INTRODUCTION
Sepsis is a life-threatening organ dysfunction that develops as a dysregulated host response to an infection and is associated with a high risk of death (1). Approximately 30 million patients suffer from severe sepsis each year, which results in potentially 6 million deaths, and the incidence rate of sepsis has increased by 1.5% to 8% worldwide (2, 3). The incidence of sepsis is high, and this syndrome remains a major cause of death worldwide. However, sepsis treatment remains an enormous problem in the medical field, and no effective therapy for sepsis has been established (4). Sepsis-associated encephalopathy (SAE) is a reversible brain dysfunction syndrome induced by a systemic response to sepsis that occurs outside the central nervous system (5). As a risk factor for disability and mortality, SAE affects approximately one-third of septic patients (6).
As the center of the energy metabolism in cells, mitochondria provide nearly 90% of the energy for cellular activities (7). Ultrastructural damage to mitochondria and mitochondrial dysfunction has been recognized as an important molecular pathology in sepsis (8). In addition, the depletion of adenosine triphosphate (ATP) and intracellular antioxidant systems, and inhibition of the respiratory chain have been observed in both septic patients and septic animal models (9). Mitochondrial biogenesis is the process through which pre-existing mitochondria grow and divide into new mitochondria. Stimulation of mitochondrial biogenesis could offer a new therapeutic approach for mitochondrial dysfunction in sepsis (10). The peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) plays a central role in the process of mitochondrial biosynthesis (10). PGC-1α, which stimulates mitochondrial biogenesis through its deacetylation or phosphorylation (10), strongly activates nuclear factor E2-related factor 2 (NRF2) and regulates the expression of mitochondrial genes encoded by the nucleus, such as mitochondrial transcription factor A (Tfam) (11). Previous studies have also reported that enhancing mitochondrial biogenesis can both improve mitochondrial function and relieve organs in sepsis (12, 13), and SR-18292 (PGC-1α inhibitor) can effectively suppress the activation of PGC-1α (14, 15).
Ohsawa et al. (16) first reported that 2% to 4% hydrogen gas (H2) exerts a therapeutic antioxidant effect by reducing the levels of two highly toxic reactive oxygen species, namely, hydroxyl radical and peroxynitrite. We have found that 2% H2 effectively alleviates sepsis-induced multiorgan dysfunction (lung, liver, kidney, heart, and brain) through its anti-inflammation, antioxidation, and anti-apoptotic effects (2, 17, 18). Moreover, the regulation of mitochondrial function and dynamics might be another mechanism that H2 protects against sepsis-induced organ injury (7).
In this study, we investigated the hypotheses that H2 alleviates sepsis-induced brain injury in mice by activating mitochondrial biogenesis and improving mitochondrial function and that the mechanism might be associated with the upregulation of PGC-1a expression.
MATERIALS AND METHODS
Animals
All experimental protocols were approved by the Animal Experimental Ethics Committee of Tianjin Medical University General Hospital, Tianjin, China. Male C57BL/6J mice purchased from the Laboratory Animal Center of the Military Medical Science Academy (Beijing, China) aged 6 to 8 weeks and weighing 20 g to 25 g were used in this study. The mice were housed under controlled conditions (room temperature of 20°C to 22°C and a 12-h light/12-h dark cycle) and fed chow and water ad libitum.
Reagents
The PGC-1a inhibitor SR-18292 (#YH-101491) was obtained from MedChemExpress (NJ, USA). Nissl staining solution (Cresyl Violet) (#G1430) and mitochondrial respiration complex I (#BC0515) and II (#BC3230) activity assay kits were purchased from Beijing Solarbio Science & Technology (Beijing, China), and the in-situ cell detection kit (AP, #11684809910) for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining was purchased from Roche Diagnostics (Minn, USA). Tissue mitochondria isolation kit (#C3606), mitochondrial membrane potential assay kit with JC-1 (#C2006), and enhanced ATP assay kit (#S0027) were provided by Beyotime Institute of Biotechnology (Nanjing, China). Antibodies against PGC-1α, NRF2, and Tfam were obtained from Abcam (Cambridge, UK), anti-acetylated-lysine was provided by Cell Signalling Technology (Mass, USA), the antibody against β-actin was purchased from Hangzhou HuaAn Biotechnology (Hangzhou, China), and goat antimouse IgG and goat anti-rabbit IgG antibodies were from KPL (Md, USA).
