In several frequently used models, such as ethanol, pylorus ligation and stress-induced GU, the ethanol-induced acute GU model is one of the widely used experimental models and similar to many characteristics of acute human peptic ulcer disease [15,16]. Gastric mucosa can be damaged by excessive intake of alcohol. In detail, ethanol ingestion causes many pathological changes in the gastric mucosa and submucosa, including hemorrhagic lesions, extensive submucosal edema, mucosal friability, and acute ulcers. In addition, ethanol can lead to direct injury of mucosa vascular endothelial cells, disrupt the cells continuity, induce the formation of reactive oxygen radicals and inflammatory cytokines, and cause local ischemia of the gastric mucosa . Here, the mice were selected as the model and absolute ethanol were administered intragastrically to simulate human GUs caused by excessive drinking. Simultaneously, we identified RUT as an agent that effectively protects against ethanol induced GU and provided several lines of evidence including assessment of ulcer index, histopathological and immunohistochemical analysis of gastric tissues and examination of the secretion of cytokines related to GU. Finally, the metabolomics strategy based on UPLC-Q-TOF/MS was used to investigate the possible biomarkers and potential complex mechanisms of RUT for the treatment of GU.
These results above demonstrated that RUT has been effective in treating GU, which can relieve the pressure on gastric mucosa caused by ethanol and reduced ulcer index. HE staining also confirmed that AR could effectively reduce the pathological changes such as hemorrhagic injury, submucosal edema, inflammatory cell infiltration and epithelial cell loss in gastric tissue. In addition, this study used kits to detect the expression of cytokines related to gastritis. The expression of antioxidases, such as SOD and CAT, play an advantageous role in protecting the stomach from ethanol damage . In addition, EGF is secreted in the gastrointestinal tract and could reduce gastric acid secretion, maintain structural integrity and promote the healing of ulcers . Inflammation is a key pathological response to ethanol-related peptic ulcers, and its main feature is the increased secretion of various pro-inflammatory factors (such as TNF-α, IL-6 and IL-1β), which showed multifaceted functions in promoting the GU formation . ET-1, as an important pro-inflammatory cytokine for the contraction of blood vessels, also plays a vital role in GU formation and is closely related to TNF-α. The increasing TNF-αpromotes the synthesis of ET-1, and the up-regulation of ET-1 results in the reduced blood supply of gastric tissue and the occurrence of serious hypoxia, acidosis and activation of neutrophils, thereby leading to the excessive release of TNF-α . Whlie a reduced ET-1 level is commonly associated with an increased NO level. As a type of endogenous vasodilator, NO could inhibit the secretion of ET-1 and regulate the secretion of gastric acid . Therefore, the determination of the above cytokines was of great significance for evaluating the effect on GU. In our study, the expression of the above factors in the model group altered significantly, while the intervention of RUT treatment could re-regulate these factors which tend to normal levels. RUT demonstrated a similar effect to OME. On one hand, RUT could significantly increase the expression of SOD, CAT, NO and EGF. On the other hand, RUT could decrease the expression of ET-1, TNF-α, IL-6, and IL-1β.
Subsequently, the metabolomic profiles of RUT in the treatment of GU were described. The results suggested that there were 7 potential biomarkers involved in 9 metabolic pathways related to GU. Among which, taurine and hypotaurine metabolism is the most influential. Taurine, a thiol-containing β-amino acid, has an established role in physiology and pharmacology, including nutrition, stabilizing cell membrane, regulating cell osmotic pressure, signaling regulation and protection against oxidant-mediated injury in various organs [23,24]. Numerous tudies have confirmed that taurine exerted a protective effect against gastric mucosal damage induced by water immersion restraint stress , ethanol damage , or others damage [24,27-29]. In addition, taurine could prevent GU induced by indomethacin through lipid peroxidation inhibition and neutrophil activation . In present study, compared with the control group, taurine of the mice administered with ethanol were significantly decreased, which suggested that the changes of taurine may imply the oxidative stress-related GU. It was in good agreement with previous reports mentioned above. In addition, the abnormal changes of taurine in GU mice were intervened by RUT treatment. So it is speculated that the regulation effects of RUT on taurine might help relive GU demage, which need further confirmation in future study.
