| ObjectivesHelicobacter pylori is one of the most common infectious pathogens worldwide, infecting up to50%of the world’s human population. H. pylori infection. H. pylori is a major pathogen of chronic gastritis and peptic ulcer, and plays an mportant role in the pathogenesis of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. The world health organization had classified H. pylori as Type I carcinogen. To date, the most effective treatment against H. pylori infection is antibiotics-based triply therapy. However, emerging resistance to antibiotics and adverse effects such as gastrointestinal dysbacteriosis have been on the rise. Hence, there is a critical need to search for alternative medicine with high safety, efficacy and selectivity to treat H.py/orz-induced gastric diseases.In our previous investigation on Lingnan genuine medicinal herb Pogostemon cablin (Blanco) Benth., its principal ingredient--patchouli alcohol (PA) was demonstrated to effectively inhibit inflammatory response in gastritic mice infected with H. pylori, and reduce H. pylori colonization. PA was further proved to exhibit highly selective antibacterial activity against H. pylori and its specific metabolic urease, without affecting intestinal bacterial flora. Urease is the remarkable feature distinguishing H. pylori from other gastrointestinal microorganisms. Urease is essential for gastric H. pylori colonization, and plays a central role in the pathogenesis of H. pylori infection by protecting the bacteria from the acid environment of the stomach.Our research was to explore the possible mechanism underlying inhibitory action of PA on H. pylori urease by virtue of enzymology regime, H. pylori urease-induced gastric mucosa epithelial cell (GES-1) model and acute gastric ulcer rat model. The present scheme should contribute to further insight into the possible mechanism underlying PA in treating H. pylori-related gastritis.Methods1. Inhibitory effect of PA on H. pylori urease and Jack bean ureaseBased on H. pylori urease and Jack bean urease, urease inhibition assay, inactivation kinetics, progress curves analysis, inhibition site analysis and molecular docking were carried out to explore the possible mechanism underlying the specific inactivation against H. pylori and its urease.2. Effect of PA in H. pylori urease-induced GES-1modelThe gastric epithelium cells (GES-1) model infected by H. pylori urease was established, mimicking symptoms as observed in H. pylori infection. Inflammatory mediators, oxidation stress and cell apoptosis was investigated to probe into the mechanism of PA in treating H. pylori-related gastritis.3. Protective effect of PA on acute gastric ulcer in rat modelFour distinct acute gastric ulcer in rat model were induced by virtue of absolute ethyl alcohol application, indometacin administration, pyloric ligation and water-immersion stress regime, respectively. Ulcer area and index, related inflammatory mediators, oxidative level, gastric mucosia blood flow and gastric mucous were analyzed to unveil the mechanism of gastroprotective potential of PA.Results1.Inhibitory effect of PA on H. pylori urease and Jack bean urease(1) Urease inhibition assaysThe obtained IC50of PA against H. pylori urease and Jack bean urease was2.67±0.798mM and2.993±0.411mM, respectively. While IC50for acetohydroxamic acid (AHA) was0.585±0.242mM and0.379±0.256mM, respectively. The inhibition efficacy of PA was inferior to the positive control AHA, however, was still within the millimole range.(2) Kinetics of urease inactivation by PA In the absence of PA, the Michaelis constant KM and the maximum velocity vmax obtained by applying nonlinear regression to the Michaelis-Menten equation were: KM=1.47±0.12mM and vmax=0.85±0.11mM/min for H. pylori urease; KM=11.12±0.12mM and vmax=0.50±0.11mM/min for Jack bean urease, respectively. Lineweaver-Burk double-reciprocal plot for PA against H. pylori urease suggested that Km value did not significantly change in the presence of PA. While vmax decreased as PA concentration increased, indicating that PA might be a non-competitive mechanism of inhibition. While the inhibition type for PA against Jack bean urease was uncompetitive inhibition.(3) Progress curves analysisThe reaction progress curves showed that the vo, vs and kapp value were1.6×10-1mM/min,1.6×10-2mM/min and1.1min-1respectively. PA inhibition against urease was concentration-dependent.