2020 Volume 26 Issue 1 Pages 153-158
Background: β-Glucans reportedly inhibit gastric ulceration; however, it is unclear which form of the polysaccharide is important for this inhibitory effect. In this study, the effect of paramylon from Euglena gracilis (β-(1,3)-glucan) on gastric ulcers was evaluated. Materials and methods: Wistar rats were exposed to water-immersion restraint stress (WIRS). Results: Dietary intake of Euglena extract powder, paramylon (insoluble), and amorphous paramylon (soluble) decreased the area of gastric ulceration in comparison with the control group. WIRS-induced increases of inducible nitric oxide synthase expression in the gastric mucosa and serum IgA and alanine aminotransferase levels were suppressed in rats fed with Euglena extract powder, paramylon, or amorphous paramylon. These results indicate that paramylon and amorphous paramylon suppressed gastric ulcers, presumably by reducing oxidative damage resulting from WIRS. In addition, our data suggest that Euglena extract powder, paramylon, and amorphous paramylon prevent the WIRS-induced influx of proinflammatory molecules from the gut.
Psychological problems, including severe stress, are associated with the prevalence of peptic ulcers (Deding et al., 2016; Lee et al., 2017). Gastric ulcers penetrate deeper than the stomach mucosal muscularis and damage the tissue. Major symptoms of gastric ulcer include abdominal swelling, nausea, vomiting, and anorexia. Water-immersion restraint stress (WIRS; acute stress model) is an experimental method for inducing stress and inflammation in laboratory animals. The water immersion process results in the formation of multiple small ulcers in the stomach (Yoshizawa et al., 2004). We have previously found that solubilized barley bran and wheat bran have suppressive effects on gastric ulcers (Yoshizawa et al., 2004; Shojo et al., 2015).
β-Glucans suppress inflammation and have antitumor properties (Vetvicka et al., 2004; Akramienė et al., 2007; Sugiyama et al., 2010; Sonck et al., 2010; Sofi et al., 2017). Additionally, they enhance protection against infection (Nakagawa et al., 2003; Sener et al., 2005) and induce wound healing (Majtan & Jesenak, 2018; Yasuda et al., 2018). β-Glucans exhibit several structures. β-Glucans in foods such as cereals and mushrooms are polymers of D-glucose in which β-(1,3), β-(1,6), and β-(1,4) are bound by glycosidic bonds. The amount and type of β-glucans differ depending on the type of food. Barley bran and wheat bran contain β-(1,3)-(1,6) and β-(1,3)-(1,4) water-soluble glucans (Yoshizawa et al., 2004; Shojo et al., 2015). Natural compounds with only β-(1,3)-glucans are not known to have the same therapeutic effects as those with other glucan structures.
Euglena gracilis possesses abundant nutrients, and its use as a functional food ingredient is expanding. It possesses approximately 60 compounds with physiological and nutritional functions (Nakano et al., 2017). Euglena produces a polyglucan with a large number of β-(1,3) bonds with CO2 by photosynthesis and accumulates a polysaccharide called paramylon, a β-(1,3)-glucan of 700–750 glucose residues that constitutes 10–40% of the dry cell weight (Takenaka et al., 1997; Kitaoka, 1987). Amorphous paramylon is a hydratable form of paramylon.
In this study, we used the WIRS method to induce an acute gastric ulcer model, and then evaluated the effect of paramylon intake on stress. In addition to the effects of paramylon (insoluble) and amorphous paramylon (water soluble) on gastric ulcers, we also evaluated the use of Euglena as a comprehensive nutritional food to suppress stress.
Experimental animals and diets Male Jcl:Wistar rats (CLEA Japan, Inc., Tokyo, Japan) were acclimated for 1 week. They were then randomly divided into four groups (six animals per group) and provided an experimental diet for 2 weeks. The experimental diet was based on AIN-93M. It was made by replacing 3% of each nutrient component with Euglena powder, paramylon, or amorphous paramylon. Since Euglena contains multiple nutrients, it was added by subtracting other nutrients based on the ingredient value. As paramylon and amorphous paramylon are β-glucans, they were replaced with 3% cellulose as dietary fiber. During the acclimation and test periods, free access to water and feed was provided. The nutritional components per 100 g of Euglena powder were as follows: water 3.6 g, protein 31.7 g, fat 14.6 g, ash 3.8 g, and carbohydrates 46.3 g. The carbohydrate content (70–80%) in Euglena was assumed to be paramylon, and paramylon isolated from E. gracilis Z was obtained from Euglena Co., Ltd. (Tokyo, Japan). For the purification of paramylon, Euglena cultures were subjected to continuous centrifugation and washing with water. Cells were deconstructed by sonication, and a paramylon mixture was collected. To remove lipids and proteins, the paramylon mixture was treated at 95 °C for 1 h with a 1% sodium dodecyl sulfate (SDS) solution and then at 50 °C for 30 min with a 0.1% SDS solution. Paramylon was obtained by continuous centrifugation; after washing with water, acetone, and ether, in turn, refined paramylon was acquired (Sugiyama et al., 2010). Amorphous paramylon was a hydratable glucan produced in which paramylon powder was dissolved with an alkali, neutralized with an acid, alcohol-precipitated, washed, and lyophilized. The nutritional components per 100 g of paramylon powder were as follows: water 3.4 g, protein 0.2 g, ash 0.4 g, and carbohydrates 96.0 g. The ratio of β/α carbohydrates in paramylon exceeded 100. Animals were housed at room temperature (23 ± 3 °C) under a 12-h light–dark cycle (08:00–20:00). This study was approved by the ethics committee of Osaka Prefecture University (26–34), and was performed in accordance with standards concerning the breeding and storage of laboratory animals.
