High-concentration hydrogen inhalation mitigates sepsis-associated encephalopathy in mice by improving mitochondrial dynamics.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a neuronal injury with poor prognosis. Mitochondrial dysfunction is critical in SAE development, and hydrogen gas (H2) has a protective effect on septic mice. This study aimed to investigate the effect of high concentration (67%) of H2 on SAE and whether it is related to mitochondrial biogenesis and mitochondrial dynamics. METHODS: A mouse sepsis model was induced by cecal ligation and puncture. The mice inhalated 67% H2 for 1 h at 1 and 6 h post-surgery, respectively. The 7-day survival rate was recorded. Cognitive function was assessed using the Y-maze test and Morris water maze test. Serum inflammatory factors, antioxidant enzymes, as well as mitochondrial function indexes including mitochondrial membrane potential (MMP) and ATP in the hippocampal tissue were evaluated 24 h after surgery. Mitochondrial dynamic proteins (DRP1 and MFN2) and biosynthetic proteins (PGC-1α, NRF2, and TFAM) in the hippocampal tissue were detected. Moreover, the morphology of mitochondria was observed by transmission electron microscopy. RESULTS: Inhalation of 67% H2 improved the 7-day survival rates and recognition memory function of septic mice, alleviated brain antioxidant enzyme activity (SOD and CAT), and reduced serum proinflammatory cytokine levels. H2 inhalation also enhanced the expression of MFN2 and mitochondrial biogenesis-related factors (PGC-1α, NRF2, and TFAM) and decreased the expression of fission protein (DRP1), leading to improvement in mitochondrial function, as evidenced by MMP and ATP levels. CONCLUSIONS: Inhalation of high concentration (67%) of H2 in septic mice improved the survival rate and reduced neuronal injury. Its mechanism might be mediated by enhancing mitochondrial biogenesis and mitochondrial dynamics.

Inhibition of Golgi stress alleviates sepsis-induced cardiomyopathy by reducing inflammation and apoptosis.

BACKGROUND: Sepsis is often accompanied by multiple organ dysfunction, in which the incidence of cardiac injury is about 60%, and is closely related to high mortality. Recent studies have shown that Golgi stress is involved in liver injury, kidney injury, and lung injury in sepsis. However, whether it is one of the key mechanisms of sepsis-induced cardiomyopathy (SIC) is still unclear. The aim of this study is to investigate whether Golgi stress mediates SIC and the specific mechanism. METHODS: Sepsis model of male C57BL/6J mice was established by cecal ligation and puncture. To observe the effect of Golgi stress on SIC, mice were injected with Golgi stimulant (Brefeldin A) or Golgi inhibitor (Glutathione), respectively. The 7-day survival rate of mice were recorded, and myocardial injury indicators including cardiac function, myocardial enzymes, myocardial pathological tissue score, myocardial inflammatory factors, and apoptosis were detected. The morphology of Golgi was observed by immunofluorescence, and the Golgi stress indices including GM-130, GOLPH3 and Goligin97 were detected by WB and qPCR. RESULTS: After CLP, the cardiac function of mice was impaired and the levels of myocardial enzymes were significantly increased. Golgi stress was accompanied by increased myocardial inflammation and apoptosis. Moreover, the expressions of morphological proteins GM-130 and Golgin97 were decreased, and the expression of stress protein GOLPH3 was increased. In addition, Brefeldin A increased 7-day mortality and the above indicators in mice. The use of glutathione improves all of the above indicators. CONCLUSION: Golgi stress mediates SIC, and the inhibition of Golgi stress can improve SIC by inhibiting apoptosis and inflammation.

THE PROTECTIVE EFFECT OF DEXMEDETOMIDINE ON THE LIVER INJURY IN SEPSIS THROUGH INHIBITION OF NECROPTOSIS.