(4) Inhibitory site of ureaseThiols and inorganic compounds against PA inactivation indicated that the acting site was probably thiol, with possible involvement of Ni in the active center. The inhibition of PA was proved to be irreversible since urease could not be reactivated by dithiothreitol (DTT).(5) Molecular dockingPA influcenced the active site of H. pylori urease, chelated Ni2+at the bottom of the cavity with hydroxy and made contact with key amino acid residuevia hydrogen bond, altered urease active conformation. PA exhibited higher affinity towards H. pylori urease than Jack bean urease, which was corroborated with aforementioned studies.2. Effect of PA in H. pylori urease-induced GES-1modelMTT and LDH results indicated that PA (5,10,20μM) arrested GES-1cell viability loss caused by H. pylori urease. PA pretreatment diminished the elevated production of tumor necrosis factor-a (TNF-a), interleukin2(IL-2), interleukin6(IL-6), and nitric oxide (NO), increased IL-13level, thus attenuating inflammation injury in H. pylori urease-stimulated GES-1cells. AnnexinV-FITC/PI FCM results suggested that PA was capable of arresting GES-1cell apoptosis, which was in concert with the Hoechst result, in which diminishing karyopyknosis, fragmentation and apoptotic body was observed. PA pretreatment increased the reduced mitochondrial membrane potential (MMP) and attenuated reactive oxygen species (ROS). Furthermore, PA exposure elevated endogenous SOD and CAT activity, and decreased the constituent MDA. Taken together, the capacity of inhibiting inflammatory reaction, reducing oxidative damage and arresting apoptosis might contribute to the protective effect of PA on H. pylori urease-induced GES-1cells.3. Protective effect of PA on acute gastric ulcer in rat modelOral administration of PA (10,20and40mg/kg) inhibited the gastric mucosal lesion induced by absolute ethyl alcohol and indometacin, decreased ulcer area and index, improved inflammatory pathologic morphology. In terms of inflammatory mediators, IL-10production rallied whereas serum levels of TNF-a and IL-6experienced varying mitigation after PA treatment. Prereatment with PA increased NP-SH and PGE2content in stomach tissue of gastric-ulcered rats, upregulated the mRNA expression of COX-1and COX-2in indometacin-induced gastritic ulcer tissue. Gastric mucosia blood flows (GMBF) and gastric mucosal lesion were improved in water-immersion stress-induced gastric ulcer rat subjected to PA administration, in contrast with model group. In addition, gastric mucous content was elevated in pyloric ligation-induced ulcer rat. To sum up, the protective effect of PA on acute gastric ulcer in rat model might be involved with the capacity to improve inflammation response, regulate oxidative balance, promoted PGE2level, gastric mucous content and GMBF, thus enhancing gastric mucosa defense function.Conclusion1. There is variation between inhibitory action of PA on bacteria-originated and plant-derived urease. Higher affinity towards H. pylori urease was observed, which was possibly associated with the selective anti-H. pylori activity. Inactivation was possibly at the active site and proximal amino acid residue.2. The gastric epithelium cells (GES-1) model infected with H. pylori urease was established and the protective effect of PA on GES-1was verified. The mechanism might involve the effect of inhibiting inflammatory reaction, reducing oxidative damage and arresting apoptosis.3. The protective effect of PA on acute gastric ulcer in rat model was demonstrated. The possible mechanism might involve the capacity of PA to improve inflammation response, regulate oxidative balance, elevate PGE2level, gastric mucous content and GMBF, thus enhancing gastric mucosa defense function. The gastroprotective potential of PA in vivo and in vitro shared the same mechanism--inflammation inhibition and oxidative stress regulation.Taken together, the mechanism underlying the effect of PA in treating H. pylori-related gastritis might involve:(1) PA inhibited H. pylori urease and colonization and subsequent gastric mucosal injury;(2) PA improved inflammation response and attenuated oxidative damage, and enhanced gastric mucosa defense function. The present scheme should contribute to an improved understanding of therapeutic basis and mechanism of Pogostemon cablin (Blanco) Benth. in treating gastrointestinal (GI) disorders, and paved the way for novel urease inhibitor and H. pylori-related gastritis medication research with Chinese medicine-originated active lead compound. |
PDF Full Text Request