A New Murine Undernutrition Model Based on Complementary Feeding of Undernourished Children Causes Damage to the Morphofunctional Intestinal Epithelium Barrier.

BACKGROUND: Complementary feeding is critical in establishing undernutrition. However, experimental undernourished diets do not represent the amount of nutrients in the complementary diets of undernourished children. OBJECTIVES: To develop, validate, and evaluate the impact of a new murine model of undernutrition on the intestinal epithelium, based on the complementary diet of undernourished children from 7 countries with low-socioeconomic power belonging to the Malnutrition-Enteric Diseases (MAL-ED) cohort study. METHODS: We used the difference in the percentage of energy, macronutrients, fiber and zinc in the complementary diet of children without undernutrition compared with stunting (height-for-age Z-score < -2) for the MAL-ED diet formulation. Subsequently, C57BL/6 mice were fed a control diet (AIN-93M diet) or MAL-ED diet for 28 d. Weight was measured daily; body composition was measured every 7 d; lactulose:mannitol ratio (LM) and morphometry were evaluated on days 7 and 28; the cotransport test and analysis of intestinal transporters and tight junctions were performed on day 7. RESULTS: The MAL-ED diet presented -8.03% energy, -37.46% protein, -24.20% lipid, -10.83% zinc, +5.93% carbohydrate, and +45.17% fiber compared with the control diet. This diet rapidly reduced weight gain and compromised body growth and energy reserves during the chronic period (P < 0.05). In the intestinal epithelial barrier, this diet caused an increase in the LM (P < 0.001) and reduced (P < 0.001) the villous area associated with an increase in FAT/CD36 in the acute period and increased (P < 0.001) mannitol excretion in the chronic period. CONCLUSIONS: The MAL-ED diet induced undernutrition in mice, resulting in acute damage to the integrity of the intestinal epithelial barrier and a subsequent increase in the intestinal area during the chronic period. This study introduces the first murine model of undernutrition for the complementary feeding phase, based on data from undernourished children in 7 different countries.

Retinoids delay cell cycle progression and promote differentiation of intestinal epithelial cells exposed to nutrient deprivation.

OBJECTIVE: Vitamin A is commonly recommended as a treatment for diarrhea and undernutrition; however, little is known about the underlying cellular mechanisms. The aim of this study was to investigate the modulation of cell cycle by vitamin A derivatives (retinyl palmitate or retinol) in undernourished intestinal epithelial crypts (IEC-6). METHODS: IEC-6 cells were exposed to nutrient deprivation (no serum and no glutamine) and supplemented with retinyl palmitate or retinol at a range of 2 to 20 µM. Proliferation, apoptosis/necrosis, cell cycle process, and gene transcription were assessed. RESULTS: Nutrient deprivation for 6, 12, 24, or 48 h decreased cell proliferation, and retinyl palmitate further decreased it after 24 and 48 h. Apoptosis rates were reduced by undernourishment and further reduced by retinyl palmitate after 48 h; whereas necrosis rates were unaltered. Undernourishment induced overall cell quiescence, increased percentage of cells in G0/G1 phase and decreased percentage of cells in S phase after 12 h and in G2/M phases at 6, 12, and 24 h after treatment. Both retinoids also showed cell quiescence induction with less cells in G2/M phases after 48 h, whereas only retinol showed significant modulation of G0/G1 and S phases. Both retinoids also increased markers of cell differentiation Fabp and Iap gene transcriptions in about fivefold rates after 42 h. Furthermore, specific gene transcriptions related to MAP kinase signaling pathway regulation of cell differentiation and cell cycle regulation were triggered by retinoids in undernourished IEC-6, with higher levels of expression for Atf2 and C-jun genes. CONCLUSIONS: These findings indicated that both vitamin A derivatives induce further survival mechanisms in undernourished intestinal epithelial crypt cells. These mechanisms include increased cell quiescence, decreased apoptosis, increased cell differentiation, and transcription of genes related to MAP kinase signaling pathway.

Intervention and Mechanisms of Alanyl-glutamine for Inflammation, Nutrition, and Enteropathy: A Randomized Controlled Trial.