Sepsis model
The classical cecal ligation and puncture (CLP) model was applied in this experiment (19). The mice were anesthetized through the intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg). In a sterile surgical environment, a 1-cm midline abdominal incision was cut to expose the caecum, and the caecum was then ligated below the ileocecal valve and punctured with a 20-gauge needle. After the needle was removed, a small amount of faeces was extruded from the puncture point. The bowel was returned to the abdomen, and the incision was closed with a sterile 6-0 silk suture. Each animal was subcutaneously injected with 1 mL of saline solution for resuscitation.
Hydrogen gas treatment
According to our previous study (7), the animals were placed in a box with inflow and outflow outlets for H2 treatment. H2 was administered through a TF-1 gas flow meter (YUTAKA Engineering Corp, Tokyo, Japan) mixed with air at a rate of 4 L/min in the box. The H2 concentration in the box was continuously monitored using a detector (HY-ALERTA Handheld Detector Model 500, H2 Scan, Valencia, Calif) and was maintained at 2% throughout the therapy. The oxygen concentration in the box was maintained at 21% and continuously monitored with a gas analyzer (Medical Gas Analyser LB-2, Model 40 M; Beckman Coulter, Inc, Fullerton, Calif). Carbon dioxide was removed from the gas mixtures in the boxes using Baralyme. The mice that were not administered the H2 treatment inhaled room air in the same box.
Experimental protocols
Experiment 1
Effects of H2 treatment on mitochondrial function and mitochondrial biogenesis in brain tissue of mice with sepsis-induced brain injury
A total of 328 mice were randomly divided into four groups (n = 82 per group): sham, sham + H2, CLP, and CLP + H2. The animals in the CLP and CLP + H2 groups were subjected to cecal ligation and puncture, whereas the animals in the sham and sham + H2 groups were only subjected to a sham operation without caecal ligation and puncture. The mice with hydrogen gas therapy inhaled 2% H2 for 1 h starting at 1 and 6 h after surgery, respectively. As controls, the mice in the sham and CLP groups inhaled air only. The 7-day survival rate of the mice in each group was observed after the operation. On days 3, 5, and 7 after the operation, the working memory of the mice was assessed using the Y-maze spontaneous alternation test. The brain histopathology, number of normal neurons, and neuronal apoptosis were observed 24 h after surgery. Twenty-four hours after CLP, the morphology of the mitochondria was observed using a transmission electron microscope. The mitochondrial membrane potential (MMP), ATP levels, and mitochondrial respiration complex (I and II) activities at 24 h after the operation were analyzed using commercial kits, and the expression of mitochondrial biogenesis-related proteins (PGC-1α, NRF2, and Tfam) was detected by Western blotting at 6, 12, and 24 h after surgery.
Experiment 2
H2 improves mitochondrial function and activates mitochondrial biogenesis through the upregulation of PGC-1α
A total of 232 mice were randomly divided into four groups (n = 58 per group): CLP, CLP + H2, CLP + SR-18292, and CLP + SR-18292 + H2. The above-described procedures were used to establish the sepsis model and for the hydrogen gas treatment. The animals in the CLP + SR-18292 and CLP + SR-18292 + H2 groups received an intraperitoneal injection of SR-18292 (45 mg/kg, dissolved in Dimethyl sulfoxide) 1 h before CLP. The 7-day survival rates of the mice in each group were observed. On days 3, 5, and 7 after the operation, we assessed the working memory of the mice through the Y-maze spontaneous alternation test. Twenty-four hours after surgery, the brain histopathology and number of normal neurons were observed. The mitochondrial morphology was assessed 24 h after CLP. Twenty-four hours after surgery, the MMP, ATP levels, and mitochondrial respiration complex I activities were analyzed, and the expression of mitochondrial biogenesis-related proteins (PGC-1α, NRF2, and Tfam) was detected by Western blotting.
Y-maze
The Y-maze consists of three arms (denoted arms A, B, and C) placed at an angle of 120° from each other. We scored the number of alternations as the number of times that each mouse visited all three arms without entering the same arm twice in a row, ie, the number of times that the mice exhibited the ABC, bicinchoninic acid (BCA), or CAB pattern. Each mouse was placed at the center of the maze and allowed to explore the three arms freely for 5 min. The ANY-maze video tracking system (Stoelting, USA) was used to record the number of line crossings and the number of alternations and analyze the movement of the mice to calculate the alternation percentages.
