2018 Volume 41 Issue 12 Pages 1874-1878
The aims of this study were to determine the effects of gamma-aminobutyric acid (GABA) on immunoglobulin A (IgA) secretion from Peyer’s patch (PP) cells; to assess rat alpha-defensin-5 (RD-5) expression in the rat small intestine; and to determine the effect of GABA on intestinal ischemia reperfusion (I/R) injury-induced intestinal innate immunity. We found that GABA caused an increase in IgA secretion in the presence and absence of lipopolysaccharide (LPS). Moreover, GABA also significantly increased the mRNA levels of RD-5 and superoxide dismutase (Sod) 1, 3. Intestinal I/R was induced by a 30-min occlusion of the superior mesenteric artery followed by a reperfusion for 60-min. This led to a significant decrease in IgA secretion, and mRNA levels of RD-5 and Sod 1-3 in the ileum. On the other hand, administration of GABA before I/R induction had a significant protective effect against oxidative injury and attenuated the effects on intestinal immunity.
Peyer’s patches (PPs) are lymphoid tissues in which mucosal immune responses such as local immunoglobulin A (IgA) production occur. There is an inverse correlation between IgA secretion levels and bacterial overgrowth and translocation.1) Therefore, intestinal IgA secretion plays an important role in the immune system, in protecting against infections.2) Moreover, in the small intestine, Paneth cells secrete a variety of antimicrobial peptides, such as alpha-defensin-5 and 6.3) Wu et al. reported that defensin-5 was potent against Escherichia coli and Staphylococcus aureus, whereas defensin-6 showed activity against neither of the two pathogens.4) Thus, we focused the current study on IgA secretion and expression of defensin-5.
Gamma-aminobutyric acid (GABA) is considered a multifunctional molecule that causes various physiological effects for human and other animals health.5–7) In the brain, regulation of the GABA receptor (GABAR) has been shown to have preventive effects against oxidative stress-induced damage.8–10) A GABA agonist has also been reported to reduce hepatic ischemia–reperfusion (I/R) injury-mediated oxidative stress.11) With regard to antioxidant defense, free radical scavenging enzymes, such as superoxide dismutase (SOD), play an important role in reducing DNA damage.12–14) Normal cells are able to defend against oxidative stress through this scavenging system.13,14) Therefore, antioxidant enzymes reduce oxidative stress-induced damage. Although it was previously reported the role of antioxidant defense in the intestine,15) little is known about them in detail. Using a Caco-2 cell line as a model of human intestinal epithelial cells, we previously reported that oxidative stress significantly decreases the mRNA levels of human α-defensin 5 (HD-5).16) In the intestine, the systemic PP-dependent immune response, changes in rat alpha-defensin (RD)-5-expression after oral administration of GABA, and the effect of GABA on intestinal I/R-induced injury and innate immunity remain to be determined. In the present study, we sought to determine the effect of a single administration of GABA on innate immunity, and to develop intestinal I/R induced injury as a model of oxidative injury. We investigated the antioxidant activities of GABA as indicators of IgA secretion from PP cells ex vivo, and on the mRNA levels of RD-5 and Sods using a rat model of I/R.
GABA was purchased from Tokyo Chemical Industry (Tokyo, Japan). Lipopolysaccharide (LPS) from Escherichia coli O111 (produced by phenol extraction) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were of the highest grade available to us, and used without further purification.
Male Wistar rats were obtained from Hokudo (Sapporo, Japan). Rats (weighing 190–220 g and aged 7 weeks) were housed in plastic cages under standard laboratory conditions of constant temperature (23±2°C) with a 12 : 12-h light–dark cycle, with ad libitum access to water and food (standard rodent chow diet). All animal experiments were conducted in accordance with the guidelines of the Care and Use of Laboratory Animals of Hokkaido University. The dosage of GABA was 1–30 mg/kg body weight. GABA was dissolved in water and 1 mL/kg was injected orally by sonde. All animals were euthanized 24 h after GABA administration.
PP cell preparation and determination of IgA levels were performed as previously described.17)
The rats were divided into 3 groups (sham-operated group, I/R group and I/R+GABA group). Intestinal ischemia–reperfusion were performed as previously described.18)
Quantitative real-time PCR was performed using a LightCycler 480 ΙΙ System (Roche Diagnostics GmbH, Mannheim, Germany) with a KAPA SYBR Green Fast qPCR kit (KAPA Biosystems, Boston, MA, U.S.A.) as previously described.17) PCR was performed using primers for rat RD-5 and Sods with the following conditions: 40 cycles at 95°C for 3 s, 95°C for 10 s, 58°C for 20 s, and 72°C for 1 s. Primer sequences are shown in Supplementary Table 1. Levels of PCR products were normalized to the internal reference gene, beta-actin.
Samples of the ileum were taken after reperfusion, and the internal portion was washed with phosphate buffered saline (PBS) (−). The tissue was immediately ﬁxed in 10% buffered formalin. Fixed tissue was embedded in parafﬁn and sectioned. Slides were stained with hematoxylin and eosin to evaluate intestinal morphology and observed under a light microscope for classiﬁcation.
Statistical significance was performed using Student’s t-test and Tukey’s test or Dunnett’s test for post hoc analysis. Differences were considered statistically significant at p<0.05.
