Hydrogen Gas Improve Sepsis-associated Encephalopathy By Regulating Mitochondrial Dynamic Balance And Function

BACKGROUND: Sepsis is a syndrome caused by systemic inflammation.In the advanced stage of sepsis,multiple organ dysfunction often endangers life and health.Worldwide,sepsis with its high morbidity and mortality brings great burden to society.The brain injury is common in sepsis,and the severity of brain injury in sepsis is closely related to the prognosis of patients.Therefore,it is of great significance to study the prevention and treatment of sepsis brain injury.Mitochondria are central of cellular energy and metabolism,which provide energy for normal physiological activities of cells by oxidative phosphorylation.The dysfunction of mitochondria is strongly associated with sepsisinduced organ dysfunction.Improving abnormal mitochondrial function in sepsis is important for the prognosis and survival rate of sepsis patients.Mitochondrial quality control,including mitochondrial biogenesis,mitochondrial autophagy,mitochondrial fusion and fission,plays a pivotal role in mitochondrial function recovery.Mitochondrial biogenesis is the process of producing new mitochondria.These newly generated functional mitochondria form mitochondrial networks by fusion and fission,while mitochondria that undergo damage and dysfunction are cleared by autophagy.The research of hydrogen medicine has become a hot spot since professor Ohsawa proved the anti-oxidative effect of low concentration hydrogen(2%~4%).Hydrogen gas have been confirmed to have antioxidant,anti-inflammatory and anti-apoptotic effects,and have a therapeutic effect on septic organ dysfunction.In addition,studies have confirmed that hydrogen can reduce lung injury in sepsis by improving mitochondrial function,promoting mitochondrial biogenesis,mitochondrial autophagy and mitochondrial fission,and inhibiting mitochondrial fusion [1-3].This experiment aims to investigate the effect of hydrogen on mitochondrial dynamics and function in hippocampal tissues of SAE mice and the mechanism of hydrogen regulation of mitochondrial biogenesis.Part 1: Hydrogen gas alleviate SAE through regulating mitochondrial dynamics balance and functional.To investigate the effects of hydrogen gas on mitochondrial function and mitochondrial dynamics in hippocampal tissue of mice with SAE.METHOD: Three hundred and sixteen wild-type adult male C57BL/6J mice,aged 6-8weeks,weighing 20-25 g,were randomized into 4 groups(n=79): Sham group,Sham+H2 group,CLP group and CLP+H2 group.The model of sepsis-induce brain injury was constructed by cecal ligation and puncture(CLP).At 1 h and 6 h after operation,the mice in Sham+H2 group and CLP+H2 group breathed in hydrogen gas for 1 h,and the rest of mice breathes in air.The 7d-survival rate of mice was observed.At 3 d,5 d and 7 d after operation,the ability of working memory of mice was detected by Y-maze test.At 24 h after operation,the pathological changes of brain tissues were observed by Nissl stain,apoptosis of hippocampal neurons was observed by TUNEL stain,morphology of mitochondria was observed by transmission electron microscope.The levels of inflammatory factor and Mitochondrial membrane potential(MMP),the concentration of ATP and the activities of mitochondrial respiratory chain complex I and mitochondrial respiratory chain complex II were measured by related kit.The expressions of proteins of mitochondrial biogenesis and mitochondrial fission/fusion were detected by Western blot.RESULTS: Hydrogen gas inhalation significantly increased 7d-survival rate in septic mice(P < 0.05).Y-maze test showed a decrease in spontaneous alternation percentage in mice with sepsis,while hydrogen gas therapy increased the spontaneous alternation percentage in septic mice(P < 0.05).The results of Nissl stain,TUNEL stain and transmission electron microscope indicated that the injury of neuronal cells and mitochondria and the apoptosis of neurons were increased in septic mice,while hydrogen gas inhalation reduced these damages.Compared with Sham group,the levels of TNF-α and IL-6 were increased in CLP group(P < 0.05);compared with CLP group,the levels of TNF-α and IL-6 were decreased in CLP+H2 