The Effects Of Enteral Nutrition On The Intestinal Mucous Barrier Functions And Intestinal Microbiota In Mice

Despite that parenteral nutrition can effectively provide nutrients for patients with gastrointestinal dysfunction, it often causes high morbidity of infectious complications, leading to sepsis and multiple organ dysfunction, which seriously affect the clinical outcomes of patients.Although catheter related infectious complications are important factors, numerous studies have indicated that the increased infectious complications are associated with intestinal barrier dysfunction induced by parenteral nutrition without intestinal stimulation. Total parenteral nutrition, while there is the non-nutritive substances to stimulate the intestine, would lead to a series of barrier dysfunctions within the lumen: 1. The intestinal epithelial barrier is damaged including the apoptosis of intestinal epithelial cell, absence of tight junctions between cells and impairment of villus; 2. Dysfunction of intestinal immunity:the lumen content of Secretory immunoglobulin decreases and also the intestinal innate immunity is partially damaged; 3. The composition of intestinal flora changes, resulting in abnormal intestinal biological barrier. Recent researches manifests that total parenteral nutrition would also significantly decrease intestinal alkaline phosphatase(IAP) activity.Clinical studies have approved that partial enteral nutrition could improve the outcomes of patients similar to those of early full-energy enteral nutrition but with fewer episodes of gastrointestinal intolerance. Meanwhile, animal experiments demonstrated that enteral nutrition supporting 20-30% of total calories was proved to maintain the intestinal sIgA (acquired immunity), protect the morphology of intestinal villus and crypt, and decrease bacterial translocation. But these results were still controversial in terms of the lowest dose to protect the gut barrier. In addition, the effects of partial enteral nutrition on the innate immunity, IAP or intestinal microbiota are still not reported. Therefore, we investigate the effect of partial EN on intestinal barrier functions, including the innate immunity, IAP and intestinal microbiota. The results potentially contribute to further explore the threshold dose and protective mechanism of partital EN on intestinal barrier.In this experiment, we establish the stable and repeatable mice model with parenteral nutrition support by optimizing the pre-existing model, providing a solid foundation for follow-up study. Through the establishments of different dose parenteral jointing enteral nutrition models and comparisons between different groups in the gut barrier function changes, we observe that by comparing with chow group, the total parenteral significantly impaired intestinal barrier, and intestinal bacteria translocation rate significantly increased, while using 20% dose of enteral nutrition support for 5 days, the mice intestinal bacteria translocation rate significantly decreased and thus there is with no difference comparing with the chow group. And further study between enteral nutrition dose and the intestinal barrier function indicates that the relationship between enteral nutrition dose and dose to the integrity of intestinal barrier function is relevant. This may provide reliable theoretical basis for critically enteral nutrition therapy of intermediate-risk and critically ill patients to reduce infectious complications which canfurther optimize clinical nutrition support in patients and reasonably guide the patient’s clinical nutrition therapy.PART ⅠEstablishment of mice model of total parenteral nutrition support and optimization of surgical skills in operationObjective:To establish a stable and repeatable mice model of total parenteral nutrition support after optimizing the existed models.Methods:Male ICR mice weighing 20-25g (aged 6-8 weeks) were used for continuous intravenous infusion model. The right jugular vein was cannulated with a catheter which was withdrawn through the root of the tail. The catheter was connected with an infusion pump and the 0.9% saline was infused continuously. After 2 days of ad libitum and infusion of 4ml/d saline, the mice were observed in terms of survival rate and capacity of activity. The observation would continue to the seventh day after the cannulation.Results:Among the 82 cases undergoing parenteral nutrition support model operation, the successful puncture rate of 75 cases after the model stabilized was 68/75(90.7%). 