| Helicobacter pylori is a gram-negative bacterial species that selectively colonizes gastric epithelium and is the common bacterial infection worldwide.The signature feature is the capacity to persist for decades.Increasing evidence indicates that H.pylori is able to send and receive signals from cellular components within the gastric mucosa,allowing host and bacteria to participate in a dynamic equilibrium Sustained interactions between H.pylori and humans significantly increase the risk for atrophic gastritis,intestinal metaplasia,and distal gastric adenocarcinoma.Pogostemonis Herba is the dried aerial part of Pogostemon cablin(Blanco)Benth.(Labiatae).It has been traditionally used in Chinese medicine to resolve dampness,whet appetite,arrest vomiting,release the exterior and expel summer-dampness.Pogostemonis Herba is a common Chinese herbal medicine frequently used to treat for common cold,nausea,diarrhea,headaches and fever.Patchouli alcoholare the major chemical constituents of the essential oil of Pogostemonis Herba.The present study was undertaken to investigate effect of patchouli alcohol on treatment effect in mouse model of H.pylori-related gastritis and its mechanism.ObjectivePatchouli alcohol effect on mechanism of anti-H.pylori acid resistance.Establishing cell model of H.pylori infection and examining protection and anti-inflammatory effects and its mechanism of patchouli alcohol in vitro experiment.Establishing mouse model of H.pylori-related chronic gastritis and examining therapeutic effect and its mechanism of patchouli alcohol in vivo experiment.Method1.Mechanism of anti-H.pylori acid resistance and virulence factors of patchouli alcohol1.1 Patchouli alcohol effect on H.pylori survival ratesIdentification of H.pylori by gram staining,Rapid urease test(RUT)and PCR method.Agar dilution method was used to detect the bacterial survival in the acidic(pH=5.3)and neutral(pH=7.0)conditions with different concentrations of patchouli alcohol..1.2 Mechanism of anti-H.pylori acid resistance of patchouli alcoholPH shock method was used to detect the bacterial survival rates in the acidic(pH=1.0)and neutral(pH=7.0)conditions;Berthelot method to detect H.pylori urease activity and RT-qPCR method to detect ureA,ureB,ureE,ureH,urel,nixA in the acidic(pH=5.3)and neutral(pH=7.0)conditions with different concentrations of patchouli alcohol.;Expression and purification of urease coenzyme protein UreG;Simulation of UreG synthesis and decomposition process in vitro,Establishing dose-response curve of patchouli alcohol.1.3 Patchouli alcohol effect on H.pylori virulence factors geneRT-qPCR method to detect cagF,oipA,cagE in H.pylori.2.Mechanism of effect in cell inflammation model of H.pylori infection2.1 Establishment and evaluation of cell inflammation model of H.pylori infectionThe cytotoxicity of MKN45 cell was determined by percentage of lactate dehydrogenase(LDH)leakage and assessing viable cell counts using non-stained trypan blue;The mitochondrial membrane potential(MMP)was analyzed by rhodamine 123 stain photofluorography and flow cytometry.H.pylori was co-cultured with RAW264.7 macrophages at a ratio of MOI=100.Content of nitric oxide(NO)and level of pro-inflammatory genes expression in the cell were detected after 6,12 and 24 h.2.2 Patchouli alcohol effect on mechanism of protection effects in MKN45 cell of H.pylori infectionEffect of patchouli alcohol on cell viability of MKN45 cells by CCK-8 reagent;Effect of patchouli alcohol on survival rate of infected MKN45 cell determined by LDH leakage and assessing viable cell counts using non-stained trypan blue;Patchouli alcohol effect on H.pylori induced ROS experssion in MKN45 cells by DCFH-DA method;Effects of patchouli alcohol on H.pylori induced loss of MMP in MKN45 cells by Rhodamine 123 method;Apoptosis rate of MKN45 was assessed by flow cytometry after staining with FITC-conjugated Annexin V and Propidium Iodide;Effects of patchouli alcohol on superoxide dismutase(SOD)and malondialdehyde(MDA)levels in MKN45 cells subjected to H.pylori by biochemical methods;Patchouli alcohol effect on H.pylori induced level of pro-inflammatory genes and bacteric virulence factors expression in MKN45 cells by RT-qPCR method;Patchouli alcohol effect on H.pylori induced level of pro-inflammatory protein secretion in MKN45 cell supernatant by ELISA method.2.3 Patchouli alcohol effect on mechanism of anti-inflammatory effects in RAW264.7 of H.pylori infectionEffect of patchouli alcohol on cell viability and nitric oxide level of RAW264.7 cells by CCK-8 reagent and Griess method;Effect of patchouli alcohol on phagocytosis of RAW264.7 cells by agar dilution method;Effects of patchouli alcohol on NO and myeloperoxidase(MPO)levels in RAW264.7 cells subjected