MAPK/ERK Signaling Pathway Regulates Nrf2 And HIF-1α To Promote IL-17D Expression In Intestinal Epithelial Cells Under Hyperoxia

Objective:Neonatal respiratory failure is a life-threatening condition for which hyperoxia therapy is a common life-saving treatment.However,hyperoxia therapy can also cause non-negligible damage to the neonatal intestine.Prolonged exposure to hyperoxia induces oxidative stress and inflammation in the intestine and impairs intestinal barrier function,thereby increasing the risk of acute gastrointestinal disease in neonates and chronic enteropathy in adulthood.Currently,there is a lack of research on the mechanisms of hyperoxia damage to the intestine,which makes the prevention and treatment of intestinal injury clinically difficult.Therefore,this study aims to investigate the molecular mechanism of intestinal injury in neonates caused by hyperoxia and to find effective targets for protecting the intestinal barrier,so as to provide a theoretical basis for the prevention and treatment of intestinal injury in neonates treated with hyperoxia.The main cause of intestinal damage under hyperoxia is excessive production of reactive oxygen species（ROS）.Excessive ROS stimulate the body to initiate self-protective programs such as activation of nuclear factor erythroid factor 2-related factor2（Nrf2）,a widespread transcription factor that is sensitive to cellular recognition of changes in oxidative stress,which neutralizes ROS to maintain redox homeostasis and plays an important role in the regulation of cellular redox responses.It was shown that Nrf2 induced the expression of interleukin-17D（IL-17D）in the intestine of neonatal rats under hyperoxia.IL-17D plays a crucial role in reducing colitis by inducing the secretion of interleukin-22（IL-22）from intestinal type 3 natural lymphocytes（ILC3s）,thereby promoting regeneration and barrier function of intestinal epithelial cells.However,it is not clear what role IL-17D plays under hyperoxia.In vitro,hyperoxia promotes the production of hypoxia-inducible factor-1α（HIF-1α）in colonic epithelial cells,and HIF-1αmainly promotes cellular adaptation to the hypoxic environment.Studies have shown that HIF-1αis involved in protecting the intestinal barrier and reducing mucosal damage in inflammatory bowel disease.However,whether HIF-1αprotects the intestinal barrier under hyperoxia remains to be investigated.ROS are important regulators of intracellular signaling pathways and regulate the expression of various cellular proteins,including Nrf2 and HIF-1α.ROS activate the Mitogen-activated protein kinase（MAPK）/Extracellular signal-regulated kinase（ERK）signaling pathway.The MAPK/ERK signaling pathway is an important cellular signaling pathway involved in cell proliferation,differentiation and death.Studies have shown that Nrf2 and HIF-1αare sensitive to redox signaling and readily regulate each other in response to redox stimuli.Mutual regulation of Nrf2 and HIF-1αhas been found in many diseases involving redox imbalance.However,it is not clear whether Nrf2 and HIF-1αare also mutually regulated during ROS-induced intestinal injury.Methodology:1.Cell culture:The intestinal epithelial cell line NCM460 cells were cultured in a cell incubator（5%CO₂,37°C）.After cell passaging,cells were treated differently under hyperoxia（5%CO₂,37°C,80%O₂）or cobaltous chloride（Co Cl₂）induced hypoxia model according to the experimental design.2.Cells were treated with ROS inhibitor N-acetylcysteine（NAC）under hyperoxia and Co Cl₂ induced hypoxia model,and using Western Blot,immunofluorescence,quantitative real-time Fluorescent Reserve Transcription Polymerase Chain Reaction（q RT-PCR）to investigate the effect of hyperoxia on MAPK/ERK signaling pathway and the expression of Nrf2,HIF-1αand IL-17D.3.Cells were treated with MAPK/ERK signaling pathway inhibitors under hyperoxia,and using Western Blot and immunohistochemistry to investigate the effect of MAPK/ERK signaling pathway on the expression of Nrf2,HIF-1αand IL-17D.4.Cells were treated with Nrf2 inhibitor under hyperoxia,and using Western Blot and immunohistochemistry to investigate the effect of Nrf2 on the expression of HIF-1αand IL-17D.5.Cells were treated with HIF-1αinhibitor under hyperoxia,and using Western Blot to investigate the effect of HIF-1αon the expression of Nrf2 and IL-17D.6.Cells were treated with HIF-1αinhibitor under hyperoxia,and using Western Blot and immunofluorescence to investigate the effect of HIF-1αon the expression of tight junction proteins Zo-1 and occludin in intestinal epithelial cell,and using flow cytometry to investigate the effect of HIF-1αon apoptosis of intestinal epithelial cells.7.Cells were treated with different concentrations of IL-17D protein,and using Western Blot to investigate the effect of IL-17D on the expression of TNF-α,IL-1β,IL-10,Zo-1,and Occludin in intestinal epithelial cells,and using immunofluorescence to investigate the effect of IL-17D on the expression of Zo-1 and occludin.Results:1.Effects of hyperoxia on MAPK/ERK signaling pathway and expression of