66 mice survived 2 days after the cannulation with the model operation rate of 88%(66/75). On the 7th day of the observation of the mice, there were still 66 mice alive, and the model survived rate was 66/66(100%).Conclusion:Skillful microsurgical procedures are the important basis to reduce hemorrhage during the canulation and increase the successful rate. Postoperative maintenance of body temperature was also a key factor to promote the survival rate of mice. This model is repeatable with a comparatively long lifetime, which is suitable for follow-up study. PART ⅡThe effect of total parenteral nutrition on mucosal barrier and intestinal microbiota in mice modelObjective:To study the effect of parenteral nutrition without intestinal stimulation on mucosal barrier and intestinal microbiota in mice model.Methods:On the basis of mice parenteral nutrition support model,24 ICR male mice were assigned to 2 groups. One of them is chow group(n=12) and the other is total parenteral nutrition (TPN group, n=12).4mL isotonic saline was infused through the catheter in the chow group. The mice in TPN group were fasting and totally supported with parenteral nutrition(4.4mL for the first day,7.7mL for the second day, and 11mL/d for the third to fifth day). Survival rates were calculated after 5 days. After the mice were killed, mesenteric lymph nodes(MLN), liver, spleen, and portal venous blood were collected aseptically for bacterial culture. The intestine samples were also collected for subsequent analysis including immunohistochemistry, western blot, periodic acid-Schiff staining and 16s rRNA pyrosequencing analysis.Results:No significant difference was observed between the TPN group and chow in terms of mortality (10/12 vs.12/12, P> 0.05). TPN group had more bacteria-positive MLN (8/10 vs.1/12, P<0.05), and a decreased numbers of goblet cells (33.0±7.8 vs.61.6±6.5,P<0.05). We could also observe that antibacterial peptides decreased significantly in TPN group compared with control group. TPN also induced the decline of lysozyme, IAP and MUC2 in ileum tissues and changed the composition of microbiota (lysozyme:0.0909±0.0289 vs.0.4786±0.0636, P<0.05; IAP:0.1390 ±0.0705 vs.1.0812±0.0732,P<0.001; MUC2:0.0679±0.0437 vs.0.6729±0.1171; P<0.001; Bacteroidetes:16.26%±1.14% vs.0.16%±0.14%, P<0.001; Tenericutes: 0.96%±0.36% vs.0.15%±0.25%, P<0.001)Conclusion:The mice receiving parenteral nutrition support would represent a higher incidence of bacterial translocation. This was associated with the impaired intestinal barrier function, including the innate immunity, IAP and intestinal microbiota.PART IIIThe effect of partial enteral nutrition accompanying supplemental parenteral nutrition on intestinal mucous barrier and intestinal microbiotaObjective:To investigate the effects and mechanism of partial enteral nutrition accompanying supplemental parenteral nutrition on intestinal mucous barrier and intestinal microbiotaMethods:60 male mice(6-8 weeks old) weighing 25-30g were randomized to receive standard chow, total parenteral nutrition,10%,20%,40% or 60% of total caloric EN supplemented with PN (chow, TPN,10%EN+90%PN,20%EN+80%PN,40%EN+ 60%PN,60%EN+40%PN, n=10 for each group).5 days later, the mice were killed.Then, the mesenteric lymph nodes (MLN), liver, spleen and portal venous blood were collected aseptically for bacterial culture. At the meantime, the intestine samples were also collected for subsequent analysis including immunohistochemistry, western blot, periodic acid-Schiff staining and 16s rRNA pyrosequencing analysis.Results:20% EN accompanying supplemental PN results in a significant increase in the concentrations of lysozyme, MUC2 and IAP and the numbers of goblet cells in the gut compared with TPN (number of goblet cells:50.2±4.3 vs.33.0±7.8, P< 0.05; lysozyme:0.4199±0.0486 vs.0.0909±0.0289, P<0.001; MUC2:0.2254±0.0114 vs. 0.0679±0.0437, P<0.001; IAP:1.0268±0.1053 vs.0.1390±0.0705, P<0.001). The percentage of Bacteroidetes and Tenericutes were significantly decreased in 20%EN mice compared with TPN group(Bacteroidetes:0.28%±0.11% vs.16.26%±1.14%, P<0.001; Tenericutes:0.08%±0.13% vs.0.96%±0.36%, P<0.001)). These changes were coincided with restored barrier function in bacterial culture(3/10 vs.8/10, P<0.05). A further increased dosage could effectively increase the therapeutic effect of EN in terms of number of goblet cells (40%EN) and levels of MUC2 (60%) (number of goblet cells:40%EN,59.2±3.94 vs. chow,61.6±6.46,.P>0.05; MUC2:60% EN, 0.6494±0.0194 vs. chow,0.6729±0.1171; P> 0.05).Conclusion:Administration of 20% EN is expected to achieve some benefits, in terms of enhancing the level of IAP, partially protecting the innate immunity and restoring the composition of intestinal microbiota.