Hyperoxia Activates The MAPK Pathway And Nrf2 In Intestinal Epithelial Cells And Induces The Expression Of IL-17D To Increase The Production Of Inflammatory Factors

Objective:providing oxygen for preterm infants is a widely used therapeutic measure in the clinic,but prolonged hyperoxia treatment causes irreversible damage to multiple organs of the body.High concentration of oxygen can damage small intestinal villous architecture,affect intestinal barrier function,and make newborns susceptible to necrotizing enterocolitis.MAPK signaling pathway is widely found in the cytoplasm.Mammalian MAPK signaling pathway mainly include ERK,p38 and JNK.MAPK signaling pathway can be activated by a variety of extracellular stimuli to participate in the pathophysiological processes such as apoptosis,differentiation,proliferation and so on.ASK1 belongs to the MAPKKK family and has a key role in a variety of mechanisms including induction of cell death,differentiation,and production of inflammatory cytokines.However,whether the ASK1/MAPK pathway is involved in the damage caused by hyperoxia on intestinal epithelial cells remains unknown.Under hyperoxia,oxidative stress is the main cause of intestinal epithelial cell damage.Nrf2 is the primary responder of cells to oxidative stress,the Nrf2-keap1pathway is activated after oxidative stress.ROS is significantly increased in intestinal epithelial cells which are exposed to hyperoxia,excessive ROS cause keap1conformational change,and free Nrf2 translocate into the nucleus,where it exerts its effects.IL-17D is expressed in a variety of tissues and cells,and its role in immune responses is complex.IL-17D can not only induce endothelial cells to produce cytokines to indirectly regulate the immune response,but also play an important role in anti-infection as well as antitumor immunity under the regulation of Nrf2.But the expression changes of IL-17D in intestinal epithelial cells in hyperoxia and whether Nrf2regulates IL-17D expression remain unclear.Therefore,the aim of this study was to clarify the mechanism of hyperoxia that damages intestinal epithelial cells by examining the effects of ROS on the ASK1/MAPK pathway in intestinal epithelial cells.To understand the regulation of Nrf2 on IL-17D expression,we examined the expressions of IL-17D,Nrf2 and keap1 in intestinal of neonatal rats in hyperoxia.At the cellular level,we further explored the regulation of Nrf2 on IL-17D and whether the Nrf2/IL-17D axis was involved in intestinal epithelial cell inflammation in hyperoxia by detecting the expressions of IL-17D,Nrf2,keap1,IL-4and IL-6 in intestinal epithelial cells in hyperoxia and the changes of these factors after the addition of Nrf2 activators and inhibitors,which provided the theoretical basis to prevent the side effects during clinical hyperoxia treatment.Methods:1.Cell culture:Caco-2 cells were cultured in a normal cell incubator（5%CO₂,37°C）and continued to be cultured for 24 h after cell passaging,incubated with different concentrations of H₂O₂（100μm,200μm and 400μm）and 85%O₂（5%CO₂,85%O₂,37°C）for 24 h;NCM460 cell were cultured in the normal cell incubation incubator（5%CO₂,37°C）,continue to culture for 24 h after cell passaging,randomly take one part into the high oxygen incubation incubator（5%CO₂,85%O₂,37°C）to continue incubate for24,48 and 72 h,and the other part into the high oxygen incubation incubator（5%CO₂,85%O₂,37°C）to continue incubate for 24,48 and 72 h after the addition of Nrf2 agonist and inhibitor.2.The expression of ASK1 in intestinal epithelial cells was detected using immunofluorescence staining.3.Western blot and q RT-PCR were utilized to detect the expression changes of ERK,p-ERK,p38,p-p38,JNK,p-JNK.4.Preparation of animal model:Neonatal SD rats were randomly divided into control（Fi O₂=20%）and model（Fi O₂=85%）groups within 12 h after birth,and small intestinal tissues were collected on postnatal days 3,7,10 and 14.5.The expression localization of IL-17D in intestinal epithelium of neonatal rats was detected using single and