ABSTRACT: Background: Sepsis-induced liver injury leads to extensive necroptosis in hepatocytes, which is the main factor of liver dysfunction. This study aims to investigate the protective effect of dexmedetomidine (DEX) on septic liver and to explore whether its molecular mechanism is related to the modulation of necroptosis. Methods: The model of septic liver injury was induced by cecal ligation and puncture (CLP) in rats. DEX and necrostatin-1(Nec-1), a specific antagonist of necroptosis, were administered 1 h before CLP. The levels of arterial blood gas, serum aspartate aminotransferase, and alanine aminotransferase were measured at 6, 12 and 24 h after CLP. The survival rate was observed 24 h after CLP. Liver pathological changes and apoptosis, the contents of IL-6 and TNF-α in liver tissue homogenates, the ROS content in liver tissue, and the expression levels of RIP1, RIP3, MLKL, and HMGB1 were detected. Results: At 6, 12, and 24 h after CLP, the levels of aspartate aminotransferase, and alanine aminotransferase levels increased, and liver enzyme levels gradually increased with the progression of sepsis. In arterial blood gas analysis, P a O 2 gradually decreased and lactic acid concentration gradually increased during these three periods. The morphological impairment of liver tissues, increased apoptosis, elevated inflammatory factors (IL-6 and TNF-α), increased ROS level, and necroptosis components (RIP1, RIP3, MLKL, and HMGB1) were all observed in sepsis rats. However, these injuries can be ameliorated by pretreatment with DEX. Meanwhile, Nec-1 pretreatment also reduced the expression of RIP1, RIP3, MLKL, HMGB1, and ROS level. Conclusion: Our study suggests that DEX alleviates septic liver injury, and the mechanism is associated with the inhibition of necroptosis.

Molecular hydrogen attenuates sepsis-induced cardiomyopathy in mice by promoting autophagy.

BACKGROUND: Approximately 40 to 60% of patients with sepsis develop sepsis-induced cardiomyopathy (SIC), which is associated with a substantial increase in mortality. We have found that molecular hydrogen (H2) inhalation improved the survival rate and cardiac injury in septic mice. However, the mechanism remains unclear. This study aimed to explore the regulatory mechanism by which hydrogen modulates autophagy and its role in hydrogen protection of SIC. METHODS: Cecal ligation and puncture (CLP) was used to induce sepsis in adult C57BL/6J male mice. The mice were randomly divided into 4 groups: Sham, Sham + 2% hydrogen inhalation (H2), CLP, and CLP + H2 group. The 7-day survival rate was recorded. Myocardial pathological scores were calculated. Myocardial troponin I (cTnI) levels in serum were detected, and the levels of autophagy- and mitophagy-related proteins in myocardial tissue were measured. Another four groups of mice were also studied: CLP, CLP + Bafilomycin A1 (BafA1), CLP + H2, and CLP + H2 + BafA1 group. Mice in the BafA1 group received an intraperitoneal injection of the autophagy inhibitor BafA1 1 mg/kg 1 h after operation. The detection indicators remained the same as before. RESULTS: The survival rate of septic mice treated with H2 was significantly improved, myocardial tissue inflammation was improved, serum cTnI level was decreased, autophagy flux was increased, and mitophagy protein content was decreased (P < 0.05). Compared to the CLP + H2 group, the CLP + H2 + BafA1 group showed a decrease in autophagy level and 7-day survival rate, an increase in myocardial tissue injury and cTnI level, which reversed the protective effect of hydrogen (P < 0.05). CONCLUSION: Hydrogen exerts protective effect against SIC, which may be achieved through the promotion of autophagy and mitophagy.

Molecular hydrogen attenuates sepsis-induced cognitive dysfunction through regulation of tau phosphorylation.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a cognitive dysfunction caused by sepsis. Hyperphosphorylated tau is considered to play a significant role in the progression of neurodegenerative disease and also contributes to cognitive dysfunction in septic mice. Molecular hydrogen (H2) plays an antioxidant and anti-inflammatory role, and plays a protective role in septic mice. This study explored the possible effects of H2 on cognition and tau phosphorylation in a mouse model of SAE. METHODS: The model of sepsis was established in C57BL/6J male mice by cecal ligation and puncture surgery. Mice treated with 2 % H2 inhalation for 60 min at 1 h and 6 h after surgery, respectively. HY-15769, the inhibitor of Tau Tubulin Kinase 1 (TTBK1), was injected 1 h before the surgery. The 7-day survival rates of the mice were recorded. Cognitive behavior was tested with both novel object recognition and the Y-maze novelty arm recognition on day 7 after surgery. Hematoxylin-eosin staining was used to observe the histological damage in CA1 region of hippocampus. The expression of inflammatory factors in hippocampus was assessed by Elisa. Western blotting was adopted to determine the tau phosphorylation levels at AT8 epitopes (pSer202 and pThr205) and T22 epitopes (neurofibrillary tangle protein oligomer), and the GSK3ß phosphorylation levels (Tyr216), as well as p-Ser422 and TTBK1 levels in the hippocampus. The number of dendritic spine and mushroom type of dendritic spines in the hippocampus were assessed by Golgi staining. RESULTS: The survival rate, visual and spatial learning ability, and memory ability were improved in septic mice treated with H2. After H2 treatment, the density of dendritic spine, mushroom type of dendritic spine, and the number of normal hippocampal neurons were progressively elevated. H2 decreased the levels of phosphorylated tau protein, tau oligomer and TTBK1, as well as the phosphorylation of tau key kinase. Furthermore, the injection of HY-15769 (a TTBK1 inhibitor) protected SAE through the similar way. CONCLUSION: The protective effect of H2 on cognitive dysfunction induced by SAE may be achieved by inhibiting tau phosphorylation, which is perhaps related with the inhibition of TTBK1.

