What Is Known/What Is New What Is Known
Alanylglutamine is a dipeptide that fuels enterocytes.
Alanylglutamine restores intestinal barrier function and prevents deterioration of gut permeability.
What Is New
Alanylglutamine healed gastric ulcers induced by indomethacin and its action was comparable to pantoprazole.
Alanylglutamine has antisecretory potential mediated by reducing H+-K+-ATPase pump expression.
Alanylglutamine enhances mucosal proliferation via the glucagon-like peptide-1 /β-catenin/cyclin-D1 cue.
Alanylglutamine inhibits apoptosis by repressing nuclear factor-kappa B/tumor necrosis factor-α/H2 O2 .
Glutamine or its more stable and soluble dipeptide, alanylglutamine (AG), are considered gut nutrients that restore the intestinal barrier function and promote the growth in malnourished enteropathic patients (1) (2) (3) (2,4) (5) (6,7) (8,9) (8,10) (8,11)
The gastric ulcer healing cascades embrace a constellation of time-dependent events and comprise the activation of an array of genes (12) (13) (14) (12,15) (12) proliferation and migration of epithelial cells and connective tissue to the denuded areas (16)
On the contrary, the commonly prescribed nonsteroidal anti-inflammatory drugs (NSAIDs) are well known for their gastropathy that limit their consumption. This adverse effect resides mainly on the inhibition of cyclooxygenase (COX) enzyme, which in turn suppresses the key protective autacoid, PGE2 (14) (17) (18) (19)
According to the above data, the present study was undertaken to delineate the conceivable effectiveness of AG in indomethacin-induced gastric ulceration in rats compared to the proton pump inhibitor pantoprazole, the reference drug as a primary objective and to unveil some of the potential molecular mechanisms of this dipeptide as a secondary objective.
METHODS
Male Wistar rats (7-week old) weighing 160 ± 10 g were purchased from the Faculty of Veterinary Medicine (Cairo University, Cairo, Egypt) and habituated to the experimental conditions for a week before experimentation. They were maintained on 12/12-hour dark/light cycles, controlled laboratory conditions (room temperature of 25°C ± 2°C; humidity of 65% ± 5%), and proper diet chow and water ad libitum. Before the induction of gastric ulcers, rats were fasted overnight with free access to water and were kept individually in wide mesh bottom cages. Handling of animals complied with the Guide for the Care and Use of Laboratory Animals (NIH, 1978) and ARRIVE guidelines. The study was approved by the Research Ethics Committee at the Faculty of Pharmacy, Cairo University (Cairo, Egypt) with the permit number (PT) 1566.
Induction of Indomethacin Gastric Ulceration and Experimental Design
Animals (n = 10 rats/group) were randomly allocated into 5 groups; viz, the control group, where rats received the vehicle (1% Tween 80), whereas those in the other groups received indomethacin (100 mg/kg; p.o.; Sigma-Aldrich, MO) suspended in the vehicle (20) (21) (22) (23) 2 O2 ) and caspase-3 activity. Of note, the protein content of the gastric mucosa was estimated using Bradford technique (24)
Determination Ulcer Index and Gastric Juice pH
The ulcer index was calculated by the summation of the length of each ulcer at its greater axis (25)
Determination of Gastric Mucosal Hydrogen Peroxide Content
H2 O2 was assessed using the colorimetric reagent kit (Sigma-Aldrich; cat CS0270).
Determination of Gastric Mucosal Caspase-3 Activity
The ApoTarget selective colorimetric assay kit (Invitrogen Corporation, MA) was used for the assessment of caspase-3 activity according to the manufacturer's instructions. The test is based on incubating the sample with DEVD-p -nitroanilide (p NA) substrate that is a synthetic upstream tetrapeptide DEVD of the caspase-3 cleavage site linked to the chromophore p NA. When the substrate is cleaved by caspase-3, p NA is released to produce a yellow color that is measured at 400 nm. For the determination of the fold elevation in the caspase-3 activity, the absorbance of p NA from apoptotic sample is compared with the control.
ELISA Technique
Serum TNF-α (cat RTA00) and GLP-1 (cat MBS723782) were assessed using the corresponding ELISA kits (MyBioSource, CA) according to the manufacturer's protocols.
