experiment,the prevent infection effects of PA and triple therapy were determined by pretreatment with those two therapeutic methods for 2 weeks,and then infected using H.pylori SS1 strains.?The study on the properties of PA anti-H.pylori:Bacterial counting and multi-step resistance induction method was adopted to analyze the post antibiotic effect and bacterial resistance degree of PA,metronidazole and clarithromycin.The next generation sequencing technology was used to compare the difference in microbiota,after treatment with PA and triple therapy.Result?The results of pharmacokinetics:Establishing the method to analyze the content of PA in serum by using cypress as the internal reference.The Cmax was 162.257±47.615 mg/l,Tmax was 0.133±0.038 h,T1/2 was 0.864±0.156 h,AUC0-t was 50.237±11.305 mg/1·h,AUCO??was 60.001±11.418 mg/1,h and MRTo?t was 60.001 ±11.418 h.?Effects and mechanisms of H.pylori invasion:The results of gentamycin protection and fluorescence assay revealed that the number of intracellular H.pylori were increased among 0 to 12 h.Besides,the invasive abilities of Hpl868,Hpl869 and Hp1870 were stronger than H.pylori standard strains NCTC11637,NCTC26695,SS1 and ICDC111001.The 2-apb,Nifedipine,GdCl3,Y27632 and M7 could inhibit the entry of NCTC11637,while the 2-apb,SKF-96365,Y27632 and M7 showed inhibition on Hp 1869 invasion.Besides,the cytochalasin B,2-apb,SKF-96365,Nifedipine and GdCl3 all could block the entry of Hp1870.H.pylori cocultured with GES-1 resulted in the high-expression of ROCK1,ROCK2,P-MYPT1 and P-MLC,and cocultured with MKN45 caused high-expression of ROCK2,P-MYPT1 and P-MLC.? Effects and mechanisms of PA on anti-intracellular H.pylori:Pre-treatment with PA decreased the number of intracellular H.pylori including NCTC11637 and Hp1869 strains in GES-1,and the H.pylori strain NCTC11637 in MKN45 cell.Also,the releasement of LDH was also reduced.PA inhibited the formation of MKN45 cell lamellipodia and filopodia,making the cell morphology change to roundness,meanwhile,the decrement of cell transepithelial resistance induced by H.pylori was restored by PA.The intracellular calcium concentration was increased with the effect of H.pylori,while PA block the progress.Vina predicted the docking affinity between PA and calmodulin was-6 to-7,besides,the surface plasmon resonance result indicated that the binding KD value is 7.444×10-6 between PA and calmodulin.PA inhibited the overexpression of ROCK2,P-MYPT1 and P-MLC in GES-1 cell caused by H.pylori.6.3×107 virus titer mRFP-LC3-GFP adenovirus infected GES-1 cell high-efficiently.H.pylori could cause the autophagy flux of GES-1,although most of the vesicle were autophagosome.Pre-treatment with PA followed by treatment with H.pylori also induced autophagy flux,however,most of the vesicle were autolysosome in this case.The mice pretreated with PA for 2 weeks would decrease the probability of H.pylori infection,indicating that PA showed the ability to prevent infection.? The post-antibiotic effect,drug-resistance,and influence on microbiota of PA:PA,metronidazole and clarithromycin showed reliable post antibiotic effect.Among them,the post antibiotic effects of 2-time MIC metronidazole,2-time MIC clarithromycin and 1.5-time MIC PA were 6.3,12.4 and 7.5 h,respectively.Treatment with 2-time MIC PA for 2 h,resulting in the loss of survival ability of H.pylori.The multi-step resistance induction method revealed that H.pylori became metronidazole-resistance easily,while it couldn't produce resistance to PA in 9 passages.The results of next generation sequencing technology indicated that the species and numbers of microbiota were markable decreased after treatment with triple therapy,while there wasn't any change with PA treatment.ConclusionPA showed high-speed drug absorption and metabolism ability,and the T1/2 was 0.864 h.The H.pylori clinical strains showed stronger invasive capacity.Also the increment of Ca2+concentration in cytoplasm and cytoskeleton rearrangement are the key factors in H.pylori invasion.PA inhibited the hyperactivity of cell membrane induced by H.pylori,resulting in anti-H.pylori invasion,also,PA increased the digestion capacity of GES-1 cell to H.pylori,presented with prevent H.pylori infection property.The further study on anti-H.pylori characteristic of PA indicated that PA showed strong post antibiotic effects,less drug-resistance for H.pylori,and less influence on microbiota.