OBJECTIVE: Determine the minimum dosage of alanyl-glutamine (Ala-Gln) required to improve gut integrity and growth in children at risk of environmental enteropathy (EE). METHODS: This was a double-blinded randomized placebo-controlled dose-response trial. We enrolled 140 children residing in a low-income community in Fortaleza, Brazil. Participants were 2 to 60 months old and had weight-for-age (WAZ), height-for-age (HAZ), or weight-for-height (WHZ) z-scores less than -1. We randomized children to 10 days of nutritional supplementation: Ala-Gln at 3 g/day, Ala-Gln at 6 g/day, Ala-Gln at 12 g/day, or an isonitrogenous dose of glycine (Gly) placebo at 12.5 g/day. Our primary outcome was urinary lactulose-mannitol excretion testing. Secondary outcomes were anthropometry, fecal markers of inflammation, urine metabolic profiles, and malabsorption (spot fecal energy). RESULTS: Of 140 children, 103 completed 120 days of follow-up (24% dropout). In the group receiving the highest dose of Ala-Gln, we detected a modest improvement in urinary lactulose excretion from 0.19% on day 1 to 0.17% on day 10 (P = 0.05). We observed significant but transient improvements in WHZ at day 10 in 2 Ala-Gln groups, and in WHZ and WAZ in all Ala-Gln groups at day 30. We detected no effects on fecal inflammatory markers, diarrheal morbidity, or urine metabolic profiles; but did observe modest reductions in fecal energy and fecal lactoferrin in participants receiving Ala-Gln. CONCLUSIONS: Intermediate dose Ala-Gln promotes short-term improvement in gut integrity and ponderal growth in children at risk of EE. Lower doses produced improvements in ponderal growth in the absence of enhanced gut integrity.

Alanyl-glutamine Protects Against Damage Induced by Enteroaggregative Escherichia coli Strains in Intestinal Cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important pathogen causing enteric infections worldwide. This pathotype is linked to malnutrition in children from developing countries. Alanyl-glutamine (Ala-Gln) is an immune modulator nutrient that acts during intestinal damage and/or inflammation. This study investigated the effect of EAEC infection and Ala-Gln on cell viability, cell death, and inflammation of intestinal epithelium cells (IEC-6). METHODS: Cells were infected with an EAEC prototype 042 strain, an EAEC wild-type strain isolated from a Brazilian malnourished child, and a commensal E coli HS. Gene transcription and protein levels of caspases-3, -8, and -9 and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL1) were evaluated using RT-qPCR, western blot analysis, and ELISA. RESULTS: Infections with both EAEC strains decreased cell viability and induced apoptosis and necrosis after 24 hours. Ala-Gln supplementation increased cell proliferation and reduced cell death in infected cells. Likewise, EAEC strain 042 significantly increased the transcript levels of caspases-3, -8, and -9 when compared to the control group, and Ala-Gln treatment reversed this effect. Furthermore, EAEC induced CXCL1 protein levels, which were also reduced by Ala-Gln supplementation. CONCLUSION: These findings suggest that EAEC infection promotes apoptosis, necrosis, and intestinal inflammation with involvement of caspases. Supplementation of Ala-Gln inhibits cell death, increases cell proliferation, attenuates mediators associated with cell death, and inflammatory pathways in infected cells.

Inhibitory effects of a standardized extract of Justicia pectoralis in an experimental rat model of airway hyper-responsiveness.

OBJECTIVE: Justicia pectoralis is a plant useful for the treatment of respiratory diseases. Here, we studied the antiasthmatic properties of a standardized extract of J. pectoralis (Jp). METHODS: Ovalbumin (OVA)-sensitized rats were actively challenged with saline or OVA to study airway hyper-responsiveness after oral treatment with saline or Jp. The ability of Jp to inhibit hyper-reactivity was evaluated in isolated trachea mounted in isolated organ bath chamber. KEY FINDINGS: Using KCl or carbachol as contractile agents, tracheal rings of OVA-challenged rats contracted with higher magnitude than trachea of rats challenged with saline. Such hyper-responsive phenotype of OVA-challenged tissues decreased with Jp administration. In Ca+ -free medium, Jp or its major constituent coumarin inhibited preferentially the contractions induced by Ca2+ addition in tissues of OVA-challenged rats stimulated with KCl or acetylcholine. In tissues depleted of their internal Ca+ stores in the presence of thapsigargin, Jp inhibited the contraction induced by capacitative Ca2+ entry. By gavage, Jp abolished the increase caused by challenge with OVA on the levels of IL-1ß and TNF-α in the bronchoalveolar fluid and also impaired the changes in gene expression of canonical transient receptor proteins. CONCLUSIONS: Jp has antiasthmatic properties in an experimental model that reproduces tracheal hyper-reactivity.

Amyrins from Protium heptaphyllum Reduce High-Fat Diet-Induced Obesity in Mice via Modulation of Enzymatic, Hormonal And Inflammatory Responses.

