| Background:Sepsis is a systemic inflammatory response syndrome caused by various infectious factors, which is the common complication of trauma and surgery and has turned into a primary cause of mortality in critical patients and a main public health burden worldwide. In septic patients, the complication in central nervous system(CNS) appears to be earlier and more frequently than those in other systems, which is related to a higher mortality. Besides, 9–71% septic patients have been found to present cognitive dysfunction of various severity. Therefore, it is of great significance for septic patients to alleviate brain injury. Hydrogen gas(H2), as a new-type gaseous signal molecule, it has obvious anti-inflammatory, antioxidant, anti-apoptosis and mediating some signal pathways. Our previous researches have indicated that inhalation of 2% H2 dramatically reduced the mortality and ameliorate crucial organ damage in septic mice, such as heart, lung and kidney. Based on these previous studies, this study was aimed to investigate the potential protective effects and the related mechanisms of H2 on brain injury in the mouse model of CLP induced by caecal ligation and puncture, which may provide theoretical basises for the clinical application of H2.Objective: To observe the effect of inhalation of 2% H2 on the survival rate and cognitive function of septic mice and investigate the effect of 2% H2 on brain injury of septic mice and to explore its possible mechanism.Methods: Adult male ICR mice, weighed 20 to 25 g, were randomly divided into 4 groups: Sham group, Sham+H2 group, CLP group and CLP+H2 group. Mice were suffered to sepsis by cecal ligation and puncture(CLP). Mice in Sham+H2 group and Sepsis+H2 group were received nhalation of 2% H2 for 1 h at 1 h and 6 h after sham or CLP operation, respectively. The 7 d survival rate of mice in four groups was observed. Y-maze spontaneous alternation test and the contextual Fear Conditioning test were used to observe short-term working memory and long-term fear memory from day 3 to 14 after sham or CLP operation. The changes of learning and memory abilities were observed by Morris water maze test at day 4 to 9 after CLP or sham operation. H2 was detected in arterial blood, venous blood and brain tissue via utilizing a needle-type Hydrogen Sensor at different points after beginning and stopping inhalation of H2. Mice were sacrificed and brain tissues were obtained at 24 h after operation. The histopathologic changes and neuron apoptosis in the hippocampus were observed by Nissl staining and TUNEL stainning. Determination of EB and water contents was used to assess the blood-brain barrier disruption. The levels of TNF-α, HMGB1, IL-1β and IL-10, the activities of SOD and CAT and the levels of MDA and 8-iso-PGF2α in plasma and hippocampus were also detected. The expression of total Nrf2, nucleus Nrf2 and cytoplasmic HO-1 were detected by Western Blot. The nuclear translocation and expression of Nrf2 were also detected by immunofluorescence. The location and expression of HO-1 were also detected by immunohistochemistry.Results: Inhalation of 2% H2 markedly meliorated the 7-day survival rate of septic mice with CLP operation(P <0.05). In Y-maze test, the percentage of alternation of septic mice was significantly increased by H2 treatment from day 3 to 14 after sham or CLP operation(P <0.01). There was no difference on the percentage of freezing time among groups in the acquisition and training phases(P ﹥ 0.05); in the assessment phase, the percentage of freezing time of septic mice was significantly increased by H2 treatment at day 3 to14 after CLP operation(P <0.01). H2 markedly shortened the escape latency of septic mice at day 4 to 8 after CLP operation(P <0.001), and there was no difference on swimming speed(P﹥0.05); the percentage of time in the target quadrant and the times of the platform crossing of septic mice were significantly increased on probe day after H2 treatment(P <0.001).At different points after beginning inhalation of H2, H2 concentration in arterial blood, venous blood and brain tissue of mice in Sham+H2 group and CLP+H2 group was much higher than that in CLP group(P <0.05); at different points after stopping inhalation of H2, H2 concentration in arterial blood, venous blood and brain tissue of mice in CLP+H2 group and CLP+H2 group was much lower than that in Sham+H2 group(P <0.05). The number of normal pyramidal neurons in hippocampal CA1 region was not significant different, and there were almost no apoptotic neurons in the hippocampal CA1 region in Sham and Sham+H2 groups at 24 h after sham operation. There were less normal pyramidal neurons, more apoptotic neurons and the extravasation of the EB and brain water content in hippocampal CA1 region in CLP group and CLP+H2 group compared with Sham group at 24 h after CLP operation(P <0.05). There were more normal pyramidal neurons, less apoptotic neurons and the extravasation of the EB and brain water content in hippocampal CA1 region in CLP+H2 group compared with CLP group at 24 h after CLP operation(P <0.05). At 24 h after sham or CLP operation, in CLP group, the levels of TNF-α, HMGB1, IL-1β and IL-10 were increased, the activities of SOD and CAT were decreased, and the levels of MDA and 8-iso-PGF2α were increased in plasma and hippocampus compared with Sham group(P <0.05). At 24 h after CLP operation, in CLP+H2 group, the levels of TNF-α, HMGB1 and IL-1β were decreased, the level of IL-10 and the activities of SOD and CAT were markedly increased, and the levels of MDA and 8-iso-PGF2α were markedly decreased in plasma and hippocampus compared with CLP group(P <0.05). At 24 h after sham or CLP operation, the expression of total and nucleus Nrf2 and cytoplasmic HO-1 were partly increased in hippocampal in CLP group compared with Sham group; the expression of total and nucleus Nrf2 and cytoplasmic HO-1 were dramatically increased in hippocampus in CLP+ H2 group compared with CLP group(P <0.05). There was no statistical difference between Sham group and Sham+H2 group(P﹥0.05).Conclusion: 2% H2 inhalation can improve the survival rate and ameliorate short-term working memory, long-term fear memory and impairment of learning and memory abilities of septic mice, and it also can alleviate the pathological damage, inhibit the apoptosis of neurons and ruduce the destruction of the BBB, which may be associated with upregulating Nrf2/HO-1 pathway to inhibiting inflammation and oxidative stress. |
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