Protective Effect Of Pogostone Against Experimental Gastric Ulcer And Its Pharmacokinetic Studies

ObjectivesPeptic ulcer occurs due to the imbalance between invasion factors(H. pylori infection, gastric acid and pepsin action, the application of non-steroidal anti-inflammatory drugs, genetic factors, gastric and duodenal motility disorders, stress and mental factors) and protective factors (gastric mucus-bicarbonate barrier, mucosal barrier, mucosal blood flow, cell renewal, prostaglandins and epidermal growth factor gastric mucus). Peptic ulcer is one of the major gastrointestinal disorders with increasing incidence and prevalence globally. It is estimated that 10% people worldwide have been affected by gastric ulcers, and 1% of the ulcer patients can be converted into the gastric cancer. Due to its great harm to human health, the disease was defined as a precancerous condition by the WHO. Hence, it is urgent and important to investigate and develop safe and efficient anti-ulcer drugs. On the other hand, the pharmacokinetic study can contribute to clarify and reveal the underlying mechanism of Chinese herbal medicine and its scientific connotation. The study of dynamic process and herbal disposal research in vivo can help to elucidate the mechanism of traditional Chinese medicine and provide scientific foundation for its theory and application.Pogostemonis Herba, which is commonly used in the clinical treatment of digestive diseases, has aromatic Huazhuo, vomiting and stomach effectiveness. Its therapeutic functions in Chinese medicine are to remove dampness, relieve summerheat and exterior syndrome, stop vomiting and stimulate appetite, which is similar to the symptoms of peptic ulcer in the modern medicine. Our research team recently has demonstrated pogostone, the main component of Pogostemonis Herba, exerted potent antibacterial, anti-fungal, anti-Candida activities and protection against endotoxic shock.Our research was to evaluate the protective effect of pogostone against ethanol and indomethacin-induced experimental gatric ulcer model in rats and explain its possible mechanism of action. The method of determining pogostone in rats plasma and tissue distribution by LC-MS/MS and UPLC were established and successfully applied to the investigation of preclinical pharmacokinetics and tissue distribution of pogostone in rats. And the pharmacokinetic parameters were calculated by DAS 3.1.5 software, and the absorption and distribution of the drug in rats was analyzed to clarify the absorption and transport characteristics of pogostone in rats.Methods1. Protective effect of pogostone against ethanol-induced gastric ulcer in ratsEthanol-induced rat gastric ulcer model was used. After oral administration of pogsotone for 7 days, ulcer index and ulcer inhibition rate were calculated by the software and HE staining were performed to evaluate the protective effects of pogsotone against ethanol-induced gastric ulcer in rats. At the same time, serum inflammatory mediators (TNF-α, IL-6 and IL-10), gatric oxidative status(SOD, GSH, CAT and MDA) as well as PGE2 and NP-SH were measured, to explain the possible protectiive mechanisms of pogostone in ethanol-induced gastric ulcer.2. Protective effect of pogostone against indometacin-induced gastric ulcer in ratsAcute gastric ulcer model in rats was induced by non-steroidal anti-inflammatory drug indomethacin. After pre-treatment with pogostone for 7 days, determination of ulcer index, ulcer inhibition rate and morphological studies were used to evaluate the possible protective effect of pogostone in indomethacin-induced acute gastric ulcer model. In addition, in order to further understand the protectiive mechanism we determined the antioxidant enzymes SOD, GSH, CAT activity. MDA level, PGE2 and expression levels of COX-1 and COX-2. Meanwhile, the expression variation of Hsp70, Bax and the Bcl-2 proteins was also examined using immunohistochemical stainning.3. Determination of pogostone in rat plasma and its application in pharmacokinetic studies by LC-MS/MSChromatographic analysis was performed on Thermo Fisher Scientific TSQ Quantum Access LC system (USA Thermo Fisher Scientific Company). Separation of the analytes was carried out on a YMC-UltraHT Pro C18 column (50 × 2.0 mm i.d.,2 mm) with a flow rate of 400 mL/min at 30 ℃ for 5 min. The mobile phase was a mixture (75:25, v/v) of methanol and water. The ionization was conducted using an electrospray ionization (ESI) interface in the negative mode. Detection of the ions was performed using in the selected reaction monitoring mode, monitoring the transition of the m/z 223.0 â†' 139.0 for pogostone and m/z 253.1 â†' 224.9 for IS. The common parameters for MS detection were as follows:sheath gas, auxillary gas and collision gas were set at 45,10 and 15 psi, respectively. Other parameters, viz., spray voltage, collision energy and capillary temperature for pogotone and IS, were 2500 V,20 V and 350 ℃, and 2500 V,31 V and 350 ℃, respectively.4. Determination and distribution study of pogostone in rat tissues by UPLCRats