Serum amino acids profile and the beneficial effects of L-arginine or L-glutamine supplementation in dextran sulfate sodium colitis

Abstract

This study was conducted to investigate serum amino acids profile in dextran sulfate sodium (DSS)-induced colitis, and impacts of graded dose of arginine or glutamine supplementation on the colitis. Using DSS-induced colitis model, which is similar to human ulcerative colitis, we determined serum profile of amino acids at day 3, 7, 10 and 12 (5 days post DSS treatment). Meanwhile, effects of graded dose of arginine (0.4%, 0.8%, and 1.5%) or glutamine (0.5%, 1.0% and 2.0%) supplementation on clinical parameters, serum amino acids, colonic tight junction proteins, colonic anti-oxidative indicators [catalase, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px)], colonic pro-inflammatory cytokines [interleukin-1 beta (IL-1β), IL-6, IL-17 and tumor necrosis factor alpha (TNF-α)] in DSS-induced colitis were fully analyzed at day 7 and 12. Additionally, the activation of signal transduction pathways, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt), and myosin light chain kinase (MLCK)-myosin light chain (MLC20), were analyzed using immunoblotting. Serum amino acids analysis showed that DSS treatment changed the serum contents of amino acids, such as Trp, Glu, and Gln (P<0.05). Dietary arginine or glutamine supplementation had significant (P<0.05) influence on the clinical and biochemical parameters (T-SOD, IL-17 and TNF-α) in colitis model. These results were associated with colonic NF-κB, PI3K-Akt and MLCK signaling pathways. In conclusion, arginine or glutamine could be a potential therapy for intestinal inflammatory diseases.

Figure 1. Arginine or glutamine supplementation affects the clinic parameters in dextran sulfate sodium colitis.

A: Body weight was calculated in each group. B: Colon weight was measured in control and DS groups at day 7, 10 and 12. C: Colon weight was measured in each group at day 7. D: Colon weight was measured in each group at day 12. E: Colon length was collected in each group at day 7. F: Colon length was collected in each group at day 12. G: Disease activity score was calculated in each group at day 7. H: Disease activity score was calculated in each group at day 12. I: Colon length was measured in control and DS groups at day 7, 10 and 12. J: Colonic morphological analyses with Hematoxylin-Eosin staining in each group at day 7. K: Colonic morphological analyses with Hematoxylin-Eosin staining in each group at day 12. Mice were treated with normal drinking (control), or distilled water containing 5% (wt/vol) dextran sulfate sodium (DS), or dextran sulfate sodium water with different dose of arginine supplementation in diet (DS+ Arg), or dextran sulfate sodium water with different dose of glutamine supplementation in diet (DS+ Gln). Data are presented as mean ± SEM, n = 6, with a-c used to indicate a statistically significant difference (P<0.05, one way ANOVA method).

Figure 2. Antioxidant enzymes activities in each group.

A: SOD activity in each group. B: Catalase activity in each group. C: GSH-Px activity in each group. Mice were treated with normal drinking (control), or distilled water containing 5% (wt/vol) dextran sulfate sodium (DS), or dextran sulfate sodium water with dietary 0.4% arginine supplementation (DSS+0.4% Arg), or dextran sulfate sodium water with dietary 0.5% glutamine supplementation (DS+ 0.5% Gln). Data are presented as mean ± SEM, n = 6, with a-c used to indicate a statistically significant difference (P<0.05, one way ANOVA method). T-SOD: total superoxide dismutase, GSH-Px: glutathione peroxidase.

Figure 3. Activation of p38 in colon.

A: Immunoblotting of total and phosphorylated p38 at day 7. B: The ratio of phosphorylated p38 to total p38 from data shown in A. C: Immunoblotting of total and phosphorylated p38 at day 12. D: The ratio of phosphorylated p38 to total p38 from data shown in C. Mice were treated with normal drinking (control), or distilled water containing dextran sulfate sodium (DS), or dextran sulfate sodium water with dietary 0.4% arginine supplementation (DSS+0.4% Arg), or dextran sulfate sodium water with dietary 1.5% arginine supplementation (DSS+1.5% Arg), or dextran sulfate sodium water with dietary 0.5% glutamine supplementation (DS+ 0.5% Gln), or dextran sulfate sodium water with dietary 2.0% glutamine supplementation (DS+ 2.0% Gln). Data are presented as mean ±SEM, n = 5.

Figure 4. Activation of JNK in colon.

