Protective Mechanism Of Hydrogen Rich Water On Hyperoxic Intestinal Injury In Mice Based On SIRT1/Nrf2/HO-1 Pathway

Objective:To investigate the protective mechanism of hydrogen rich water（HRW）on hyperoxia-induced intestinal injury in mice by regulating silent information regulator 1/nuclear factor-E2 related factor 2/heme oxygenase 1（SIRT1/Nrf2/HO-1）pathway.Methods:Twenty-four C57BL/6 mice were divided into normoxia group（group N）,hyperoxia group（group O）,hydrogen rich water group（group H）and hydrogen rich water combined with SIRT1 inhibitor group（group HE）,6 mice in each group.Mice in group N were exposed to indoor environment（FiO₂=21%）,and mice in groups O,H,HE were exposed to hyperoxia environment（FiO₂=85%）for 7 days.Mice in group H was given 0.1ml/10g hydrogen rich water by gavage,twice a day for 7 days;Mice in group HE,on the basis of the group H,the SIRT1 inhibitor EX527 10mg/kg was injected intraperitoneally once a day for 7 days;Mice in group N and group O were given equal volume of normal saline.The mice were weighed and recorded daily.After7 days,the mice were euthanized and the specimens were collected.The ileum tissue was taken to prepare sections and Chiu’s score was performed.The ultrastructure of ileum was observed by transmission electron microscope.The levels of malondialdehyde（MDA）,superoxide dismutase（SOD）were compared among the four groups.The expression levels of SIRT1,Nrf2,HO-1 protein were detected by using Western blot.Results:1.Compared with group N,the weight of mice in group O,H and HE was decreased after the experiment;compared with group O,the weight of mice in group H was increased after the experiment;compared with group H,the weight of mice in group HE was decreased after the experiment（P<0.05）.2.Under the microscopic,HE staining showed that the intestinal mucosal epithelium of group N mice was structurally intact,with neatly arranged villi and no epithelial shedding;in group O,the structural integrity of the intestinal mucosa epithelium was destroyed and the villi structure was severely damaged with blurred boundaries;in group H,the villus structure gradually returned to normal,and the structure of each layer was relatively clear and complete;compared with the group H,the group HE had disorganized villi arrangement,atrophy and deformation,and some epithelial necrosis and detachment;microvilli and tight junctions in group H were close to normal;Chiu’s score:Compared with group N,Chiu’s score of group O,group H,and group HE was increased;compared with group O,Chiu’s score of group H was decreased;compared with group H,Chiu’s score of group HE was increased（P<0.05）.3.The tight junctions between the intestinal epithelial cells in group N were clear and intact,with abundant microvilli and normal morphology,and no widening of the cell gap was seen under transmission electron microscopy;in group O,the tight junctions between intestinal epithelial cells were blurred and reduced in number,the microvilli were disorganized and partially detached,and the cell gaps were significantly widened;compared with group H,the microvilli in the group HE were more disorganized,with fuzzy tight junctions and slightly widened cell gaps.4.The levels of SOD,MDA in ileum tissue:Compared with group N,SOD activity was decreased and MDA content was increased in groups O,H,HE;compared with group O,SOD activity was increased and MDA content was decreased in group H;compared with group H,SOD activity was decreased and MDA content was increased in group HE（P<0.05）.5.Compared with group N,the expression of SIRT1was down-regulated,Nrf2 and HO-1 were up-regulated in group O,and the expression of SIRT1,Nrf2,HO-1 were up-regulated in group H and HE;compared with group O,the expressions of SIRT1,Nrf2 and HO-1 in group H were up-regulated;compared with the group H,the expressions of SIRT1,Nrf2 and HO-1 were down-regulated in group HE（P<0.05）.Conclusions:Hydrogen rich water pretreatment can protect mice from hyperoxic intestinal injury by up-regulating the expression of SIRT1,Nrf2 and HO-1,increasing the activity of SOD and reducing the content of MDA in ileum tissue.