| The clinical application of total parenteral nutrition (TPN) has been developed for half a century. TPN is not only able to provide the nutrients and energy required for the maintenance of normal physiological activities, but also improves nutritional condition and promotes recovery. To date, TPN has been used extensively in the treatment of patients with intestinal failure, especially the nutritional support for short bowel syndrome. However, long-term TPN support will induce intestinal mucosal atrophy and damage and immune dysfunction, and thereby increase the risks of intestinal bacterial translo cation and infection complications, even induce gut-derived sepsis. However, the mechanisms remain unknown. How to effectively avoid or reduce TPN treatment-induced complications is an important issue in the area of surgical nutritional treatment.Glucagon-like peptide-2 (GLP-2) is an intestine-specific growth factor. It enhances intestinal absorption of nutrients, improves intestinal blood supply, promotes the repair of intestinal mucosal damage and maintains the integrity of intestinal mucosal structure. In recent years, increasing attention and studies are being laid on GLP-2. Compared with other polypeptide growth factors, GLP-2 has a potent physiological function or pharmacological action and is tissue-specific (only limited to the intestine). It might be used extensively in the treatment of intestinal barrier dysfunction associated diseases.Teduglutide is a human GLP-2 analog. Phase III clinical trials were conducted in the treatment of short bowel syndrome and inflammatory bowel disease and favorable efficacy was achieved. It was revealed that exogenous supply of GLP-2 analog might reduce the dependence of patients with short bowel syndrome on PN and improve the life quality. The end goal of supportive treatment with intestinal and parenteral nutrition for patients with short bowel syndrome is to live independent of PN and maintain the life via intestinal nutrition even oral feeding. However, long-term TPN treatment might cause the risks of intestinal mucosal barrier dysfunction, even intestinal bacterial translocation and infection complications. A new drug for short bowel syndrome, GLP-2 analog, is able to repair intestinal epithelial cells specifically and maintain the integrity of intestinal mucosal barrier. Therefore, it is of great importance to explore the effects of GLP-2 on TPN-induced intestinal mucosal barrier dysfunction.Long-term TPN support is not only able to induce the dysfunction of intestinal mucosal mechanical barrier, but also might cause the damage of intestinal mucosal immune barrier. It was studied that GLP-2 was able to protect intestinal mucosal immune barrier and might play an important role in the immune response of intestinal mucosa. GLP-2 increased sIgA level in rat bile in a rat model of shock. It was found in GLP-2-treated severe acute pancreatitis mice that CD3+, CD4+and CD8+ lymphocytes and IgAλ expression were increased in mucosa of small intestine, which further indicated that GLP-2 exerted certain protective effects on intestinal mucosal immune barrier. However, studies on the influence of GLP-2 on intestinal mucosal immune barrier in TPN animal model are still not found in literature.Considering that GLP-2 had relatively exact protective effects on the intestinal mucosal epithelial cells and no reports on the protective effects of GLP-2 on TPN-induced intestinal mucosal barrier dysfunction in animal model were found, we designed the animal study to explore the complications derived from TPN-induced intestinal mucosal barrier dysfunction, which was a common issue in clinical practice. Firstly, we established a TPN mouse model by external jugular vein catheterization to observe if GLP-2 was able to prevent TPN-induced intestinal mucosal atrophy. The further study was to observe if GLP-2 was able to protect TPN-induced intestinal mucosal mechanical barrier and to explore related mechanisms. Besides, we preliminarily explored if GLP-2 was able to improve TPN-induced intestinal mucosal immune dysfunction.Part I. Preventive Effects of GLP-2 on Intestinal Atrophy in TPN MiceObjectiveThe study was to explore the