Obesity remains a global problem. In search of phytochemicals that have antiobesity potential, this study evaluated α,ß-amyrin, a triterpenoid mixture from Protium heptaphyllum, on high-fat diet-induced obesity in mice. Groups of mice (n = 8) were fed a normal diet or a high-fat diet, and were orally treated or not treated with either α,ß-amyrin (10 or 20 mg/kg) or sibutramine (10 mg/kg) for 15 weeks. Variables measured at termination were body weight, visceral fat accumulation, adipocyte surface area, peroxisome proliferator-activated receptor gamma, and lipoprotein lipase expressions in adipose tissue, the levels of plasma glucose and insulin, the satiety hormones ghrelin and leptin, the digestive enzymes amylase and lipase, and the inflammatory mediators TNF-α, interleukin-6, and MCP-1. Results showed that α,ß-amyrin treatment resulted in lower high-fat diet-induced increases in body weight, visceral fat content, adipocyte surface area, peroxisome proliferator-activated receptor gamma, and lipoprotein lipase expressions, and blood glucose and insulin levels. Additionally, the markedly elevated leptin and decreased ghrelin levels seen in the high-fat diet-fed control mice were significantly modulated by α,ß-amyrin treatment. Furthermore, α,ß-amyrin decreased serum TNF-α and MCP-1. These results suggest that α,ß-amyrin could be beneficial in reducing high-fat diet-induced obesity and associated disorders via modulation of enzymatic, hormonal, and inflammatory responses.

Antinociceptive Effect of the Essential Oil Obtained from the Leaves of Croton cordiifolius Baill. (Euphorbiaceae) in Mice.

Croton cordiifolius Baill. is a shrub known as "quebra-faca" and is used to treat inflammation, pain, wounds, and gastrointestinal disturbances in the semiarid region in the northeast of Brazil. In an ethnobotanical survey in the state of Pernambuco, "quebra-faca" use was cited in 33% of the interviews. Thus, we decided to evaluate the antinociceptive effects of the essential oil from C. cordiifolius (CcEO). Chemical analysis by gas chromatography-mass spectrometry revealed 1,8-cineole (25.09%) and α-phellandrene (15.43%) as major constituents. Antinociceptive activity was evaluated using murine models of chemically induced pain (writhing induced by acetic acid, formalin, capsaicin, and glutamate tests). Opioid and central nervous systems (CNS) involvement were also investigated. Regarding antinociceptive activity, CcEO (50 and 100 mg/kg) reduced the number of writhing responses induced by acetic acid and decreased the licking times in both phases of the formalin test. CcEO also was evaluated in capsaicin- and glutamate-induced nociception. While no effect was observed in the capsaicin test, CcEO (100 mg/kg) was effective in the glutamate test. Naloxone, an opioid antagonist, did not affect the antinociceptive activity of CcEO in writhing test. In conclusion, the antinociceptive effect of CcEO could be explained, at least in part, by inhibition of the glutamatergic system.

The Resin from Protium heptaphyllum Prevents High-Fat Diet-Induced Obesity in Mice: Scientific Evidence and Potential Mechanisms.

Herbal compounds rich in triterpenes are well known to regulate glucose and lipid metabolism and to have beneficial effects on metabolic disorders. The present study investigated the antiobesity properties of resin from Protium heptaphyllum (RPH) and the possible mechanisms in mice fed a high-fat diet (HFD) for 15 weeks. Mice treated with RPH showed decreases in body weight, net energy intake, abdominal fat accumulation, plasma glucose, amylase, lipase, triglycerides, and total cholesterol relative to their respective controls, which were RPH unfed. Additionally, RPH treatment, while significantly elevating the plasma level of ghrelin hormone, decreased the levels of insulin, leptin, and resistin. Besides, HFD-induced increases in plasma levels of proinflammatory mediators TNF-α, IL-6, and MCP-1 were significantly lowered by RPH. Furthermore, in vitro studies revealed that RPH could significantly inhibit the lipid accumulation in 3T3-L1 adipocytes (measured by Oil-Red O staining) at concentrations up to 50 µg/mL. These findings suggest that the antiobese potential of RPH is largely due to its modulatory effects on various hormonal and enzymatic secretions related to fat and carbohydrate metabolism and to the regulation of obesity-associated inflammation.

CB1 and CB2 contribute to antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint.

Electroacupuncture (EA) and cannabinoids have been reported to have anti-inflammatory and antinociceptive effects in animal models of arthritis. Male Wistar rats were injected with saline or zymosan (2 mg) into the temporomandibular joint (TMJ). EA (10 Hz, 30 min) was performed 2 h after or 1 h before zymosan administration. AM251 or AM630 (3 mg/kg, i.p.)were administered before EA treatment. Mechanical hypernociception was accessed after zymosan administration. Rats were sacrificed 6 h after zymosan administration and the joint was removed for histopathological analysis. The gene expression of CB1 and CB2 receptors was assessed after sacrifice of the TMJ arthritic animals. EA inhibited zymosan-induced hypernociception (p < 0.05). AM251 reversed significantly the antinociceptive effect of EA, suggesting that the CB1 receptor is involved in this effect. AM630 reversed the anti-inflammatory effect of EA. CB1 and CB2 receptor gene expression was upregulated 6 h after zymosan-induced arthritis in the EA-treated group. We observed downregulation of CB2 receptor gene expression in the EA group at the 24th hour compared with the 6th hour. Higher CB1 receptor gene expression was also found compared with the 6th hour. EA produced antinociceptive and anti-inflammatory effects, and these effects appeared to be mediated through CB1 and CB2 receptor activation.