Nissl staining
Twenty-four hours after the operation, the mice were deeply anesthetized, and their brains were obtained for the assessment of hippocampal damage. The mice were perfused transcardially with 4% paraformaldehyde, and the samples were post-fixed with 10% formalin for 24 h and embedded in paraffin. After deparaffinization and rehydration with dimethylbenzene and ethanol, coronal sections with a thickness of 5 μm were used for Nissl staining. The histopathological changes in the hippocampal CA1 region were observed under a light microscope (CKX41, Tokyo, Japan) at ×400 magnification.
TUNEL staining
Twenty-four hours after the operation, neuronal apoptosis was observed using the TUNEL assay. The nucleotide-labeling mix (TUNEL Label) contains fluorescein-dUTP and fluorescein-dNTPs, which are both needed for the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) reaction for the in situ detection of apoptosis. The nucleotide-labeling mix was used in combination with the TUNEL enzyme to prepare the TUNEL reaction mixture. DNA-strand breaks were labeled using this reaction mixture to detect and quantify the degree of apoptotic cell death at a single-cell level in cells and tissues. The apoptotic cells were stained green, and the nuclei in all the cells were stained blue by DAPI.
Mitochondrial morphology
Twenty-four hours after CLP, the mitochondrial morphology was observed by transmission electron microscopy. Hippocampal tissues were cut into 1-mm cubes, fixed with 2.5% glutaraldehyde, and stored at 4°C for 24 h. The cubes were embedded in Spurr's resin, cut into 0.12-μm-thick sections, and stained with 0.2% lead citrate and 1% uranyl acetate. The images were examined with a transmission electron microscope (JEM-1200X, Shimadzu, Japan).
Preparation of isolated mitochondria
Intact mitochondria from the mice hippocampus were isolated with a commercial kit following the instructions provided (Beyotime, Shanghai, China). Twenty-four hours after the operation, the mice were sacrificed, and the hippocampus was cut into small pieces. Precooled mitochondria isolation reagent A and the tissue pieces were added to a homogenizer, and the mixture was fully homogenized. The homogenate was transferred into centrifuge tubes, and the homogenate was centrifuged at 600 g and 4°C for 5 min. The supernatant was collected and centrifuged at 11,000 g and 4°C for 10 min. The supernatant was discarded, and the resulting precipitates, which consisted of isolated mitochondria, were gently resuspended in mitochondria storage medium. All the steps were completed at 0°C to 4°C. The protein concentration was determined using the BCA protein assay reagent.
JC-1 assay of the mitochondrial membrane potential
The mitochondria membrane potential was detected using the mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China) per the manufacturer's instructions. If mitochondria have a high membrane potential, JC-1 can be aggregated in the mitochondrial matrix to form a polymer (J-aggregates), which can produce red fluorescence. However, if the mitochondrial membrane potential is low, JC-1 cannot be aggregated and remains in monomer form, which exhibits green fluorescence. The red and green fluorescence intensities can be detected using a multifunctional enzyme labeling instrument (EnSpire, PerkinElmer, Mass). A higher ratio of red to green fluorescence indicates a higher mitochondrial membrane potential.
ATP content determination
The ATP content was determined using an enhanced ATP assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Firefly luciferase catalyzes the generation of fluorescence by a luciferin, and this reaction requires energy provided by ATP. In the presence of excessive firefly luciferase and luciferin, the fluorescence production is proportional to the concentration of ATP in a certain concentration range. The fluorescence intensity can be detected using a multifunctional enzyme labeling instrument (EnSpire, PerkinElmer, Mass).
Mitochondrial-respiration chain complex activity
The activities of mitochondrial-respiration chain complexes I and II were determined using assay kits (Solarbio, Beijing, China) per the manufacturer's instructions. Complex I can catalyze the dehydrogenase of 1,4-dihydronicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide, and the oxidation rate of NADH can be measured at 340 nm to calculate the activity of the enzyme. The catalytic product of complex II, reduced coenzyme Q, can further reduce 2,6-dichloroindole phenol, which has a characteristic absorption peak at 605 nm. The enzyme activity was calculated by detecting the reduction rate of 2,6-dichloroindoleol.
Western blotting analysis
Specimens were homogenized in precooled radioimmunoprecipitation assay buffer, sodium dodecyl sulphate, and protease inhibitors and centrifuged at 12,000 rpm for 15 min, and the supernatant was the total protein. Equal amounts of protein lysates were separated by dodecyl sulfate,sodium salt (SDS)-Polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk at room temperature for 1 h and then incubated overnight with the following primary antibodies at 4°C: PGC-1α (1: 1,000), NRF2 (1: 1,000), Tfam (1: 1,000), and β-actin (1: 2,000). The membranes were then washed five times with TBS-T and incubated with horseradish peroxidase -conjugated secondary antibody (1:8,000) for 1 h. The Western blotting bands were imaged with Protein Simple (USA) and analyzed with ImageJ software.