Western Blot
Samples were washed twice with RIBA lysis buffer PL005 (Bio Basic; cat L3R 8T4) composed of NaCl (150 mmol/L), Triton X-100 (0.1%), sodium deoxycholate (0.5%), sodium dodecyl sulfate (0.1%), Tris-HCl (50 mmol/L), protease inhibitor buffer, and phosphatase inhibitor buffer. The cell lysates were centrifuged and equal amounts of protein extracted from the gastric mucosa were separated by 4% sodium dodecyl sulfate polyacrylamide gel electrophoresis TGX Stain-Free FastCast Acrylamide kit (Bio-Rad, CA; cat 161-0181) and transferred to nitrocellulose membranes. Blots were blocked for 1 hour in Tris-buffered saline that contained 3% bovine serum albumin, then incubated using the primary antibodies (Thermo Fisher Scientific) for p S133-cAMP response element-binding protein (CREB; 1:2000; cat 700129), p S473-protein kinase B (Akt; 1:2000; cat 700256), p S9-glucagon synthase kinase 3 beta (GSK3β; 1:2000; cat MA5-15023), p S536-nuclear factor kappa B (NFκB; 1:2000; cat 51-0500), p S45-β catenin (1:2000; cat PA5-17685), and β-actin (1:2000; cat MA1-300). Incubation was done with the horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin secondary antibody (Novus Biologicals, CO; cat NBP2-30348H), then visualized by the enhanced chemiluminescent substrate (Clarity TM Western ECL; Bio-Rad; cat 170-5060). For the quantitative analysis, the mean intensity of each band (mean pixel) was compared with β-actin band using stain-free blot technology and ChemiDoc MP Imager (Bio-Rad).
Gene Expression Using Real-time Polymerase Chain Reaction Analysis
In alliance with the manufacturer's protocol, the total RNA was extracted using SV Total RNA Isolation System (Promega, WI) that was determined at 260 nm. RNA was reverse transcribed into cDNA using random primers (250 ng/μL), deoxyribonucleotide triphosphate (10 mmol/L), and the SuperScript II Reverse Transcriptase kit (Invitrogen). The following primer set was used for the quantitative determination of the gene expression of claudin-1 (forward primer: 5′-CTATCGCTCCGTTACTGGCAT-3 and reverse primer: 5′-ACAGCTGGGACCGTATGTGG-3), proton pump H+ /K+ ATPase (forward primer: 5′-GCAGCGAGTGACTGGAACA-3′, reverse primer:5′-CCAGCCACGTTGCATTGTAG-3′), cyclin-D1 (forward primer: 5′-TGGAGCCCCTGAAGAAGAG-3′ and reverse primer: 5′-AAGTGCGTTGTGCGGTAGC-3′), and the housekeeping gene β-actin (forward primer: 5′-GGTCGGTGTGAACGGATTTGG-3 and reverse primer: 5′- ATGTAGGCCATGAGGTCCACC-3). The cDNA synthesis was performed at 37°C for 60 minutes, then 95°C for 10 minutes for reverse transcriptase inactivation. The resulting cDNA was mixed with SYBR green and other polymerase chain reaction mix components, then incubated in programmed thermocycler that was set at 40 cycles of denaturation step (95°C for 15 seconds), primer annealing (60°C for 1 minute), and extension step (72°C for 1 minute). The relative quantitation was calculated using an Applied Biosystem with software version 3.1 (StepOne, Thermo Fisher Scientific).
Histological Analysis
The stomach from 4 representative animals from each group was fixed in 10% formalin and embedded in paraffin. The blocks were sectioned (5 μm) and stained with hematoxylin and eosin and examined under a light microscope blindly.
Statistical Analysis
Data are expressed as a mean of 6 to 10 rats ± standard deviation. The GraphPad Prism v7.0 (GraphPad Prism, CA) was used to analyze and present all data. Multiple comparisons were performed using 1-way analysis of variance, followed by Tukey's multiple comparison post-hoc test; P < 0.05 was considered as the significance limit for all comparisons.
RESULTS
Effect of Different Treatments on the Indomethacin-induced Gastric Ulceration
As depicted in Figure 1 A, AG post-treatment prohibited the indomethacin-induced gastric ulceration in a manner comparable to that of pantoprazole; however, DEXA in combination with AG partly reverted its protective effect. Compared to the (Fig. 1 B) control rat stomach photomicrograph, (Fig. 1 C–E) indomethacin reveals ulceration of the gastric mucosa (u) with the presence of necrotic mucosal cells, edema, congested capillaries (c), and dense inflammatory infiltrate (i). Moreover, inflammatory cellular infiltrate were noted in the submucosa in gastropathic rats. Section of (Fig. 1 F) pantoprazole shows an average mucosal thickness, intact mucus covering, and normal gastric pits, mucosal neck cells, parietal and peptic cells in the mucosa, with very few inflammatory cells (i) in the submucosa. Section of (Fig. 1 G) AG shows gastric mucosa with average mucosal thickness and intact mucus covering with normal crowded gastric pits ending by branched tubular glands. Finally, the gastric mucosal section of rats treated with (Fig. 1 H) DEXA and AG shows patchy mucosal ulceration (u) indicating its antagonistic effect.
