基于SIRT1/Nrf2/HO-1通路探讨富氢水对小鼠高氧肠损伤的保护机制

Protective mechanism of hydrogen rich water on hyperoxic intestinal injury in the mice based on SIRT1/Nrf2/HO-1 pathway

目的研究富氢水(HRW)通过调节沉默信息调节因子1/核因子E2相关因子2/血红素加氧酶-1(SIRT1/Nrf2/HO-1)通路对小鼠高氧肠损伤发挥保护作用的机制。方法C57BL/6小鼠24只,随机分为常氧组(N组)、高氧组(O组)和富氢水组(H组)和富氢水联合SIRT1抑制剂组(HE组),每组6只。N组小鼠置于室内环境(FiO_(2)=21%)中,O组、H组和HE组小鼠置于高氧环境(FiO_(2)=85%)中,持续7 d。H组给予0.1 mL/10 g富氢水灌胃,2次/d,连续7 d;HE组在H组基础上每天腹腔注射SIRT1抑制剂EX52710 mg/kg 1次,连续7 d;N组和O组给予等体积生理盐水。每天称质量并记录小鼠体质量,7 d后安乐死小鼠并留取标本。取回肠组织制备切片并行Chiu病理评分,透射电镜观察回肠组织超微结构,检测回肠组织超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,Western blot检测回肠组织SIRT1、Nrf2、HO-1表达水平。结果与N组比较,O组、H组、HE组实验后小鼠体质量减轻,Chiu病理评分、MDA含量升高,SOD活性降低;与O组比较,H组实验后小鼠体质量增加,H组、HE组Chiu病理评分、MDA含量降低SOD活性升高;与H组比较,HE组实验后小鼠体质量减轻,Chiu病理评分、MDA含量升高,SOD活性降低[体质量(g):N组23.17±1.70,O组17.60±1.58,H组20.87±1.24,HE组17.20±1.27;Chiu病理评分(分):N组0.33±0.27,O组3.44±0.37,H组2.11±0.25,HE组3.06±0.23;MDA含量(nmol/mg):N组59.15±3.77,O组86.18±3.88,H组68.12±2.27,HE组76.94±5.26;SOD活性(U/mg):N组105.51±7.67,O组64.58±4.62,H组86.45±5.51,HE组72.43±4.27;均P<0.05]。与N组比较,O组SIRT1表达下调,Nrf2、HO-1表达上调,H组、HE组SIRT1、Nrf2、HO-1表达上调;与O组比较,H组SIRT1、Nrf2、HO-1表达上调,HE组SIRT1、Nrf2表达上调;与H组比较,HE组SIRT1、Nrf2、HO-1表达下调(SIRT1:N组0.17±0.01,O组0.13±0.01,H组0.66±0.04,HE组0.36±0.01;Nrf2:N组0.07±0.01,O组0.23±0.02,H组0.54±0.02,HE组0.38±0.02;HO-1:N组0.37±0.01,O组0.42±0.02,H组0.56±0.02,HE组0.44±0.02;均P<0.05)。结论富氢水预处理可通过激活SIRT1/Nrf2/HO-1信息通路,对小鼠高氧肠损伤发挥保护作用。

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),HRW group(group H)and HRW combined with SIRT1 inhibitor group(group HE),6 mice in each group.Mice in group N were exposed to indoor environment(FiO_(2)=21%),and mice in groups O,H,HE were exposed to hyperoxia environment(FiO_(2)=85%)for 7 days.Mice in group H was given 0.1 mL/10 g HRW by gavage,twice a day for 7 days;Mice in group HE,on the basis of the group H,the SIRT1 inhibitor EX52710 mg/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.After 7 days,the mice were euthanized and the specimens were collected.The ileum tissue was taken to prepare the sections and Chiu pathological 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 were detected by using Western blot.Results Compared with group N,the weight of mice in groups O,H and HE was decreased after the experiment,Chiu pathology score,MDA content were increased,and SOD activity was decreased(P<0.05);Compared with group O,the weight of mice in group H was increased after the experiment,Chiu pathological scores,MDA content of group H and HE were decreased,and SOD activity was increased(P<0.05);Compared with group H,the weight of mice in group HE was decreased,Chiu pathological score,MDA content were increased,and SOD activity was decreased[weight(g):group N 23.17±1.70,group O 17.60±1.58,group H 20.87±1.24,group HE 17.20±1.27;Chiu pathology score(score):group N 0.33±0.27,group O 3.44±0.37,group H 2.11±0.25,group HE 3.06±0.23;MDA content(nmol/mg):group N 59.15±3.77,group O 86.18±3.88,group H 68.12±2.27,group HE 76.94±5.26;SOD activity(U/mg):group N 105.51±7.67,group O 64.58±4.62,group H 86.45±5.51,group HE 72.43±4.27;all P<0.05].Compared with group N,the expression of SIRT1 was down-regulated,Nrf2,HO-1 were up-regulated in group O,SIRT1,Nrf2,HO-1 in group H and HE were up-regulated;Compared with group O,the expressions of SIRT1,Nrf2,HO-1 were up-regulated in group H,and SIRT1,Nrf2 in group HE were up-regulated;Compared with group H,the expression of SIRT1,Nrf2 and HO-1 were down-regulated in group HE(SIRT1:group N 0.17±0.01,group O 0.13±0.01,group H 0.66±0.04,group HE 0.36±0.01;Nrf2:group N 0.07±0.01,O组0.23±0.02,group H 0.54±0.02,group HE 0.38±0.02;HO-1:group N 0.37±0.01,group O 0.42±0.02,group H 0.56±0.02,group HE 0.44±0.02;all P<0.05).Conclusions HRW pretreatment can protect mice from hyperoxic intestinal injury by activating SIRT1/Nrf2/HO-1 pathway.