To investigate the innate intestinal immuno-stimulatory activity of GABA, we first evaluated the levels of IgA secretion from rat PP cells. We found that GABA increased IgA secretion with and without LPS, in a concentration-dependent manner (Fig. 1A), and had no effect on cell viability (Supplementary Fig. 1A). This shows that GABA activates natural immunity in the small intestine. Next, we measured mRNA levels of RD-5 in the rat ileum in response to GABA (30 mg/kg) and found that RD-5 expression was significantly increased (Fig. 1B) compared to that in the jejunum (Supplementary Fig. 1B). It has been reported that Paneth cells, which are the primary producers of RD-5, are present in higher numbers in the ileum than in the jejunum.19) Thus, our results correlate with the occurrence of a higher number of Paneth cells in the ileum.
(A) Rats received a single oral administration of GABA (1, 10, 30 mg/kg), and PP cells were cultured with or without LPS (100 µg/mL) for 72 h. Bar graphs (n=5) show mean with standard deviation for more than two independent experiments. *,**; Significantly differences (vs. controls) were calculated by Dunnett’s test (* p<0.05, ** p<0.01). (B, C) RD-5 (B) and Sods (C) mRNA levels in the rat ileum after a single oral administration of GABA (30 mg/kg). Bar graphs (n=5) show mean with standard deviation for more than two independent experiments. *,**; Significantly differences (vs. controls) were calculated by Student’s t-test (* p<0.05, ** p<0.01).
It has been reported that GABA prevents oxidative stress-induced damage.8–10) We previously reported that oxidative stress causes a significant decrease in the mRNA levels of human α-defensin 5 (HD-5) in Caco-2 cells, which serve as a model of human intestinal epithelial cells.16) We therefore hypothesize that the mechanism by which GABA induces these changes is related to the activity of antioxidant enzymes, such as SOD. To test this, we assessed the expression levels of Sods in the rat intestine. We found that the mRNA levels of Sods 1 and 3 were significantly increased in response to treatment with 30 mg/kg GABA (Fig. 1C). These results correlate with IgA production and RD-5 mRNA levels. On the other hand, we examined the effect of another factor such as Toll-like receptor (TLR) 2, 4, tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β). GABA had no effect on TLR2, 4, TNF-α and TGF-β mRNA level (Supplementary Table 2). Since GABA induced the secretion of IgA as well as changes in the expression levels of mRNA in RD-5 and Sods, we speculate that GABA attenuates oxidative stress-induced alterations in innate immunity.
Intestinal I/R occurs in a number of clinical settings, including small intestine transplant, intestinal surgery, circulatory shock, and strangulation ileus. It has been shown that the small intestine is highly sensitive to I/R.20) Reactive oxygen species (ROS) play an important role in acute intestinal I/R injury.21,22) ROS induce membrane lipid peroxidation, which is accompanied by a loss of intestinal barrier function. We previously reported that intestinal I/R injury causes an increase in malondialdehyde levels.18,23) Therefore, in the present study, we used a rat model of I/R. IgA secretion from rat PP cells and RD-5 mRNA levels in the rat ileum were significantly decreased compared with the sham-operated group (Figs. 2A, B). Moreover, Sods mRNA levels were also significantly decreased in the rat ileum (Fig. 2C). These results suggest that the ROS-induced reduction in innate immunity is associated with mRNA levels of Sods in the rat’s small intestine.
Rats received a single oral administration of GABA (30 mg/kg). (A) IgA production in cultured rat PP cells. (B, C) RD-5 (B) and Sods (C) mRNA levels in the rat ileum. Bar graphs (n=5) show mean with standard deviation for more than two independent experiments. *,†; Significantly differences (vs. *, sham-operated groups; †, I/R groups) were calculated by Tukey’s test (* p<0.05, † p<0.05).
Finally, we investigated the effect of GABA on I/R-induced injury and innate immunity. In addition to our findings that GABA suppresses I/R, induces IgA secretion, and affects RD-5 mRNA levels (Figs. 2A, B), we also found that pre-treatment with GABA prevents damage to the crypt (Fig. 3). There were no significant differences in the mRNA levels of Sods between the I/R plus GABA treatment group and the sham-operated group (Fig. 2C). These results are consistent with a previous report showing that GABA reduces hepatic I/R injury-mediated oxidative stress.11) Li et al. reported that a functional GABA signaling system existed in the intestinal epithelium.24) GABA content has an antioxidant capacity.25) Next, we examined direct effect of GABA on I/R-induced IgA reduction in cultured rat PP cells. Intestinal concentration of orally administered GABA was predicted according to the previous report.26) Based on these results, we selected 5 mM GABA concentration in vitro study. A direct treatment of 5 mM GABA in PP cells significantly increased IgA secretion. Although I/R significantly reduced IgA secretion, there were no significant differences in IgA secretion between I/R plus 5 mM GABA treatment group and the sham-operated group (data not shown). We therefore suggest that the protective effect of GABA on I/R induced injury can be caused by inducing an antioxidant enzyme such as Sods and a direct immuno-stimulatory activity. The results of this study provide further scientific evidence of the value of GABA in treating oxidative injury and attenuating alterations in intestinal immunity.
Hematoxylin and eosin staining of sham-operated ileum (A), I/R-treated ileum (B), and I/R-treated ileum pretreated with GABA (30 mg/kg) via oral administration (C). Images represent typical data. (Color figure can be accessed in the online version.)
In conclusion, we focused on the protective effect of GABA on intestinal I/R-induced alterations in innate immunity. We showed that GABA, an antioxidant, significantly reduced oxidative injury and relieved the effects of I/R on innate immunity.
This study was supported in part by a Grant from the Mishima Kaiun Memorial Foundation Research Grant and Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant number 16K08388). This study was partly supported by Hokkaido University, Global Facility Center (GFC), Pharma Science Open Unit (PSOU), and funded by Ministry of Education, Culture, Sports, Science and Technology (MEXT) under “Support Program for Implementation of New Equipment Sharing System.”
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.