Next is sulfur metabolism, in which sulfur is an essential nutrient for all life forms. It exists in a plethora of metabolites of primary and secondary metabolism, most notably in the amino acids cysteine and methionine, as well as cofactors such as iron sulfur clusters, lipoic acid, and CoA [31,32]. Some compounds contain sulfur in its oxidized form of sulfate, which plays an important role in numerous biochemical and cellular processes in mammalian physiology. It is necessary to provide sufficient sulfate from the circulation and the intracellular pathways for maintaining healthy growth and development . Although, we have not find the direct proof to illustrate the relationship between sulfur and GU, in this study, we observed that compared with the control group, sulfate was significantly decreased in the model group and RUT treatment reversed this reduction.
LysoPCs are the basic components of cell membranes and signaling molecules that regulate cell functions, including energy storage, cell proliferation and death, stress response and inflammation . Alterations in lipids metabolism are associated and suggested as causative for the pathophysiology of inflammation-related diseases such as atherosclerosis, diabetes, cancer and dyslipidemia [35,36]. In addition, previous studies have also shown that they are associated with the mechanism of ulcer induced by ethanol . In the present study, two kinds of lipid metabolism including glycerophospholipid and ether lipid metabolisms were found. LysoPC(18:1(9Z)) in glycerophospholipid metabolism could induce gastric injury and ulceration by causing impairment of the gastric mucosal barrier , along with the increased PCs. LysoPC(O-18:0) is an intermediate in ether lipid metabolism, which is associated with activating phospholipid and inflammation . Arachidonic acid (AA) is one of the most biologically active n-6 polyunsaturated fatty acids and can be used to produce prostaglandins (PGs) by cyclooxygenase. AA plays a vital role in the process of inflammatory responses and is related to GUs [40,41]. It can be hydrolyzed, generated and released into multifarious active substances. 5,6-EET is one of them and has interesting beneficial effects such as such as vasodilation, anti-inflammation, anti-platelet aggregation and maintaining tissue homeostasis [42,43]. In this study, the three inflammation-associated metabolites above were found abnormally changed compared with control group, indicating that sustained inflammatory response is the key to the progress of GU. However, RUT can re-regulate their expression, demonstrating the gastric protective effects on GU mice.
Tricarboxylic acid (TCA) cycle is the major pathway of energy production for universal organisms. Acetyl CoA and Citrate are important intermediary metabolite in TCA cycle and participate in the synthesis of adenosine triphosphate (ATP) [44,45]. Herein, we found a marked drop of citrate in the model group compared with the control ones, which indicated the down-regulation of TCA cycle. The alteration of this intermediate in the TCA cycle might suggest the disturbance of energy metabolism in GU . Interestingly, citrate increased in RUT treatment mice that could be due to elevated energy consuming to protect against gastric damage. Pyruvate transports into mitochondria as a master fuel input undergirding citric acid cycle carbon flux to drive ATP production by oxidative phosphorylation and multiple biosynthetic pathways intersecting the citric acid cycle . Pyruvate may be either oxidized to carbon dioxide producing energy or transformed into glucose. Pyruvate oxidation requires oxygen supply and the cooperation of pyruvate dehydrogenase, the tricarboxylic acid cycle, and the mitochondrial respiratory chain. Enzymes of the gluconeogenesis pathway sequentially convert pyruvate into glucose. Congenital or acquired deficiency on gluconeogenesis or pyruvate oxidation, including tissue hypoxia, may induce lactate accumulation . In the study, r-lactate was observed to be over aggregated in the model mice compared with the control group, indicating that ethanol may prevent the stomach from getting oxygen, which may lead to the accumulation of lactate and disorder of energy metabolism. However, the level of r-lactate in RUT group was lower than those observed in model group.
It was demonstrated that RUT treatment ameliorated gastric mucosal injury and serum metabolism in ethanol-induced GU mice. 7 endogenous metabolites in serum involved in 9 metabolic pathways were found as biomarkers to explain the underlying mechanism of GU. The effects of RUT on GU might involve in regulating the energy metabolism, oxidative stress, and inflammation. These findings not only provided a new insight for the synthesis of GU induced by ethanol but also revealed the possible action mechanism of RUT for the treatment of GU. This study suggested that RUT might be a promising candidate drug in the application of GU treatment and the selected metabolites might be served as potential drug targets for the diagnosis or treatment of GU. Future studies are needed to explore the potential roles of RUT in the regulation of the selected endogenous metabolites related to GU.