Water-immersion restraint stress (WIRS) The gastric ulcerogenic response was examined as described previously (Yoshizawa et al., 2004; Shojo et al., 2015; Ohta et al., 2009). After fasting for 12 h, rats were inserted into wire cages and immersed in a water bath (23 °C) for 18 h or 3.5 h to induce WIRS. After 18 h of WIRS, the rats were killed by an overdose of isoflurane. Their stomachs were removed and scored for hemorrhagic damage. Scores were based on measurements of the area of all lesions in mm2. The values were summed to obtain an overall gastric lesion index. After 18 h of WIRS, blood samples and various organs were collected. The excised stomach was fixed in 10% formalin and the site of mucosal lesion was measured. After 3.5 h of WIRS, the gastric mucosa was collected using glass slides, and oxidative damage stress-related gene expression was evaluated by RT-PCR (Brzozowski et al., 2000; Szlachcic et al., 2013; Konturek et al., 1997).
RT-PCR Total RNA was extracted from the gastric mucosa using Sepasol-RNA Super G (Nacalai Tesque, Tokyo, Japan) and reverse-transcribed with dNTPs and ReverTra Ace (TOYOBO, Osaka, Japan). cDNA was used as a template for RT-PCR with PrimeStar GXL (Takara Bio, Shiga, Japan). The primers were as follows: Forward, 5′-CAGTGGCAACATCAGGTC-3′, Reverse, 5′-GGTCTCGGACTCCAATCT-3′ (439 bp) for inducible nitric oxide synthase (iNOS); and Forward, 5′-TGTCACCAACTGGACGATA-3′,Reverse, 5′-GGGGTGTTGAAGGTCTCAAA-3′ (165 bp) for β-actin. For the amplification of iNOS, the PCR conditions were as follows: 95 °C for 1 min, 60 °C for 45 s, and extension at 72 °C for 2 min, for a total of 35 cycles. To amplify β-actin, the PCR conditions were as follows: 95 °C for 1 min, 55 °C for 30 s, and extension at 72 °C for 1 min, for a total of 28 cycles. The signals for iNOS mRNA were standardized against the β-actin signal for each sample, and the results are expressed as the iNOS/β-actin mRNA ratio (Konturek et al., 1998; Konturek et al., 2004).
Measurement of IgA by enzyme-linked immunosorbent assay (ELISA) IgA levels in rat serum were determined using a Rat IgA ELISA Kit (E111–102; Bethyl Laboratories, Montgomery, USA) according to the manufacturer's instructions. Absorbance was determined using a microplate reader at 450 nm.
Measurement of serum alanine amino transferase (ALT) levels ALT levels in rat serum were determined using a Transaminase CII Test Kit (Wako, Osaka, Japan) according to the manufacturer's instructions. Absorbance was determined using a microplate reader at 540 nm.
Statistical analysis Data were analyzed using 4 Steps
Excel Tokei (OMS Publishing Inc., Saitama, Japan). Results are expressed as mean ± Standard error (SE). The mean differences between groups were analyzed by one-way or mixed between–within subjects analysis of variance (ANOVA), followed by Tukey-Kramer post hoc tests. Normality was verified using the chi-squared goodness-of-fit test. A p-value < 0.05 was considered statistically significant.
Effects of Euglena, paramylon, or amorphous paramylon intake on WIRS-induced gastric ulceration There were no differences in dietary intake (mean ± SE; Control group 25.2 ± 0.70 g/d; Euglena group 25.4 ± 0.56 g/d; Paramylon group 25.5 ± 0.91 g/d; Amorphous paramylon group 22.7 ± 0.71 g/day) or body weight gain (mean ± SE; Control group 91.2 ± 3.92 g; Euglena group 91.3 ± 2.33 g; Paramylon group 84.7 ± 8.07 g; Amorphous paramylon group 88.8 ± 3.29 g) among groups before WIRS.
After 18 h of WIRS, a black ulcer lesion was observed in the control group, and the ulcer areas were significantly lower in the Euglena, paramylon, and amorphous paramylon groups than in the control group (p < 0.01) (Figs. 1, 2).
Images of gastric stress ulcers. Gastric ulcer formation after WIRS for 18 h is shown. Black lesion is a gastric ulcer. The red arrow indicates the site of the gastric ulcer lesion.