FIGURE 1: Effect of AG postadministration with or without DEXA and PANT on ulcer index (A) and microscopical alterations (B–H) in INDO-induced gastric ulceration. Data are expressed as means ± standard deviation (SD) (n = 10). As compared to CONT (σ), INDO (∗), PANT (ω), and AG (•) groups at P < 0.05 using 1-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. On the microscopic level, sections of (C–E) INDO reveals ulceration of the fundic gastric mucosa (u) with dense inflammatory infiltrate (i) and necrotic mucosal cells with edema and congested capillaries (c), compared to the (B) CONT section. However, treatment with (F) PANT or (G) AG improved the gastric structure, an effect that was worsened in the combined section of (H) AG and DEXA to show patchy mucosal ulceration (u). AG = alanylglutamine; DEXA = dexamethasone; INDO = indomethacin; PANT = pantoprazole.
Effect of Different Treatments on Gastric Acid Secretion in Indomethacin-induced Gastric Ulceration
As presented in Figure 2 , indomethacin increased the basal hydrogen ion concentration, as depicted by a reduction in the (Fig. 2 A) gastric juice pH and (Fig. 2 B) an upregulation of the mRNA expression of H+ /K+ - ATPase. These effects were almost equally hindered by AG and pantoprazole, but reverted when DEXA was combined with AG. Administration of indomethacin caused 9.3- and 11.5-fold increase in (Fig. 2 C) p S9-GSK3β, and (Fig. 2 D) p S133-CREB, respectively, relative to the control rats. Treatment with AG, however, brought them back to normal, an effect that surpassed that of pantoprazole and was partially hindered by the coadministration of DEXA.
FIGURE 2: Effect of AG postadministration with or without DEXA and PANT on gastric juice pH (A), mRNA expression of H+ /K+ - ATPase (B), p S9-GSK3β (C), and p S133-CREB (D) in INDO-induced gastric ulceration. Data are expressed as means ± standard deviation (SD) (n = 6). As compared to CONT (σ), INDO (∗), PANT (ω), and AG (•) groups at P < 0.05 using 1-way analysis of variance (ANOVA) followed by Tukey's post hoc test. AG = alanylglutamine; DEXA = dexamethasone; INDO = indomethacin; PANT = pantoprazole; p S133-CREB = phosphorylated cAMP response element binding protein at serine 133; p S9-GSK3β = phosphorylated glycogen synthase kinase 3 beta at serine 9.
Effect of Different Treatments on Indomethacin-induced Impairment of Gastric Mucosal Proliferation and Tight Junction
Figure 3 illustrates the suppressing effect of indomethacin on (Fig. 3 A) p S473-Akt, (Fig. 3 B) cyclin-D1, and (Fig. 3 E) GLP-1, effects that were associated with the marked increase in (Fig. 3 C) p S45-β catenin and (Fig. 3 D) the tight junction protein claudin-1, as compared to the control rats. These alterations were reverted by AG and pantoprazole in a comparable pattern; however, coadministration of DEXA with AG abolished its beneficial effects on all assessed parameters.
FIGURE 3: Effect of AG post-administration with or without DEXA and PANT on gastric mucosal p S473-Akt (A), cyclin-D1 (B), p S45-β catenin (C), claudin-1(D), and serum GLP-1 (E) in INDO-induced gastric ulceration. Data are expressed as mean ± standard deviation (SD) (n = 6). As compared to CONT (σ), INDO (∗), PANT (ω), and AG (•) groups at P < 0.05 using 1-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. AG = alanylglutamine; DEXA = dexamethasone; GLP-1 = glucagon-like peptide-1 ; INDO = indomethacin; PANT = pantoprazole; p S473 and p S45 = phosphorylation sites at serine 473 and 45, respectively.
Effect of Different Treatments on Indomethacin-induced Gastric Mucosal Inflammation, Oxidative Stress, and Apoptosis
As shown in Figure 4 , indomethacin increased the contents/activity of (Fig. 4 A) p S536-NF-κB p65, (Fig. 4 B) H2 O2 , (Fig. 4 C) caspase-3, and (Fig. 4 D) TNF-α, and, as compared to the control rats. AG and pantoprazole, abated the inflammatory, oxidative, and apoptotic markers, whereas combination with DEXA, obliterated the effect of AG to different degrees.
FIGURE 4: Effect of AG postadministration with or without DEXA and PANT on gastric mucosal p S536-NF-κB p65 (A), H2 O2 (B), caspase-3 (C), and serum TNF-α (D) in INDO-induced gastric ulceration. Data are expressed as mean ± standard deviation (SD) (n = 6). As compared to CONT (σ), INDO (∗), PANT (ω), and AG (•) groups at P < 0.05 using 1-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. AG = alanylglutamine; DEXA = dexamethasone; H2 O2 = hydrogen peroxide; INDO = indomethacin; PANT = pantoprazole; p S536-NF-κB p65 = phosphorylated nuclear factor-kappa B at serine 536; TNF-α = tumor necrosis factor alpha.