were randomly divided into two groups (intravenous administration group and oral administration group) and given a single dose of 10 mg/kg pogostone by intravenous administration and oral administration, respectively. After intravenous injection, the rats were sacrificed at 15,60 and 360 min. While rats, after oral administration, were euthanasized at 30,90 and 360 min, respectively. For the analysis of the preparation, optimal chromatographic conditions were determined using Acquity UPLC BEH C18 column with acetonitrile-water containing 0.1% formic acid (55:45, v/v) as the mobile phase, at a flow rate of 400 μL/min. UV detection wavelength was set at 310 nm with temperature maintained at 30 ℃ and the injection volume was set at 5 μL.Results1. Protective effects of pogostone against ethanol-induced gastric ulcer in ratsAdministration of three different doses of pogostone prior to ethanol ingestion effectively protected the stomach from ulceration. The gastric lesions and ulcer index were significantly ameliorated compared to the vehicle group and pathologic morphology was also improved. Pre-treatment with pogostone elevated the decreased IL-10 level, while markedly mitigated the increased IL-6 and TNF-a levels; and SOD, CAT and GSH levels in gastric tissue were increased, while MDA content was decreased, meanwhile NP-SH and PGE2 content were also significantly improved. Taken together, these results strongly indicate that pogostone could exert a gastro-protective effect against gastric ulceration, and the underlying mechanism might be associatedwith the stimulation of PGE2, improvement of antioxidant and anti-inflammatory status, as well as preservation of NP-SH.2. Protective effects of pogostone against indometacin-induced gastric ulcer in ratsOral administration of pogostone (10,20 and 40 mg/kg) inhibited the gastric mucosal lesion, decreased the ulcer area and index, as well as improved pathologic morphology. Pogostone pretreatment significantly raised the depressed activities of SOD, GSH and CAT, while reduced the elevated MDA level compared with indomethacin-induced group. Pogostone-pretreated group induced a significant increase in the gastric mucosal PGE2 level and obvious up-regulation of mRNA expressions of COX-1 and COX-2. Furthermore, immunohistochemical staining for pogsotone showed up-expression of Hsp70 and Bcl-2 protein, and suppression of Bax protein expression in the ulcerated tissues. To sum up, pogostone was demonstrated to exert gastroprotective effect against indomethacin-induced gastric injury, and the mechanisms of this protective effect were potentially associated with stimulation of COX-mediated PGE2, enhancement of the cellular antioxidant mechanism by replenishing gastric SOD, CAT and GSH levels along with reducing lipid peroxidation, depression of Bax protein as well as activation of Bcl-2 and Hsp70.3. Determination of pogostone in rat plasma and its application in pharmacokinetic studies by LC-MS/MSThe calibration curve was linear over the concentration range 0.05-160 mg/mL (r 0.9996). The intra- and inter-day accuracy and precision were within 10%. The validated method was successfully applied to the preclinical pharmacokinetic investigation of pogostone in rats after intravenous (5,10 and 20 mg/kg) and oral administration (5,10 and 20 mg/kg). Finally, the oral absolute bioavailability of pogostone in rats was calculated to be 70.39,78.18 and 83.99% for 5,10 and 20mg/kg, respectively.4. Determination and distribution study of pogostone in rat tissues by UPLCExcellent linear relationship of calibration curve (r>0.9984) was achieved over the range of 0.1～40 μg/mL for all the tissue samples. The limit of quantification (LOQ) and the limit of detection (LOD) were found to be 0.1 and 0.05 μg/mL, respectively. This method was proved with great precision, accuracy, stability, extraction recovery and matrix effect to for tissue distribution study of pogostone in rats.ConclusionThe current study showed pogostone exerted gastric-protective effect against experimental gastric ulcer induced by ethanol and indometacin. Administration of pogostone could decrease the gastric lesions and ulcer index, ameliorate pathologic morphology, raise the depressed activities of SOD, GSH, and CAT as well as PGE2, while reduced the elevated MDA level. For the experimental ulcer induced by ethanol, pogostone could also improve the inflammatory reaction, reduce TNF-a and IL-6 concentration in serum, increase IL-10 level and NP-SH levels in gastric tissue. For the indomethacin-induced gastric ulcer model, pogostone group was able to up regulate mRNA expressions of COX-1 and COX-2, increase Hsp70, Bcl-2 protein expressions and reduce the expression of Bax in gastric tissue.Moreover, the LC-MS/MS method and UPLC method were developed for studying the pharmacokinetics and tissue distribution of pogostone in rats. The methods, which were specific with proper matrix effects, and have good linearity, sensitivity, accuracy and stability, were successfully applied to pharmacokinetic and tissue distribution study.