A: Immunoblotting of total and phosphorylated JNK at day 7. B: Immunoblotting of total and phosphorylated JNK at day 12. C: The ratio of phosphorylated JNK to total JNK from data shown in A. D: The ratio of phosphorylated JNK to total JNK from data shown in B. Mice were treated with normal drinking (control), or distilled water containing dextran sulfate sodium (DS), or dextran sulfate sodium water with dietary 0.4% arginine supplementation (DSS+0.4% Arg), or dextran sulfate sodium water with dietary 1.5% arginine supplementation (DSS+1.5% Arg), or dextran sulfate sodium water with dietary 0.5% glutamine supplementation (DS+ 0.5% Gln), or dextran sulfate sodium water with dietary 2.0% glutamine supplementation (DS+ 2.0% Gln). Data are presented as mean ±SEM, n = 5. JNK: c-Jun N-terminal kinase.

Figure 5. Abundance of p65 in the colon.

A: Immunoblotting of colonic nuclear p65 at day 7. B: Immunoblotting of colonic nuclear p65 at day 12. C: Quantification of relative colonic nuclear p65 abundance from data shown in A. D: Quantification of relative colonic nuclear p65 abundance from data shown in B. Mice were treated with normal drinking (control), or distilled water containing dextran sulfate sodium (DS), or dextran sulfate sodium water with dietary 0.4% arginine supplementation (DSS+0.4% Arg), or dextran sulfate sodium water with dietary 1.5% arginine supplementation (DSS+1.5% Arg), or dextran sulfate sodium water with dietary 0.5% glutamine supplementation (DS+ 0.5% Gln), or dextran sulfate sodium water with dietary 2.0% glutamine supplementation (DS+ 2.0% Gln). Data are presented as mean ±SEM, n = 5, with a-b used to indicate a statistically significant difference (P<0.05, one way ANOVA method). PCNA: proliferating cell nuclear antigen.

Figure 6. Activation of colonic PI3K-Akt pathway.

A: Immunoblotting of colonic PI3K and p-Akt at day 7. B: Quantification of relative PI3K abundance from data shown in A. C: Quantification of relative P-Akt abundance from data shown in A. D: Immunoblotting of colonic PI3K and p-Akt at day 12. E: Quantification of relative PI3K abundance from data shown in D. F: Quantification of relative P-Akt abundance from data shown in E. Mice were treated with normal drinking (control), or distilled water containing dextran sulfate sodium (DS), or dextran sulfate sodium water with dietary 0.4% arginine supplementation (DSS+0.4% Arg), or dextran sulfate sodium water with dietary 1.5% arginine supplementation (DSS+1.5% Arg), or dextran sulfate sodium water with dietary 0.5% glutamine supplementation (DS+ 0.5% Gln), or dextran sulfate sodium water with dietary 2.0% glutamine supplementation (DS+ 2.0% Gln). Data are presented as mean ±SEM, n = 5, with a-b used to indicate a statistically significant difference (P<0.05, one way ANOVA method). PI3K: Phosphatidylinositide 3-kinases, Akt: protein kinases B.

Figure 7. Activation of colonic MLCK-MLC20 pathway.

A: Immunoblotting of colonic MLCK-MLC20 at day 7. B: Quantification of relative MLCK abundance from data shown in A. C: Quantification of relative MLC20 abundance from data shown in A. D: Immunoblotting of colonic MLCK and MLC20 at day 12. E: Quantification of relative MLCK abundance from data shown in D. F: Quantification of relative MLC20 abundance from data shown in E. Mice were treated with normal drinking (control), or distilled water containing dextran sulfate sodium (DS), or dextran sulfate sodium water with dietary 0.4% arginine supplementation (DSS+0.4% Arg), or dextran sulfate sodium water with dietary 1.5% arginine supplementation (DSS+1.5% Arg), or dextran sulfate sodium water with dietary 0.5% glutamine supplementation (DS+ 0.5% Gln), or dextran sulfate sodium water with dietary 2.0% glutamine supplementation (DS+ 2.0% Gln). Data are presented as mean ±SEM, n = 5, with a-c used to indicate a statistically significant difference (P<0.05, one way ANOVA method). MLCK: Myosin light-chain kinase. MLC20: Myosin light chain 20.

Figure 8. Tight junctions expression in the colon.

Claudin1, occludin, and ZO-1 were analyzed with immunohistochemistry analyses in DSS-induced colitis model at day 7 and 12. Mice were treated with normal drinking (control), or distilled water containing dextran sulfate sodium (DS), or dextran sulfate sodium water with dietary arginine supplementation (Arginine), or dextran sulfate sodium water with dietary glutamine supplementation (Glutamine). The dosage of arginine supplementation at day 7 is 1.5%, while it is 0.4% at day 12. The dosage of glutamine supplementation at day 7 is 2.0%, while it is 0.5% at day 12. ZO-1: zonula occluden-1.