Protective effect of Acer mono Max. sap on water immersion restraint stress-induced gastric ulceration

Abstract

Acer mono Max. sap (AmMs) is called 'Gol-Li-Su' or 'Go-Lo-Soe' in Korean, which means 'water beneficial to the bones'. It is reported that the sap contains several types of minerals and sugars. In particular, the calcium concentration of the sap is 36.5 times higher than that of commercial mineral water. Apart from its anti-osteoporosis effect, no reports have addressed the biological activities of AmMs against degenerative diseases. In the present study, we investigated whether AmMs alleviates gastric ulcer-related symptoms in a stress-induced mouse model. To assess the effect of AmMs on gastric ulcer-like symptoms, we carried out a water immersion restraint (WIRE) test and found that AmMs has potential in alleviating gastric ulcers in a concentration-dependent manner. These results indicate that the nutritional factors of the sap mitigate the gastric ulcer-related symptoms caused by stress-induced gastric lesions in mice. AmMs-treated mice exhibited a significant decrease in the ulcer index as compared to those treated with omeprazole or L-arginine. To examine one potential mechanism underlying this effect, we performed reverse transcription-polymerase chain reaction to ascertain whether molecular markers were associated with the mitigation of the gastric lesions. Epithelial and/or tissue nitric oxide synthase (NOS) was assessed to determine whether or not the genes were down-regulated dose-dependently by the sap. The levels of these enzymes were found to be lower in the tissue samples treated with AmMs compared with the levels in the control samples. These findings collectively suggest that AmMs significantly protects the gastric mucosa against WIRE stress-induced gastric lesions, at least in part, by alleviating inducible NOS and/or neuronal NOS expression.

Figure 1.

Gastroprotective effects of AmMs on WIRE stress-induced ulcer damage in mice. One hour prior to the initiation of WIRE stress, (A) control mice received a single saline administration p.o. (0.02 ml); (B) positive control mice received a single dose of L-arginine (300 mg/kg) or (C) omeprazole (300 mg/kg); and (D and E) experimental mice received a single p.o. administration of AmMs (30 and 150 mg/kg, respectively). After 6 h of exposure to water immersion, mice were euthanized and their stomach tissues were anatomized for damage. The images are representative of a set of classical examinations visualized under a microscope (Nikon ECLIPSE 90i; Tokyo, Japan). The arrows denote disrupted or intact forms of the ulcerative tissues as shown and scored according to the ulcer index (Fig. 2).

Figure 2.

Decrease in the ulcer index of WIRE stress-induced ulcer tissue samples from mice treated with AmMs. The ulcer index was calculated as described in Materials and methods. Data are presented as the means ± SD (6 mice/group) of triplicate experiments by the Student-Newman-Keuls method for independent means, using the Sigma Plot. The critical level for significance was set at p<0.05; p<0.05 was considered significant.

Figure 3.

mRNA expression levels of various target proteins in AmMs-treated ulcerative tissues. mRNA expression levels of CSE, BTC, EGF, COX-1, COX-2, MMP-2, MMP-3, MMP-9 and 18s rRNA are shown. Lane 1, control group; lane 2, L-arginine-treated group (300 mg/kg, i.p.); lane 3, omeprazole-treated group (3 mg/kg, i.p.); lanes 4 and 5, AmMs-treated experimental groups (30 and 150 mg/kg, p.o., respectively).

Figure 4.

Comparison of the mRNA expression levels of NOSs. (A) The expression levels of iNOS, eNOS and nNOS mRNA were normalized with that of 18s rRNA, using Quantity One Version 4.6.1. One hour prior to the start of WIRE-induced stress, control mice received a single saline administration p.o.; positive control mice received a single i.p. injection of L-arginine (300 mg/kg) or omeprazole (300 mg/kg); and experimental mice received a single p.o. administration of AmMs (30 and 150 mg/kg). Lane 1, control group; lane 2, omeprazole-treated group (3 mg/kg, i.p.); lane 3, L-arginine-treated group (300 mg/kg, i.p.); lanes 4 and 5, AmMs-treated experimental groups (30 and 150 mg/kg, p.o., respectively). Relative band intensity (%) of (B) iNOS and (C) nNOS comparing the expression levels in the various treatment groups. The data are the representative results of three independent experiments.