The Mechanism Of PHD2/HIF-1α Signal Pathway On Regulating Intestinal Mucosal Barrier Function In Response To Exercise

ObjectiveThe intestinal mucosal barrier is the first line of defense against the invasion of pathogen.Moderate-intensity of exercise relieves the intestinal mucosal barrier dysfunction caused by chronic stress.However,heavy-load exercise disrupts the intestinal mucosal homeostasis,causing bacterial translocations and inflammatory responses.The body’s blood redistributes during exercise.The visceral blood flow is reduced,reducing the oxygen partial pressure,resulting in a hypoxic environment.Hypoxia induced factor（HIF）-1 plays a key role in the transcriptional regulation response induced by changes in oxygen partial pressure.HIF-1 consists of anαsubunit and aβsubunit.Under normoxia,theαsubunit of HIF-1 is mainly degraded by hydroxylation of its proline residue by prolyl hydroxylase（PHD）2.Under hypoxia,PHD2 activity is reduced,so that theαsubunit is stable.HIF-1αenters the nucleus,binding to HIF-1β,and initiating a series of transcriptional regulation such as inflammation,metabolism,and angiogenesis.What has happened in the redistribution of blood and HIF-1αin internal organs during exercise?Whether the PHD2/HIF-1αsignal pathway is involved in the protection of intestinal mucosal barrier by moderate-intensity exercise?Whether the PHD2/HIF-1αsignaling pathway is involved in the intestinal mucosal barrier dysfunction caused by the heavy-load exercise?Based on the above arguments,we hypothesize that:（1）Blood redistribution causes intestinal hypoxia during exercise,resulting in changes in the oxygen partial pressure of intestine,thereby changes HIF-1αexpression and redistribution.（2）The PHD2/HIF-1αsignaling pathway is involved in alleviating intestinal mucosal injury caused by chronic stress during moderate-intensity exercise.（3）By regulating the PHD2/HIF-1αsignaling pathway,it can alleviate the damage of intestinal mucosal barrier caused by heavy-load exercise.This study was to observe the hypoxia of organs and the redistribution of HIF-1αin the abdominal region after exercise.The Cre-LoxP system was used to construct Hif1a or Phd2 intestinal epithelial-specific knockout mice.By using a chronic stress model and different load exercise methods,we explored the regulation mechanism of PHD2/HIF-1αsignaling pathway on intestinal mucosal barrier function.MethodROSA26 ODD-Luc/⁺transgenic mice were used to observe the hypoxia of visceral organs and the redistribution of HIF-1αin the abdominal region by hypoxyprobe and bioluminescence imaging in vivo.A Hif1a or Phd2 intestinal epithelial-specific knockout mouse model was constructed by Vil-Cre,Hif1a^flox/flox and Phd2^flox/floxlox/flox transgenic mice.The effect of intestinal epithelial-specific knock-out of Hif1a or Phd2on mucosal immune-related gene expression was examined by RT² Profiler PCR Array.The mouse intestinal mucosal barrier dysfunction model was established by repeated restraint stress,and Hif1a or Phd2 knockout mice were used to explore the role of PHD2/HIF-1αsignaling pathway by moderate-intensity swimming exercise intervention.To construct two heavy-load exercise models of heavy-intensity weight-bearing forced swimming and long-duration swimming,and to explore the role of PHD2/HIF-1αsignaling pathway in intestinal mucosal barrier dysfunction caused by heavy-load exercise by using Hif1a or Phd2 knockout mice.ResultExercise aggravated the hypoxia in the small intestine,inducing the redistribution of HIF-1αin the small intestine.The phenotype and intestinal morphological structure of intestinal epithelial-specific Hif1a or Phd2 knockout mice were normal compared with the control group.The data of RT² Profiler PCR array showed that intestinal epithelial-specific Hif1a or Phd2 gene knockout mice expressed differently in genes associated with innate immune responses.Compared with Hif1a^flox/flox mice,Hif1a^VKOKO mice showed lower expression of Bpi,Cxcl1,Tlr2,Tnf（P<0.05）,and higher expression of Irak3,Nlpr1a,Tirap（P<0.05）.Phd2^VKO mice decreased in Birc3,Ccl5,Irf7,Jun,Lbp,Nfkbia,Ticam1 and Tirap expression（P<0.05）compared with Phd2^flox/flox mice,and increased in Camp and Ticam2（P<0.05）.Chronic stress lead to the destruction of the morphological structure of intestinal mucosa,higher intestinal barrier permeability,and bacterial translocation.Hif1a^VKO exacerbated this phenomenon,and Phd2^VKOKO mitigated this phenomenon.Moderate-intensity exercise intervention had played a relief role.Heavy-load exercise led to the destruction of intestinal tissue morphology and the permeability of intestinal barrier.Hif1a^VKO exacerbated this phenomenon,and Phd2^VKO mitigated this phenomenon.ConclusionThe small intestine is an important effector of hypoxia and HIF-1αredistribution during exercise.The redistribution of HIF-1αchanged after exercise.Intestinal epithelial cells specifically knock out Hif1a or Phd2 gene has changed in response to the bacteria and innate immunity.The PHD2/HIF-1αsignaling pathway is involved in moderate-intensity exercise to alleviate intestinal barrier dysfunction caused by chronic stress and is also involved in intestinal barrier dysfunction caused by heavy-load exercise.The regulation mechanism of exercise on intestinal mucosal barrier dysfunction and the application of PHD2/HIF-1αsignaling pathway in protecting the intestinal mucosal barrier have been explored by using the intestinal epithelial cells specifically Hif1a or Phd2 gene knock out mice.