Relative area of the gastric stress ulcer. The degree of gastric ulcer is expressed as a ratio as follows. Relative gastric ulcer area (%) = area of gastric ulcer/total area of the gastric epithelium × 100. Mean ± SE (%). Tukey-Kramer: ab p < 0.05.
Effects of Euglena, paramylon, or amorphous paramylon intake on iNOS expression in the gastric mucosa We evaluated the expression of iNOS, which is involved in gastric ulcers, after 3.5 h of WIRS (Konturek et al., 1998; Konturek et al., 2004). iNOS is expressed during stress and inflammatory responses. Compared with levels in healthy animals (rats not subjected to WIRS), iNOS levels were higher in the control group. The increase in iNOS induced by WIRS was suppressed in the Euglena group, and was significantly decreased in the paramylon and amorphous paramylon groups (p < 0.001) (Fig. 3).
Ratio of iNOS/β-actin mRNA in the intact gastric mucosa. After WIRS for 3.5 hours, genes altered along with gastric ulcer formation were measured by RT-PCR. Mean ± SE. Tukey-Kramer: ab p < 0.001.
Effects of Euglena, paramylon, or amorphous paramylon intake on serum IgA levels Levels of serum IgA, which is a negative marker of gut integrity, were significantly lower in the paramylon and amorphous paramylon groups than in the control group (p < 0.05). In addition, the level tended to be reduced in the Euglena group (Fig. 4).
Serum IgA levels. Formation of serum IgA on gastric ulcers after WIRS for 18 h is shown. Mean ± SE. Tukey-Kramer: ab p < 0.05.
Effects of Euglena, paramylon, or amorphous paramylon intake on serum ALT levels We also evaluated serum ALT concentrations. WIRS-induced serum ALT levels were significantly lower in rats fed Euglena, paramylon, and amorphous paramylon than in rats in the control group (p < 0.05) (Fig. 5).
Serum ALT levels. Formation of serum ALT on gastric ulcers after WIRS for 18 h is shown. Mean ± SE. Tukey-Kramer: ab p < 0.05.
We previously found an inhibitory effect of dietary fiber in barley and wheat bran on gastric ulcer formation (Yoshizawa et al., 2004; Shojo et al., 2015). It is possible that the β-(1,3) polymer can suppress gastric ulcers using an enzymatically digested fraction of β-(1,3)-(1,4)-glucan. However, the effects of pure β-(1,3)-glucan have not been evaluated. Using paramylon consisting of β-(1,3), we observed the same effect. Further, paramylon was subjected to alkali treatment, and similar effects were obtained using water-solubilized amorphous paramylon. The cellulose used in the control group was a β-(1,4)-glucan.
β-Glucans in foods are polymers of D-glucose in which β-(1,3), β-(1,6), and β-(1,4) are bound by glycosidic bonds, and their quantity and type vary among foods (Sofi et al., 2017). The water-soluble and alkaline-extracted β-glucan of Agaricus also effectively suppresses ulcers (Vetvicka & Yvin, 2004). We previously found that solubilized barley bran and wheat bran have a suppressive effect on gastric ulcers mediated by β-glucan (Yoshizawa et al., 2004; Shojo et al., 2015). As described above, β-D-glucan is a biological response modifier, but its clinical use requires solubility in aqueous media.
In the intestinal tract, an increase in iNOS is a hallmark of ulcerative disease (e.g., ulcerative colitis) and is involved in the pathogenesis of inflammation by inducing NO (Schreiber et al., 2013; Soufli et al., 2016). Dietary supplementation with β-(1,3)-(1,4)-glucan (oats) and β-(1,3)-(1,6)-glucan (shiitake) suppresses dextran sodium sulfate-induced ulcerative colitis by reducing iNOS (Liu et al., 2015; Shi et al., 2016). Stress increases gut permeability (Calarge et al., 2019), and enhancement by increasing claudin-1 and occludin is a proposed mechanism underlying the suppression of pro-inflammatory factors by β-glucan (Shao et al., 2013). Similar to ulcerative colitis, iNOS levels are positively correlated with WIRS-induced gastric ulcers, and its expression is suppressed by the dietary intake of Euglena, paramylon, and amorphous paramylon. In addition, serum IgA levels are increased by xenobiotics such as lipopolysaccharides (Maes et al., 2007; Dong & Yuan, 2018), and these compounds also cause liver damage (Han et al., 2004). We found that both serum IgA and ALT (a marker of liver injury) levels were suppressed by the dietary intake of Euglena, paramylon, and amorphous paramylon. However, serum corticosterone, a stress marker, did not differ among groups (data not shown). Taken together, these results suggest that Euglena, paramylon, and amorphous paramylon suppress the influx of xenobiotics and both serum IgA and ALT levels. β-(1,3)-Glucan may suppress WIRS-induced ulceration by the enhancement of gastric barrier function.
In conclusion, our results demonstrate that Euglena extract powder, paramylon, and amorphous paramylon suppress gastric ulcers by reducing oxidative damage caused by excessive stress. Furthermore, since amorphous paramylon has been useful in suppressing gastric ulcers, there is a possibility that it may be widely used as a functional food supplement in the future.