to H.pylori by biochemical methods;Patchouli alcohol effect on H.pylori induced level of pro-inflammatory genes expression and protein secretion in RAW264.7 cell by RT-qPCR and ELISA method.3.Establishing mouse model of/H.pylori-related gastritis and examining therapeutic effect and its mechanism in vivo experiment3.1 Establishment and evaluation of mouse model of H.pylori-related gastritisC57BL/6 mice were oral gavage with H.pylori suspension and the blank control was administrated with BHI equally.Two week later,PCR and RUT experiments were adopt to validate the H.pylori infection status.Next,the infected mice were randomly divided into 3 groups including control,6-week and 12-week infected groups.After 6 weeks and 12 weeks'infection,the stomach was removed for pathological examination and pro-inflammatory genes expression detection.3.2 Patchouli alcohol effect on mechanism of treatment effect in H.pylori-related gastritisEstablishing mouse model of H.pylori-related gastritis and giving medicine for 2 weeks in 10 week.The stomach is divided into gastric antrum and the gastric body tissue for pathological examination;Effects of patchouli alcohol on gastric oxidative stress level in H.pylori infected mice by biochemical methods;Patchouli alcohol effect on level of pro-inflammatory genes expression and protein secretion in mice with H.pylori infected gastritis by RT-qPCR and Cytometry Beads Array method.Effects of patchouli alcohol on level of urease genes expression in mice with H.pylori infected gastritis by RT-qPCR.Result1.Mechanism of anti-H.pylori urease activity and virulence factors of patchouli alcoholSurvival rate of H.pylori has not significantly changed in neutral and acidic(pH=5.3)conditions,however survival rate has significant reduction in acidic(pH=K0)conditions.Further research in pharmacological mechanism of patchouli alcohol,Urease activity and ureAy ureB,ureE,ureH,urel,nixA have decreased expression in H.pylori under the acidic(pH=5.3)and neutral conditions with patchouli alcohol;However,Patchouli alcohol have no effect on UreG synthesis and decomposition process;cagE and oipA have obviously decreased with patchouli alcohol.2.Mechanism of protection and anti-inflammatory effects in cell inflammation model of H.pylori infection2.1 Establishment and evaluation of cell inflammation model of H.pylori infectionThe cytotoxicity was obviously increased with dissipation of MMP after 12 and 24 hour co-culture in infeceted MKN45 cell;The content of NO in supernatant and expressions of tnf-a,inos and cox-2 in cell were obviously increased after 6,12 and 24 h co-culture in infected RAW264.7 macrophage.In conclusion,Cellular inflammation is more serious in 24 hour post-infection.2.2 Patchouli alcohol effect on mechanism of protection effect in MKN45 cell of H.pylori infectionPatchouli alcohol have effect on promoting survival rate,inhibiting apoptosis,reducing pro-inflammatory genes and proteins,increasing the activity of antioxidant enzyme,restoring MMP and decreasing bacterial virulence factor genes in infected MKN45 cell.2.3 Patchouli alcohol effect on mechanism of anti-inflammatory effect in RAW264.7 of H.pylori infectionPatchouli alcohol have effect on inhibiting pro-inflammatory genes and proteins,reducing the level of NO,decreasing the activity of oxidase enzyme and promoting phagocytosis in infected RAW264.7 cell.3.Establishing mouse model of H.pylori-related gastritis and examining therapeutic effect and its mechanism in vivo experiment3.1 Establishment and evaluation of mouse model of H.pylori-related gastr:itisThe stomach mucosa layer was observed with monocytes infiltration.In 12-week infected group,the reduction of gastric inherent glands,the increment of fibrous,atrophy and intestinal metaplasia were all observed in the rat stomach.The gene expressions of Cox-2,Inos,Tnf-? and Il-1? in 6-week and 12-week infected group were significantly increased.In conclusion,12-week infected group has greater inflammation than 6-week infected group.3.2 Patchouli alcohol effect on mechanism of treatment effect in H.pylori-related gastritisPatchouli alcohol have effect on reducing pro-inflammatory genes and proteins,inhibiting urease genes expression,increasing the activity of antioxidant enzyme and decreasing the activity of oxidase enzyme in the stomach of infected mice.In pathological examination,patchouli alcohol have effect on removing H.pylori and reducing inflammation.ConclusionPatchouli alcohol have therapeutic effect in mouse model of H.pylori-related gastritis by removing H.pylori and anti-inflammatory effects.Its mechanism may be related to anti-H.pylori and inhibiting pro-inflammatory factors and RNS/ROS expression. |
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