double staining of immunofluorescence.6.The expression changes of IL-17D,Nrf2 and keap1 in intestinal epithelium of neonatal rats were detected by immunohistochemical staining and Western blot.7.The expressions of IL-17D,Nrf2,keap1,IL-4 and IL-6 in NCM460 cells cultured under hyperoxia were detected using immunocytochemical staining,Western blot and q RT-PCR.8.Western blot was utilized to detect the expression of IL-17D,Nrf2,keap1,IL-4 and IL-6 in NCM460 cells cultured in hyperoxia after the addition of Nrf2 agonist and inhibitor.Results:1.Effects of ROS on the ASK1/MAPK pathway in intestinal epithelial cells:（1）after treatment with different concentrations of H₂O₂and hyperoxia,ASK1 migrated from the cytoplasm to the nucleus,and H₂O₂had a dose-dependent effect on ASK1 expression.ASK1 expression was higher in hyperoxia treated cells compared with untreated or H₂O₂treated cells.（2）The results of Western blot and q RT-PCR showed that compared with the control group,the expressions of ERK,p-ERK,JNK,p-JNK,P38 and p-P38 protein and m RNA were gradually elevated after treatment with different concentrations of H₂O₂and hyperoxia（P<0.05 or P<0.01）,and the expressions of ERK,p-ERK,JNK,p-JNK,P38and p-P38 were increased in a dose-dependent manner after treatment with H₂O₂,but were still lower than the expression levels after hyperoxia induction（P<0.01）.2.Effects of hyperoxia on the expression of IL-17D,Nrf2 and keap1 in the small intestine of neonatal rats:（1）The results of immunohistochemical staining and Western blot showed that compared with the control groups,the expressions of Nrf2 and IL-17D in the model group on day 7 increased and reached a peak（P<0.01）;and gradually decreasing and significantly lower on days 10 and 14 than that in the control groups（P<0.01）;There was no obvious difference in keap1 expression between every control groups,but on days 3 and 7,the expression of keap1 in the model group was significantly lower than that in the control group（P<0.01）.（2）The results of immunofluorescence staining showed that IL-17D was expressed in the intestinal epithelial cells as well as CD4⁺T cells and CD19⁺B cells in the model and control groups.3.Effects of hyperoxia on the expression of IL-17D,Nrf2,keap1,IL-4 and IL-6 in intestinal epithelial cells:compared with the control group,the expression of IL-17D,Nrf2,IL-4 and IL-6 in the hyperoxia groups was increased in a time-dependent manner（P<0.01）,while the expression of keap1 in the hyperoxia group was lower than that in the control group（P<0.01）,and the expression of keap1 decreased gradually with the extension of the time of hyperoxia.4.Effect of different concentrations of t BHQ and ML385 on intestinal epithelial cell viability:the cell viability of NCM460 cells did not change significantly after treatment with different concentrations of t BHQ（10μM,20μM,50μM,100μM）and ML385（5μM,10μM,20μM,100Μm）.5.Effect of t BHQ on the expressions of IL-17D,Nrf2,IL-4 and IL-6 in intestinal epithelial cells in hyperoxia:compared with the hyperoxia group,the expression of IL-17D,Nrf2,IL-4 and IL-6 gradually increased with the extension of t BHQ action time（P<0.01 or P<0.05）,but the expression of keap1 was no obvious change compared with the hyperoxia group.6.Effect of ML385 on the expressions of IL-17D,Nrf2,IL-4 and IL-6 in intestinal epithelial cells in hyperoxia:compared with the hyperoxia group,the expression of IL-17D,Nrf2 showed a decreasing trend with the prolonged action time of ML385（P<0.01）;However,there was no obvious difference in keap1 protein expression in hyperoxia and ML385 group;The overproduction of IL-6and IL-4 was significantly inhibited by ML385 at 24h and 48h,but at 72h,the inhibition was partially lost and the expression of IL-6 and IL-4 went up again.Conclusions:1.Hyperoxia damages intestinal epithelial cells through ROS activation of the ASK1/MAPK signaling pathway.2.Hyperoxia activates Nrf2 through MAPK pathway,and Nrf2 increases the expression of IL-17D,which aggravates the inflammatory response of intestinal epithelial cells.