group(P < 0.05).Compared with Sham group,the level of MMP,the concentration of ATP and the activities of mitochondrial respiratory chain complex I were decreased in CLP group(P < 0.05);compared with CLP group,the level of MMP,the concentration of ATP and OBJECTIVE:the activities of mitochondrial respiratory chain complex I were increased in CLP+H2 group(P < 0.05).There were no significant differences in the activities of mitochondrial respiratory chain complex II(P > 0.05).Compared with Sham group,the expressions of PGC-1α、NRF2 and Tfam were increased in CLP group(P < 0.05);compared with CLP group,the expressions of PGC-1α、NRF2 and Tfam were further increased in CLP+H2 group(P < 0.05).Compared with Sham group,the expressions of Drp1 were increased,while the expressions of Mfn2 were decreased in CLP group(P < 0.05);compared with CLP group,the expressions of Drp1 were decreased and the expressions of Mfn2 were increased in CLP+H2 group(P < 0.05).CONSLUSION: Hydrogen gas can improve the survival rate of sepsis mice,improve the cognitive function,alleviate the brain injury caused by sepsis,and play an anti-apoptotic and antiinflammatory role.The protective effect of hydrogen on SAE is associated with its improvement of mitochondrial function,regulation of mitochondrial biogenesis and mitochondrial fusion/fission.Part 2: Hydrogen gas alleviates SAE by promoting mitochondrial biogenesis through the activation of PGC-α.OBJECTIVE: To investigate the effects of hydrogen gas on mitochondrial biogenesis in hippocampus of mice with SAE and its mechanism.METHOD: Two hundred and forty-four wild-type adult male C57BL/6J mice,aged 6-8weeks,weighing 20-25 g,were randomized into 4 groups(n=61): CLP group,CLP+H2 group,CLP+SR-18292 group and CLP+SR-18292+ H2 group.The model of sepsis-induce brain injury was constructed by cecal ligation and puncture(CLP).At 3 d before operation,the mice in CLP+SR-18292 group and CLP+SR-18292+ H2 group were injected SR-18292(45 mg/kg)into abdominal cavity [4].At 1 h and 6 h after operation,the mice in CLP+H2 group and CLP+SR-18292+H2 group breathed in hydrogen gas for 1 h,and the rest of mice breathes in air.At 24 h after operation,the expressions of mitochondrial biogenesis related proteins were detected by Western blot.The 7 dsurvival rate of mice was observed.At 3 d,5 d and 7 d after operation,the ability of working memory of mice was detected by Y-maze test.At 24 h after operation,apoptosis of hippocampal neurons was observed by TUNEL stain.The levels of inflammatory factor and Mitochondrial membrane potential(MMP),the concentration of ATP and the activities of mitochondrial respiratory chain complex I were measured by related kit.RESULTS: Compared with CLP group,the expressions of acetylated PGC-1α in hippocampal tissue of mice was decreased,and the expressions of NRF2 and Tfam were increased in CLP+H2 group(P < 0.05);compared with CLP+H2 group,the expressions of acetylated PGC-1α in hippocampal tissue of mice was increased,and the expressions of NRF2 and Tfam were decreased in CLP+SR-18292+H2 group(P < 0.05).Compared with CLP group,the 7d-survival rate of mice in CLP+H2 group was increased(P < 0.05);compared with CLP+H2 group,the 7d-survival rate of mice in CLP+SR-18292+H2 group was decreased(P < 0.05).Y-maze test showed that hydrogen gas therapy increased the spontaneous alternation percentage in septic mice,while the use of the PGC-1α inhibitor SR-18292 reversed the effect of hydrogen gas.The TUNEL stain showed that hydrogen gas therapy reduced neuronal cell apoptosis,whereas neuronal cell apoptosis was evident in the CLP+SR-18292+H2 group.Compared with CLP group,the level of MMP,the concentration of ATP and the activities of mitochondrial respiratory chain complex I in hippocampal tissue of mice were increased in CLP+H2 group(P < 0.05);compared with CLP+H2 group,the level of MMP,the concentration of ATP and the activities of mitochondrial respiratory chain complex I in hippocampal tissue of mice were decreased in CLP+SR-18292+H2 group(P < 0.05).CONSLUSION: Hydrogen gas alleviate brain injury induced by sepsis by promoting mitochondrial biogenesis and improving mitochondrial function through activating PGC-1α in hippocampal tissue.