Hyperoxia Inhibits The Expression Of Intestinal Tight Junction Proteins And Intestinal Stem Cell Markers

Objective:Oxygen is indispensable for the continuous generation of energy in the body.Hyperoxia therapy refers to inhale more than 60%concentration oxygen.Hyperoxic therapy is widely used in the emergency treatment of severe neonates,which can improve blood oxygen partial pressure and oxygen saturation to meet the oxygen demand of tissue organ,eliminate or reduce the adverse effects of hypoxia on the body.It is an indispensable component in clinical emergency treatment of severe neonates.Although hyperoxic therapy can improve the survival rate of sick children,improper use may also cause many adverse reactions,including intestinal damage,which must be highly valued.TJ is a kind of multi-protein complex,which widely exists at the top of all epithelial or endothelial cell junctions,including Claudins,Occludin and Zos.Intestinal TJ is an important physical barrier,damaged TJ can lead to overactivation of immune system and inflammation,and is closely related to the pathogenesis of various intestinal and systemic inflammatory diseases.Bmi1 and Lgr5 are well-recognized markers of intestinal stem cells,intestinal stem cells can move from intestinal crypt to villus axis,produce a series of specialized epithelial cells,maintain intestinal epithelial self-renewal and repair,and are the key factors of intestinal function changes and pathological changes.The inappropriate use of hyperoxia treatment is likely to cause tissue and organ damage,mainly because of the increased level of reactive oxygen species（ROS）.There are two sources of ROS production:mitochondria and nicotinamide adenine dinucleotide phosphate oxidase（NADPH-oxidase）.The antioxidant Apocynin is mainly directed to ROS derived from NADPH-oxidase.The antioxidant NAC can react with hydroxyl radicals,causing them to be inactivated and directly used as antioxidants to scavenge ROS.It can also act as an indirect antioxidant by increasing the synthesis of glutathione.In this study,we aim to understand the mechanism of intestinal damage caused by hyperoxia’s effects on intestinal tight junction proteins and stem cells,explore the protective effect of antioxidant NAC on intestinal tight junction protein by detect the changes of tight junction protein of NCM460 cells in hyperoxia after adding antioxidant NAC.This study can provide a theoretical basis for clinical treatment of oxygen therapy in neonates with hypoxemia.Methods:1.Establishment of the hyperoxic animal model:Within in 12h after birth,the newborn SD rats（with mother rats）were randomly divided into control group（FiO2=21%）and hyperoxia group（FiO₂=80%-85%）.The hyperoxia group was placed in a glass oxygen chamber and oxygen was continuously supplied to maintain FiO₂=80%-85%.The control group was placed in the air（FiO₂=21%）,and the other control factors were the same as the hyperoxia group.2.Detection of rat intestinal tight junction protein and intestinal stem cell markers:RT-qPCR,immunohistochemistry and Western blot was used to detect the expression of tight junction proteins Zo-1,Occludin,Claudin-4 and intestinal stem cells Bmi1 and Lgr5.3.Cell culture experiment:NCM460 cells were cultured in a normal cell incubator（5%CO₂,37°C）,then the cells were seeded at 1x10⁶cells/ml.After 24 hours,different concentrations of antioxidants NAC and Apocynin were added.Then them were randomly placed in normal cell incubator and hyperoxia cell incubator（5%CO2,85%O2,37°C）.4.The effects of different concentrations of antioxidants NAC and Apocynin on the viability of NCM460 cells cultivated in hyperoxia were investigated by cell proliferation assay（MTT）.The effect of antioxidant NAC on the expression levels of Zo-1,Occludin and Claudin-4 in NCM460 cells under hyperoxia were detected by Western Blot and RT-qPCR.Results:1.Hyperoxia inhibits the expression of intestinal tight junction proteins and intestinal stem cell markers in the neonatal rats:（1）Western blot was used to detect the tight junction proteins Zo-1,Occludin and Claudin-4.Compared with the control group,the protein expression of Zo-1,Occludin and Claudin-4 was decreased in each hyperoxia group（P<0.05 or P<0.01）.（2）Immunohistochemistry was used to detect tight junction proteins Zo-1,Occludin and Claudin-4.These three proteins were mainly located in the membrane and cytoplasm of intestinal epithelial cells.In the control group,the intestinal staining was yellow or brown,and the intestinal villi were neat and the shape was normal.The intestinal staining of the hyperoxia group was pale yellow,and the morphology and continuity of the intestinal villi were destroyed.Compared with the control group,the protein expressions of Zo-1,Occludin and Claudin-4 were decreased in each hyperoxia group（P<0.01）.（3）The RNA content of the tight junction proteins Zo-1,Occludin and Claudin-4 were detected by RT-qPCR.Compared with the control group,the mRNA expression levels of Zo-1,Occludin and Claudin-4 were decreased in each hyperoxia group（P<0.05 or P<0.01）.（4）Western blot was used to detect intestinal stem cell markers Bmi1 and Lgr5.Compared with the control group,the protein expression of Bmi1 and Lgr5 was decreased in each hyperoxia group（P<0.05 or P<0.01）.（5）The intestinal stem cell markers Bmi1 and Lgr5 were detected by immunohistochemistry,which were mainly located in the intestinal crypt.The stains in the intestinal crypts of the control group were yellow or tan,and the hyperoxia group was pale yellow.Compared with the control group,the protein expression of Bmi1 and Lgr5 was decreased in each hyperoxia group（P<0.05 or P<0.01）.（6）RT-qPCR was used to detect the RNA content of the intestinal stem cell markers Bmi1 and Lgr5.Compared with the control group,mRNA expression of Bmi1 and Lgr5 was decreased in each hyperoxia group（P<0.05 or P<0.01）.2.Effects of different concentrations of antioxidants on NCM460 cells in hyperoxia:（1）Effects of different concentrations of antioxidants NAC and Apocynin on the viability of NCM460 cells in hyperoxia,20μM NAC significantly increased the survival rate of NCM460 cells in hyperoxia（P<0.01）,while Apocynin did not（P>0.05）.（2）Different concentrations of antioxidants affect NCM460 cell tight junction protein in hyperoxia:Western blot analysis showed that compared with the control group,the protein expression levels of Zo-1,Occludin and Claudin-4 in the hyperoxia group were significantly decreased（P<0.01）.The protein expression levels of Zo-1,Occludin and Claudin-4 were increased in the hyperoxia plus20μM NAC group compared with the hyperoxia group（P<0.05 or P<0.01）.（3）RT-qPCR results showed that compared with the control group,the mRNA expression levels of Zo-1,Occludin and Claudin-4 in the hyperoxia group were significantly decreased（P<0.05 or P<0.01）.The mRNA expression levels of Zo-1,Occludin and Claudin-4 were increased in the hyperoxia plus 20μM NAC group compared with the hyperoxia group（P<0.01）.Conclusion:1.Hyperoxia reduces the expression of intestinal tight junction proteins Zo-1,Occludin,Claudin-4 and intestinal stem cells Bmi1,Lgr5.2.The antioxidant NAC can protect NCM460 cells in the hyperoxic environment.