| Appropriate exercise is not only the foundation of health,but also the inherent requirement for promoting national health and reducing the incidence of chronic diseases in the"Outline of Healthy China 2030"and"Healthy China Action(2019-2030)".But unscientific or excessive exercise,beyond their own load,will bring injuries.According to the exercise medical researches,high-intensity exercise,particularly the endurance exercise(running),would induce the occurrence of gastrointestinal syndrome(GIS)which was characterized by diarrhea,gastric pain,vomit,nausea.The GIS would not only reduce the exercise capacity,but also affect the recovery after exercise.Generally,the occurrence of GIS is closely related to the intestinal barrier dysfunction.In recent years,with the development of immunology,we realized that the intestinal regional immune was closely related to the occurrence of some diseases.A variety of cells including intestinal intraepithelial or lamina propria lymphocytes and the cytokines participate in the regulation of intestinal immune homeostasis and the maintenance of intestinal barrier function.However,the effects and mechanisms of high-intensity exercise on the intestinal barrier function,particular the role of various immune cells and cytokines in the intestine,are still unclear.It is important for paying attention to the mechanism and prevention or treatment strategies for the high-intensity exercises induced intestinal immune homeostasis dysregulation.The mechanical barrier,including the intestinal epithelial cells(IECs)and the intercellular junction,such as tight junctions(TJ)and adhesive junctions(AJ),is an important aspect in preventing the occurrence of intestinal endotoxemia.In recent years,the researches of the immunology have found that the intestinal intraepithelial lymphocytes(IELs)and the protective cytokines such as IL-10,KGF and the injurious cytokines such as IFN-γand TNF-α,play a vital role in the maintaining the intestinal mucosal barrier function interacting with IECs.The changes of numbers and function in IELs are related to the occurrence of various diseases.In addition,type 3 innate lymphoid cells(ILC3)and IL-22in the intestinal lamina propria are also important components of intestinal regional immunity.IL-22 could regulate the expression of tight junction protein and maintain the intestinal barrier function by binding to IL-22R on the surface of IECs.In the past,it was believed that excessive high-intensity exercise will temporarily reduce the immune function following the"J"curve and the"opening window"theory,but there were many controversies about the mechanism,especially the role of the important immune cells and cytokines such as IELs,ILC3 and IL-22.In our previous studies,high-intensity exercise would induce the the intestinal barrier dysfunction and the change of the inflammatory factors such as TNF-αand IL-10.Therefore,our aim in this study is to further investigate the immune-related mechanism and prevention strategies by animal experiments of high-intensity exercise.The researches on the effects and mechanism of plant foods which contain many phytochemicals have become a hot issue.Dihydromyricetin(DHM)is a natural polyphenolic compound with many biological effects such as anti-oxidation,anti-tumor,anti-inflammatory,and improvement of glucose and lipid metabolism.Our previous researches showed that DHM can regulate the transformation of skeletal muscle fiber types through the FLCN-FNIP1/AMPK pathway and improve exercise capacity under hypoxic conditions at high altitudes.It can also regulate mitochondrial metabolic dynamics through the PGC-1α-SIRT3-OPA1 pathway to improve skeletal muscle micro-damage.However,it is unclear whether DHM can improve high-intensity exercise induced GIS.Many studies have shown that aryl hydrocarbon receptor(AhR)is a key nuclear receptor and transcription factor of some intestinal immune cells such as IELs and ILC3,involving in the regulation of a variety of cytokines.AhR is also a natural receptor of polyphenolic compound.Our previous study showed that DHM could activate AhR in vitro,and in our opinion,DHM may be the natural ligand of AhR.However,more researches are needed in whether DHM could regulate intestinal region immune function such as the immune cells or cytokines.According to the relevant researches,we propose that"various immune cells colonized in the intestine and cytokines,especially IELs,ILC3 and IL-22 may play a key role in the high intensity exercise induced intestinal barrier dysfunction.DHM may ameliorate intestinal mucosal barrier function and high-intensity exercise-induced GIS through intestinal regional immune pathways".1.Objective:To clarify the role of intestinal regional immune pathways especially various immune cells colonized in the intestine and cytokines such as IELs,ILC3 and IL-22 in the high intensity exercise induced intestinal barrier dysfunction.To determine the improvement of DHM on high-intensity exercise-induced intestinal mucosal barrier dysfunction and the mechanism of intestinal regional immune pathways.2.Methods:(1)An over-training model of male C57BL/6 mice was established in our studies.histological examinations and ultrastructure observation were used to show the change of intestinal villus,IECs and microvillus by HE staining and transmission electron microscope.The molecular biology ways and permeability experiments were used to observe the intestinal barrier dysfunction of mice to determine the establishment of this model.Based on the detection of IL-22 expression in intestinal tissues,we further analyzed the IL-22expression levels of intestinal mucosal lamina propria lymphocytes and CD4+T cells after the high intensity exercise by intracellular cytokine staining technique.Then multi-color flow cytometry ways was used to investigate the proportions of each subgroup on IELs and the ILC3 in intestinal lamina propria.Finally we would clarify the role of intestinal regional immune pathways in the occurrence of high-intensity exercise induced GIS.(2)In addition,a high-intensity exercise model was used.We further elucidated the improvement after two weeks’supplement of DHM(100mg/kg·bw)on the intestinal barrier function by ELISA,HE staining,qRT-PCR and Western blot.We also analyzed the effects of DHM on the number and phenotypes of IELs as well as the migration and activation marker expression of CD8αα~+IEL by multi-color flow cytometry.We further detected the IFN-γand IL-10 expression levels of CD8αα+IEL by intracellular cytokine staining technique.and immune functions in the small intestine by flow cytometry.Detection of the AhR activation marker CYP1A1 and mimicking molecular docking were to clarify the target of DHM.Finally we revealed the effect and the role of intestinal regional immune pathway on DHM improving high-intensity exercise induced intestinal barrier dysfunction.3.Results:(1)High-intensity exercise could significantly induce the intestinal barrier dysfunction and the imbalance of multiple immune cells and cytokine expression in the intestine.The results demonstrated that high-intensity exercise could significantly reduced the expression of IL-22 and increased the expression of IFN-γ、TNF-αin the small intestine tissues.The change of some immune cells in intestinal epithelium or lamina propria such as ILC3(especially NCR~+ILC3)and IELs involved in that.(2)DHM supplement could significantly improve the intestinal barrier dysfunction following high-intensity exercise in a mice model,which was manifested by reducing intestinal mucosal inflammation,permeability and endotoxemia,increasing expression of intestinal epithelial tight junction protein and intestinal mucosal integrity.(3)DHM supplement may improve intestinal barrier dysfunction following the high-intensity exercise through immune-related pathways.The results showed that DHM decreased the number of IELs and the frequency of CD8αα~+IEL following the high-intensity exercise,and regulated the expression of pro-inflammatory factors IFN-γand IL-10,as well as increased the expression of IL-22.These effects may be related to the AhR activation.4.Conclusion:High-intensity exercise induced-GIS is related to the dysregulation of the intestinal regional immune homeostasis,especially the dysregulated frequency of IELs as well as the decreased expression of IL-22 and ILC3 in the intestine.DHM supplement can effectively improve intestinal mucosal barrier dysfunction following high-intensity exercise;And this effect may be mainly related to the increased IL-22 and reduction of IELs cell number(especially CD8αα~+IEL frequency and phenotypic regulation).This study provides scientific evidence for elucidating the immune mechanism and nutrition intervention strategies of high-intensity exercise on intestinal barrier function. |
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