Nrf2,HIF-1α,and IL-17D:（1）Western Blot results showed that MAPK/ERK signaling pathway was activated compared with the normoxia group（P<0.001）;The activation of MAPK/ERK signaling pathway was inhibited in the hyperoxia+NAC group compared with the hyperoxia group（P<0.001）;（2）Western Blot and immunofluorescence results showed that the expression of HIF-1αunder hyperoxia was higher than normoxia（P<0.05）and lower than hypoxia（P<0.001）;Western Blot results showed that the total protein expression of HIF-1αwas increased at the 12th,24th,and 48th hours under hyperoxia compared with the control group（P<0.05）,the expression of HIF-1αat the 48th hour was lower than that at the 24th hour（P<0.05）;The expression of nuclear HIF-1αat the 12th,24th,and 48th hours was increased under hyperoxia（P<0.05）,and the expression of nuclear HIF-1αat the 48th hour was lower than that at the 24th hour（P<0.05）;The expression of cytoplasmic HIF-1αincreased only at the 24th hour（P<0.05）.The q RT-PCR results showed that HIF-1αm RNA expression was increased at the 12th,24th,and 48th hours under hyperoxia compared with the control group（P<0.001）,and HIF-1αm RNA expression at the 48th hour was higher than that at the 24th hour（P<0.001）.（3）Western Blot results showed that the expression of Nrf2,HIF-1α,and IL-17D was elevated in the hyperoxia group compared with the normoxia group（P<0.05 or P<0.001）;The elevated expression of Nrf2,HIF-1α,and IL-17D was inhibited in the hyperoxia+NAC group compared with the hyperoxia group（P<0.05 or P<0.001）.2.Effect of MAPK/ERK signaling pathway on the expression of Nrf2,HIF-1α,and IL-17D:（1）Immunohistochemistry results showed that the expression of p-ERK,Nrf2,HIF-1α,and IL-17D was elevated in the hyperoxia group compared with the normoxia group（P<0.001）;The expression of p-ERK,Nrf2,HIF-1α,and IL-17D was inhibited in the hyperoxia+PD98059 group compared with the hyperoxia group（P<0.001）;（2）Western Blot results showed that the expression of p-ERK,Nrf2,HIF-1α,and IL-17D expression were inhibited in the hyperoxia+PD98059 group compared with the hyperoxia group（P<0.001）;（2）Western Blot results showed that the expression of p-ERK,Nrf2,HIF-1α,and IL-17D were inhibited in the hyperoxia+PD98059 group compared with the hyperoxia group（P<0.05 or P<0.001）.3.Effect of Nrf2 on the expression of HIF-1αand IL-17D:（1）Immunohistochemistry showed that the expression of Nrf2,HIF-1α,and IL-17D was elevated in the hyperoxia group compared with the normoxia group（P<0.001）;The elevated expression of Nrf2,HIF-1α,and IL-17D was inhibited in the hyperoxia+ML385 group compared with the hyperoxia group（P<0.001）.（2）Western Blot results showed that the expression of Nrf2,HIF-1α,and IL-17D were inhibited in the hyperoxia+ML385 group compared with the hyperoxia group（P<0.001）;（3）Western Blot results showed that the expression of Nrf2,HIF-1α,and IL-17D were increased in the hyperoxia+TBHQ group compared with the hyperoxia group（P<0.05or P<0.001）;The upregulation of Nrf2,HIF-1α,and IL-17D expression was inhibited in the hyperoxia+TBHQ+PD98059 group compared with the hyperoxia+TBHQ group（P<0.05）;The expression of HIF-1αand IL-17D was inhibited in the hyperoxia+PD98059+ML385 group compared with the hyperoxia+PD98059 group（P<0.001）.4.Effect of HIF-1αon the expression of Nrf2 and IL-17D:Western Blot results showed that the expression of HIF-1α,Nrf2,and IL-17D were inhibited in the hyperoxia+LW6 group compared with the hyperoxia group（P<0.05 or P<0.001）.5.Effect of HIF-1αon apoptosis and tight junction protein expression of intestinal epithelial cells:（1）Western Blot and immunofluorescence results showed that the expression of Zo-1 and Occludin was significantly reduced in the hyperoxia+LW6group compared with the hyperoxia group（P<0.001）.（2）Flow cytometry results showed that the apoptosis rate was increased in the hyperoxia group compared with the normoxia group（P<0.001）,and was higher in the hyperoxia+LW6 group compared with the hyperoxia group（P<0.001）.6.Effect of IL-17D on inflammation and tight junction protein expression of intestinal epithelial cells:（1）Western Blot results showed that TNF-αand IL-1βexpression increased（P<0.001）and IL-10,Zo-1,and Occludin expression decreased（P<0.05 or P<0.001）in the hyperoxia group compared to the normoxia group;TNF-αand IL-1βexpression decreased with increasing IL-17D concentration in the hyperoxia+IL-17D（50ng/ml,100ng/ml,200ng/ml）group compared to the hyperoxia group（P<0.05 or P<0.001）,and IL-10,Zo-1,and Occludin expression increased with increasing IL-17D concentration（P<0.05 or P<0.001）.（2）Immunofluorescence results showed that the expression of Zo-1 and Occludin decreased in the hyperoxia group compared with the normoxia group,and the expression of Zo-1 and Occludin increased in the hyperoxia+IL-17D group compared with the hyperoxia group.Conclusion:The ROS-activated MAPK/ERK pathway regulates the reciprocal regulation of Nrf2 and HIF-1α.Nrf2 and HIF-1αsignaling is interdependent and promotes IL-17D expression.HIF-1αand IL-17D expression protects the tight junctions of intestinal epithelial cells.