The role of Nrf2 in the hydrogen treatment for intestinal injury caused by severe sepsis

OBJECTIVE To investigate the role of Nrf2 on hydrogen treatment for intestinal injury caused by severe sepsis. METHODS 152 male ICR mice were randomly divided into four groups: sham operation group, hydrogen control group, sepsis group, and hydrogen treatment group, each n=38. Sepsis model was reproduced by cecal ligation and puncture (CLP). The mice in sham operation group and hydrogen control group did not receive CLP, and the operative procedure was the same as follows. The mice in hydrogen control group and hydrogen treatment group received 1-hour inhalation of 2% hydrogen 1 hour and 6 hours after sham operation or CLP. Twenty animals in each group were selected and observed for 7-day survival rate.Eighteen animals in each group were selected and sacrificed at 6, 12 and 24 hours after CLP. The intestinal tissues were obtained to determine the expression of Nrf2 and high mobility group B1 (HMGB1) protein by Western Blot, and the expression of Nrf2 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The middle portion of jejunum was obtained to evaluate the degree of septic injury by light microscope after hematoxylin and eosin (HE) staining. RESULTS There was no statistical significance in variables between sham operation group and hydrogen control group. Compared with sham operation group, the 7-day survival rate was significantly decreased in sepsis group (0 vs. 100%, P<0.05); compared with sepsis group, the 7-day survival rate was significantly increased in hydrogen treatment group (55% vs. 0, P<0.05). Compared with sham operation group, the expression of Nrf2 protein (gray value) and Nrf2 mRNA were up-regulated in sepsis group at 6, 12 and 24 hours after CLP (Nrf2 protein 6 hours: 1.973 ± 0.350 vs. 1.000 ± 0.000, t=4.411, P=0.002; 12 hours: 2.367 ± 0.186 vs. 1.000 ± 0.000, t=10.210, P=0.000; 24 hours: 2.517 ± 0.280 vs. 1.000 ± 0.000, t=9.521, P=0.000; Nrf2 mRNA 6 hours: 1.606 ± 0.271 vs. 1.000 ± 0.000, t=3.631, P=0.002; 12 hours: 1.692 ± 0.399 vs. 1.000 ± 0.000, t=3.233, P=0.005; 24 hours: 1.784 ± 0.341 vs. 1.000 ± 0.000, t=3.894, P=0.001), and it was also the expression of HMGB1 (gray value) at 24 hours after CLP operation (1.507 ± 0.220 vs. 1.000 ± 0.000, t=3.948, P=0.004). Compared with sepsis group, the expression of Nrf2 protein and Nrf2 mRNA in intestines were up-regulated at 6, 12 and 24 hours after CLP in hydrogen treatment group (Nrf2 protein 6 hours: 2.583 ± 0.395 vs. 1.973±0.350, t=2.765, P=0.024; 12 hours: 2.725 ± 0.235 vs. 2.367 ± 0.186, t=2.674, P=0.028; 24 hours: 2.930 ± 0.212 vs. 2.517 ± 0.280, t=2.595, P=0.032; Nrf2 mRNA 6 hours: 2.008 ± 0.400 vs. 1.606±0.271, t= 2.405, P=0.029; 12 hours: 2.188 ± 0.475 vs. 1.692 ± 0.399, t= 2.317, P=0.034; 24 hours: 2.333 ± 0.406 vs. 1.784 ± 0.341, t= 2.728, P=0.015). Compared with sepsis group, the expression of HMGB1 was down-regulated significantly at 24 hours after CLP in hydrogen treatment group (1.147 ± 0.152 vs. 1.507 ± 0.220, t=2.805, P=0.023). HE staining showed that there was significantly aggravated intestinal pathological injury in the mice of sepsis group; compared with sepsis group, the pathology was significantly less marked in hydrogen treatment group. CONCLUSIONS Through activation of Nrf2-antioxidant response element (ARE) pathway, hydrogen may increase the level of Nrf2, which is a kind of protective protein, in the intestine of mice, thus decreases the level of late pro-inflammatory factor, HMGB1, and it may protect the intestinal tissues in septic mice and increase the survival rate significantly.