DISCUSSION
The current study verified that AG has an ulcer-healing activity that was comparable to the reference proton pump inhibitor. The merit of the dipeptide in healing indomethacin-induced gastric ulceration and some of the potential molecular mechanisms underlying its effect are addressed for the first time in this study. The AG healing capacity relies on the interplay between multiple pathways.
In the present work, sundry mechanisms gather to substantiate the AG antisecretory character; the first is its ability to increase GLP-1 level, which in turn reduces acetylcholine release and consequently gastric acid secretion (26) (27)
Meanwhile, GLP-1 activates its subsequent downstream phosphatidylinositol 3 kinase/Akt signaling pathway, which plays also a role in suppressing acid secretion via stimulating the serum and glucocorticoid regulated kinase 3 (28)
Although GLP-1 is reported to activate Akt, which in turn phosphorylates/inactivates GSK-3β, findings of the present study showed an opposite result. AG has activated GSK-3β instead, to consequently reduce gastric acidity. Notably, GSK-3β phosphorylation at S9 residue was reported to enhance histamine release to increase acid secretion by binding to the H2 receptor (29) (30) p- CREB, possibly by inhibiting TNF-α and H2 O2 formation, as documented in the present work. Both mediators are reported to activate p38 MAPK (31) (32) p- CREB has been reported to play a role in the transcription of the α2 subunit of the proton pump (33) p -CREB protein expression, can explain the current downregulation of the H+ /K+ -ATPase pump to afford an explanation of the elevated gastric juice pH value. The aforementioned molecular alterations induced by the NSAID point to additional new mechanisms of indomethacin ulcerogenic activity, beside its known COX-2 inhibition.
Apart from its antisecretory effect, AG increased gastric mucosal proliferation by activating the GLP-1/β-catenin/cyclin-D1 cue to verify its gastric healing capacity. In the present work, AG decreased the phosphorylation of β-catenin at the S45 to prevent its ubiquitination and to permit its nuclear translocation. As a transcription factor, salvaged β-catenin enhances the formation of cyclin-D1, a Wnt target gene (34) (30) (34,35) (36)
In the present work, AG succeeded to inactivate NF-κB p65 to signify its anti-inflammatory and antiapoptotic properties that play a role in enhancing the healing of gastric ulcer. The indomethacin-induced activation of NF-κB p65 is again linked to the previously mentioned protein, PP-2A, as well as to increased TNF-α and H2 O2 (30,37,38) (30,39) (40,41) 2 O2 . On the contrary, in the present work AG had curtailed the excessive production of H2 O2 and TNF-α in the indomethacin model, which in turn inactivate NF-κB p65, nailing down its anti-inflammatory effect. In addition, these markers entail their effect to initiate apoptosis in the indomethacin untreated rats, where NF-κB/TNF-α/H2 O2 cycle ends up with the activation of caspase-3, as reported here and hitherto (41)
In addition, the current activation of NF-κB upregulated the gene expression of the tight junction protein claudin-1 through TNF-α activation (42) (43) proliferation process triggered by the GLP-1/β-catenin/cyclin D1 cue, seen with AG post-treatment to verify its gastric healing capacity.
In the existing work, DEXA was able to oppose AG healing ability to reach that of indomethacin alone on most of the examined markers. Corticosteroids are known to act mainly through inhibiting phospholipase A2 to curtail the production of PGE2 (44) p- GSK-3β and p- CREB, while decreasing p -Akt and GLP-1. Unexpectedly, in the present model, DEXA also has canceled the AG anti-inflammatory/-apoptotic and its proliferative effects, where NF-κB/TNF-α/H2 O2 , caspase3, and claudin-1 were elevated once again, whereas cyclin-D1 was abated. Indeed, previous studies using different experimental models, showed that DEXA reduced GLP-1 (45) p S473-Akt (46) acid secretion (47) (48) 2 O2 (49) (46)
In conclusion, AG proved its healing efficacy against indomethacin-induced gastric ulceration via manipulating several intersecting pathways; viz, NF-κB/TNF-α/H2 O2 /claudin-1, GLP-1/β-catenin/cyclin-D1, and p- CREB- p -Akt/ H+ -K+ -ATPase pump and p -GSK-3β–GLP-1/pH. These mechanisms were reverted, when DEXA was coadministered with AG, results that shed some light on the involvement of other mechanisms in the ulcerogenic effects of both indomethacin and DEXA.
Acknowledgments
The authors would like to thank Prof Dr. Kawkab Ahmed (Pathology Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt) for her valuable assistance in the histopathological examination.
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