effects of GLP-2 on TPN-induced intestinal mucosal atrophy.MethodsHealthy ICR mice (n=24,33.6±1.1), aged 6-8 weeks, were randomized into 3 groups:chow group (with access to food ad libitum), TPN group and TPN+GLP-2 group. The animals were anesthetized by intraperitoneal ketamine injection.2 days after external jugular vein catheterization, the animals in TPN group were given TPN solution, while TPN+GLP-2 group were given equivalent volume of TPN solution plus external jugular vein injection of GLP-2 (30ug twice daily, a total of 60ug/day). The animals were weighed at specific time points each day and sacrificed on day 7. The small intestine was removed and weighed. The terminal ileum tissue was sampled to detect associated proteins and mRNA expression. The intestinal mucosa was observed for morphological changes.ResultsCompared with chow group, significant weight decrease, obvious reduction in small intestinal length, shortened intestinal villi and microvilli and decreased crypt depth were observed in mice in TPN group (all P<0.05). However, all the above parameters in TPN+GLP-2 group were improved (all P<0.05). GLP-2 was able to increase the expression of proliferating cell nuclear antigen (PCNA) protein and mRNA and reduce cleaved caspase-3 expression in the intestine of TPN mice.ConclusionsGLP-2 is able to prevent intestinal mucosal atrophy in TPN mice. The mechanism might be related to stimulating the expression of PCNA protein and mRNA in intestinal tissue and reducing the expression of cleaved caspase-3 protein.Part Ⅱ. Effects of GLP-2 on Intestinal Mechanical Barrier in TPN MiceObjectiveThe study was to explore the effects of GLP-2 on intestinal mechanical barrier in TPN mice and the possible mechanisms.MethodsThirty-six healthy ICR mice were randomized into 3 groups:chow group (with access to food ad libitum), TPN group and TPN+GLP-2 group. The animals were anesthetized by intraperitoneal ketamine injection.2 days after external jugular vein catheterization, the animals in TPN group were given TPN solution, while TPN+GLP-2 group were given equivalent volume of TPN solution plus external jugular vein injection of GLP-2 (30ug twice daily, a total of 60ug/day). The animals were sacrificed on day 7. Venous blood was collected from the eyeball. The terminal ileum tissue was sampled to detect associated proteins and mRNA expression. The intestinal mucosa was observed for morphological changes using transmission electron microscope.ResultsCompared with chow group, tissues IL-6 and TNF-a levels were elevated while IL-10 was reduced in mice in TPN group (all P<0.05). Compared with TPN group, tissues IL-6 and TNF-a levels were reduced while IL-10 was elevated in mice in TPN+GLP-2 group (all P<0.05). The expressions of IL-6. TNF-a and IL-10 mRNA were consistent with serum level changes in TPN and TPN+GLP-2 groups (all P<0.05). Compared with chow group, elevated plasma endotoxin and D-lactic acid levels were observed in TPN group (all P<0.05), however, the changes in TPN+GLP-2 group were not obvious (all P>0.05). Compared with chow group, increased MPO level and Caspase-3 activity were found in TPN group, while the changes in TPN+GLP-2 group were not evident (all P>0.05). Electron microscopy revealed that rare microvilli, microvilli malalignment, lodging, defluxation and damaged tight junction proteins were found in mice in TPN group, while slight aligned microvilli and unobvious damage of tight junction proteins were identified in TPN+GLP-2 group. Immunofluorescence showed that, compared with chow group, less and discontinuous fluorescence was observed for tight junction proteins Occludin, Caudin-1 and ZO-1 in the intestine of mice in TPN group. However, the changes in TPN+GLP-2 were not obvious. Western blot indicated that, compared with chow-group, reduced expression of tight junction proteins Occludin and Caudin-1 were found in the intestine of mice in TPN group (all P<0.05). However, there were no statistically significant differences between TPN+GLP-2 and chow groups (all P>0.05). Reduced PCNA and Bcl-2 protein expression were observed in the intestine of mice in TPN group (all P<0.05), while Bax protein expression was increased (all P<0.05). On