Clemastine Fumarate Attenuates Myocardial Ischemia Reperfusion Injury Through Inhibition of Mast Cell Degranulation.

Mast cell (MC) activation is associated with myocardial ischemia reperfusion injury (MIRI). Suppression of MC degranulation might be a target of anti-MIRI. This study aimed to determine whether clemastine fumarate (CLE) could attenuate MIRI by inhibiting MC degranulation. A rat ischemia and reperfusion (I/R) model was established by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. Compound 48/80 (C48/80) was used to promote MC degranulation. The protective effect of CLE by inhibiting MC degranulation on I/R injury was detected by cardiac function, 2,3,5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, arrhythmia, and myocardial enzyme detection. Inflammatory factor mRNA levels, such as TNF-α, interleukin (IL)-1ß, and IL-6, were detected. Cultured RBL-2H3 mast cells were pretreated with CLE and subjected to C48/80 treatment to determine whether CLE suppressed MC degranulation. Degranulation of MCs was visualized using tryptase release, Cell Counting Kit-8 (CCK-8), and cell toluidine blue (TB) staining. RBL cells were conditionally cultured with H9C2 cells to explore whether CLE could reverse the apoptosis of cardiomyocytes induced by MC degranulation. Apoptosis of H9C2 cells was detected by CCK-8, the LDH Cytotoxicity Assay Kit (LDH), TUNEL staining, and protein expression of BAX and Bcl-2. We found that CLE pretreatment further inhibited cardiac injury manifested by decreased infarct size, histopathological changes, arrhythmias, MC degranulation, and myocardial enzyme levels, improving cardiac function compared with that in the I/R group. C48/80 combined with I/R exacerbated these changes. However, pretreatment with CLE for C48/80 combined with I/R significantly reversed these injuries. In addition, CLE pretreatment improved the vitality of RBL cells and reduced tryptase release in vitro. Similarly, the supernatant of RBL cells pretreated with CLE decreased the cytotoxicity, TUNEL-positive cell rate, and BAX expression of conditioned H9C2 cells and increased the cell vitality and expression of Bcl-2. These results suggested that pretreatment with CLE confers protection against I/R injury by inhibiting MC degranulation.

Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in vitro by inhibiting NLRP3 Inflammasome activation.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is the most common cause of death worldwide. The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the inflammatory response to MIRI. Dexmedetomidine (DEX), a specific agonist of α2-adrenergic receptor, is commonly used for sedation and analgesia in anesthesia and critically ill patients. Several studies have shown that dexmedetomidine has a strong anti-inflammatory effect in many diseases. Here, we investigated whether dexmedetomidine protects against MIRI by inhibiting the activation of the NLRP3 inflammasome in vitro. METHODS: We established an MIRI model in cardiomyocytes (CMs) alone and in coculture with cardiac fibroblasts (CFs) by hypoxia/reoxygenation (H/R) in vitro. The cells were treated with dexmedetomidine with or without MCC950 (a potent selective NLRP3 inhibitor). The beating rate and cell viability of cardiomyocytes, NLRP3 localization, the expression of inflammatory cytokines and NLRP3 inflammasome-related proteins, and the expression of apoptosis-related proteins, including Bcl2 and BAX, were determined. RESULTS: Dexmedetomidine treatment increased the beating rates and viability of cardiomyocytes cocultured with cardiac fibroblasts. The expression of the NLRP3 protein was significantly upregulated in cardiac fibroblasts but not in cardiomyocytes after H/R and was significantly attenuated by dexmedetomidine treatment. Expression of the inflammatory cytokines IL-1ß, IL-18 and TNF-α was significantly increased in cardiac fibroblasts after H/R and was attenuated by dexmedetomidine treatment. NLRP3 inflammasome activation induced the increased expression of cleaved caspase1, mature IL-1ß and IL-18, while dexmedetomidine suppressed H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. In addition, dexmedetomidine reduced the expression of Bcl2 and BAX in cocultured cardiomyocytes by suppressing H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. CONCLUSION: Dexmedetomidine treatment can suppress H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts, thereby alleviating MIRI by inhibiting the inflammatory response.