Homocysteine-mediated intestinal epithelial barrier dysfunction in the rat model of irritable bowel syndrome caused by maternal separation

Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is characterized by altered bowel evacuation, bloating, and visceral pain in the absence of anatomical or biochemical abnormalities [1]. Up to date, the etiologies of these symptoms are still not fully understood. The maternal separation (MS) model, rodent pups separated from the dam (3 h/day) during the neonatal period, has been found to display greater anxiety-like behavior, visceral hypersensitivity, increased fecal output, and higher levels of proinflammatory cytokines in adulthood [2], which may constitute a valuable experimental model to investigate the pathophysiology of IBS and to identify novel pharmacological targets. The rat model of IBS caused by MS was established in this study. All of the investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Homocysteine (Hcy), a cytotoxic sulfur-containing amino acid, is an intermediary metabolite in methionine/cysteine metabolism. The elevated plasma level of Hcy is termed as hyperhomocysteinemia (HHcy). And the regulation of Hcy is critically important for health maintenance since several diseases are related to HHcy [3,4]. Augmentations in Hcy are due to enzyme abnormalities in metabolic pathways or to nutritional deficits, including folate and Vitamin B6 and B12 deficiencies [5]. The gastrointestinal tract is a significant site of sulfur amino acid metabolism, which accounts for 25% of whole body transmethylation and transsulfuration pathways and is a prominent site of net Hcy release [6]. The demonstration of a relationship among Hcy, inflammation, and autoimmunity intriguingly expands the spectrum of the possible pathogenetic implications for HHcy. However, there is a paucity of studies investigating its role in the context of IBS. In our study, we found that plasma Hcy levels in IBS rats were significantly greater than that of control (7.26+1.84 vs. 3.6+1.0 mmol/l, P , 0.01, Fig. 1A), which was increased 1-fold. After administration of folate, Vitamins B6 and B12, Hcy levels were remarkably decreased (5.02+ 1.48 mmol/l, P , 0.05), which reduced 30% of the IBS. To our knowledge, this was the first report showing that MS stress could lead to Hcy accumulation in the circulation, and there was a significant association between Hcy levels and the severity of IBS symptoms including visceral hyperalgesia and stress-induced increase in colonic motility. Thus, Hcy may be implicated in the onset and progression of the tissue injury underlying IBS. Recently, researchers have found that a low-grade colonic inflammation may play a role in the pathogenesis of IBS [7]. Disruption of the intestinal epithelial barrier is a feature of gut inflammation in humans and is a pathogenic factor in IBS [8,9]. Previous studies have shown that MS predisposed adult rats to colonic barrier dysfunction in response to mild stress as a stress factor owing to the exaggerated expression of cytokines [10]. Our results showed that plasma TNF-a levels in IBS rats were significantly higher than those in control (443.6+8.18 vs. 383.0+11.13 pg/ml, P , 0.01, Fig. 1B). After treatment with Hcy-lowering reagent, the level of plasma TNF-a was markedly declined (411.0+7.8 pg/ml, P , 0.05), but still higher than that in the control. These data indicated that Hcy probably acted as a proinflammatory molecule triggering inflammatory effects in IBS course. Furthermore, intestinal permeability was assessed using a sugar probes test including sucralose, lactulose, and mannitol by HPLC-tandem mass spectrometry analysis in this study. Lactulose and sucralose permeate the intestinal epithelial barrier through paracellular junctions, and mannitol is a marker of transcellular permeation. There is no difference for urine mannitol excretion in 0–5 h collection following administration of sugar probes among three groups (control: 1601+48.85 mg; IBS: 1598+122.3 mg; IBS þ Vit B: 1541+155.3 mg, Fig. 1C), while the ratio of lactulose to mannitol in 0–5 h collection was significantly increased by 47% in IBS rats compared with that in control (0.22+0.07 vs. 0.15+0.03, P , 0.01, Fig. 1D). After Hcy-lowering treatment, the ratio of lactulose to mannitol was markedly declined (0.17+0.03, P , 0.05). These results suggested that paracellular permeability of small intestine was increased based on urine mannitol and lactulose excretion analysis. Meanwhile, urine sucralose excretion in 6–24 h collection Acta Biochim Biophys Sin 2014, 46: 917–919 | a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu076. Advance Access Publication 3 September 2014