the contrary, the differences between TPN+GLP-2 and chow groups were of no statistical significance (all P>0.05).Conclusions1. Dysfunction of intestinal mucosal mechanical barrier might be induced in mice after 5-day TPN support, manifested by increased proinflammatory responses and plasma endotoxin and damage of tight junction proteins.2. GLP-2 is able to improve TPN-induced intestinal mucosal mechanical barrier dysfunction, manifested by relief of pathological intestinal lesion, disruption of tight junction protein, reduced permeability of intestinal mucosa and expression of inflammatory factors.3. The protective effects of GLP-2 on TPN-induced intestinal mucosal mechanical barrier might be realized by promoting the proliferation of intestinal mucosal epithelial cells, inhibit apoptosis and relieve intestinal mucosal inflammatory responses.Part Ⅲ. Effects of GLP-2 on Intestinal Immune barrier in TPN MiceObjectiveThe study was to explore the effects of GLP-2 on intestinal immune barrier in TPN mice.MethodsThirty healthy ICR mice, aged 6-8 weeks, were equally randomized into 3 groups:chow group (with access to food ad libitum), TPN group and TPN+GLP-2 group.2 days after external jugular vein catheterization, the animals in TPN group were given TPN solution, while TPN+GLP-2 group were given equivalent volume of TPN solution plus external jugular vein injection of GLP-2 (30ug twice daily, a total of 60ug/day). The animals were sacrificed on day 7. Portal venous blood was collected to detect bacterial translocation rate. The wash solution of enteric cavity of small intestine was sampled to detect slgA level. The terminal ileum tissue was sampled to detect IL-4, IL-13, associated proteins and mRNA expression. The slgA level of terminal ileum tissue was also determined. Immunofluorescent staining was performed for associated proteins. PAS staining was performed for terminal ileum tissue to observe the change in number of goblet cells in small intestine.ResultsThe number of mouse intestinal goblet cell in the TPN+GLP-2 group were significantly increaded when compared with chow group (P<0.05). ELISA revealed that, compared with chow group, reduced sIgA, IL-4 and IL-13 levels were observed in the intestine and intestinal wash solution in TPN group (all P<0.05). However, compared with TPN group, increased sIgA, IL-4 and IL-13 levels were identified in the intestine and intestinal wash solution in TPN+GLP-2 group (all P<0.05). TPN significantly reduced intestinal sPLA2 and cryptdin-4 levels relative to chow group (all P<0.05). However, compared with TPN group, intestinal sPLA2 and cryptdin-4 levels were improved after supplement of GLP-2 (all P<0.05). Western blot indicated that increased lysozyme and PIgR expression were found in TPN+GLP-2 group compared with TPN group (all P<0.05). qRT-PCR showed that, compared with TPN group, increased IL-4 mRNA, IL-13 mRNA, Lysozyme mRNA, pIgR mRNA, SPLA2 mRNA、Mucin2 mRNA and Cryptdin 4 mRNA expression were found in TPN+GLP-2 group (all P<0.05). however, there was no significant difference between Tff3 mRNA and Reg Ⅲ-γ mRNA expression. The plasma level of arginine and citrulline in the TPN+ GLP group were significantly higher than that in the TPN group, respectively (all P<0.05). Immunofluorescence showed that, compared with TPN group, more and relatively continuous fluorescence was observed for Lysozyme and PIgR protein in TPN+GLP-2 group. In addition, the overall bacterial translocation rate in portal venous blood, liver and spleen was significantly reduced in TPN+GLP-2 group relative to TPN group (all P<0.05).Conclusions1. TPN might induce intestinal mucosal immune barrier dysfunction, including inherent immune and acquired immune dysfunctions.2. GLP-2 might reduce the rate of TPN-induced bacterial translocation, which is possibly related to the improvement of intestinal mucosa.3. GLP-2 might improve TPN-induced intestinal mucosal immune dysfunction, manifested by increased secretion of intestinal sIgA, lysozyme, SPLA2 and Cryptdin 4. The mechanism might be related to the improvement of the arginine, citrulline levels, and intestinal IL-4 and IL-13 levels. |
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