Effects of hypobaric and hypoxia on barrier function of intestinal mucosa and expression of LINE-1 endonuclease variant GCRG213
Background Long interspersed nuclear element-1(LINE-1 or L1)is currently the only active autonomous retrotransposon in the genome,and its activity is suppressed by high levels of methylation.Hypobaric and hypoxia environments can affect the methylation level of L1.Objective To investigate the impact of hypobaric and hypoxia environments on the mechanical barrier function of intestinal mucosa as measured by expression levels of L1 endonuclease variants GCRG213,Occludin,and Claudin-1.Methods The expression of GCRG213 in normal human small intestine,colon and mouse colon tissues were detected by immunohistochemical staining(IHC)to observe the distribution pattern of the GCRG213 protein in the intestines.Male C57BL/6 mice were randomly divided into control group and experimental group.The hypobaric hypoxia mouse model was established by exposing them to a simulated high-altitude environment of 6000m for 7 days.NCM-460 cells derived from human normal colon epithelial cells were randomly divided into normal oxygen group and hypoxic groups for 24 h and 48 h.The normal oxygen group was cultured at normal oxygen concentration environment while the low oxygen groups were cultured at 0.3%O2 concentration for 24 h and 48 h,respectively.qPCR and Western blot techniques were employed to detect the expression levels of GCRG213,Occludin,and Claudin-1 in mouse colon tissues and NCM-460 cells.Results The IHC results consistently showed that GCRG213 was expressed in the cytoplasm of intestinal epithelial cells but not in goblet cells in normal human small intestine,colon,and mouse colon tissues.In animal experiments,compared to the control group,the mRNA and protein expression levels of GCRG213 in mouse colon tissues were significantly upregulated in the experimental group(P＜0.05 and P＜0.01 respectively).Meanwhile,Occludin exhibited a significant downregulation at the protein level(P＜0.05).In cell experiments,NCM-460 cells showed an upregulation of GCRG213 at both mRNA(P＜0.001)and protein(P＜0.01)levels after exposure to hypoxia,and the expression level of GCRG213 was lower in the 24-hour low oxygen group compared to the 48-hour low oxygen group.Occludin and Claudin-1 demonstrated a downregulation in mRNA expression(P＜0.01;P＜0.001)and the expression level was lower in the 24-hour low oxygen group compared to the 48-hour low oxygen group.Occludin exhibited a significant decrease in protein expression(P＜0.05)after 48-hour hypoxic culturing.Conclusion The L1-EN variant GCRG213 is expressed in the cytoplasm of human and mouse normal colonic mucosal columnar epithelial cells.In vivo and in vitro models,GCRG213 expression levels are observed to be increased in colonic mucosal epithelial cells under hypobaric hypoxia exposure,while the hypobaric hypoxia exposure disrupts the integrity of colonic mucosal tight junction as indicating by the expression level of Occludin and Claudin-1 proteins.
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