Statistical analysis
The statistical analyses were performed using GraphPad Prism statistical software. We performed power analysis to calculate the number of experimental mice. The survival rates of the different groups are reported as percentages (%), and the other data are reported as the means ± standard deviations. The survival rates were analyzed using the log-rank (Mantel-Cox) test. The statistical significance of the differences in the data, except for the survival rate, was evaluated by one-way analysis of variance. P < 0.05 was considered to indicate significance.
RESULTS
Hydrogen gas improved the survival rate and alleviated the pathological changes and apoptosis of neurons
The 7-day survival rate of the mice belonging to the sham and sham + H2 groups was 100%, and hydrogen gas clearly promoted the survival rate of septic mice (Fig. 1A; P < 0.05). The analysis of the sham and sham + H2 groups revealed closely arranged pyramidal neurons in the hippocampal CA1 region, and abundant Nissl bodies in the cytoplasm of the neurons (Fig. 1B). However, the pyramidal neurons exhibited a sparse arrangement with dissolved Nissl bodies in the mice belonging to the CLP group. After treatment with hydrogen gas, the pyramidal neurons in the CLP + H2 group showed more Nissl bodies than those in the CLP group (Fig. 1B). To visualize the effect of hydrogen or sepsis on neurons, we calculated the number of normal neurons in all the groups. The numbers of normal neurons in the CLP groups were significantly decreased, and hydrogen gas treatment reduced the damage to normal neurons induced by sepsis (Fig. 1C; P < 0.05). Neuronal apoptosis in the hippocampal CA1 region was detected by TUNEL staining (Fig. 1D). The results revealed few apoptotic neurons in the sham and sham+H2 groups, whereas many TUNEL-positive cells were observed in the CLP group, and fewer TUNEL-positive cells were found after treatment with hydrogen gas.
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Fig. 1:
Hydrogen gas improved the survival rate and alleviated the pathological changes and apoptosis of neurons. A, Hydrogen gas inhalation improved the 7-day survival rate of septic mice. B, Nissl bodies in neurons were observed by Nissl staining, and hydrogen gas improved the pathological changes of hippocampal tissue. C, The number of normal neurons was calculated based on Nissl staining. D, Apoptotic neurons were observed by TUNEL staining. The apoptotic cells were stained green, and the nuclei were stained blue. The values in (A) are expressed as the survival rates (n = 20, log-rank test). ∗ P< 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. The data in (C) are expressed as the means ± SDs (n = 6, one-way analysis). ∗ P< 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. CLP indicates cecal ligation and puncture; SD, standard deviation.
Hydrogen gas treatment ameliorated damage to cognitive function and mitochondrial dysfunction
To evaluate the cognition function of mice, we tested the working memory of the mice through the Y-maze spontaneous alternation test (Fig. 2, A and B). At days 3 to 7 after the operation, the percentage of alternation obtained for the mice belonging to the sham group was significantly decreased compared with those found for the CLP and CLP + H2 groups, whereas H2 treatment (CLP + H2 group) clearly increased the percentage of alternation compared with that found for the CLP group (Fig. 2A; P< 0.05). However, no significant difference in the number of line crossings was found among the four groups (Fig. 2B; P> 0.05).
Fig. 2:
Hydrogen gas treatment ameliorated damage to cognition function and mitochondrial dysfunction. A, B, The cognition function at days 3 to 7 after the operation was tested using the Y-maze test. The percentage of alternation was assessed using the spontaneous alternation test, and the number of line crossings was unchanged. C, The JC-1 aggregate/monomer ratio was used to assess the MMP. D, The ATP content was detected based on fluorescein. E, F, The activities of mitochondrial-respiration chain complexes I and II were detected 24 h after the operation. The data are expressed as the means ± SDs (n = 8 in (A, B), n = 6 in (C, F), one-way analysis). ∗ P< 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. CLP indicates cecal ligation and puncture; MMP, mitochondrial membrane potential; SD, standard deviation.
One of the functions of the mitochondria is to produce ATP. Moreover, the MMP and mitochondrial-respiration chain complex activity (I and II) play important roles in the process of ATP production. We found that the MMP, ATP content, and mitochondrial-respiration chain complex I activity were decreased in septic mice (Fig. 2, C–E; P< 0.05). The MMP, ATP content, and mitochondrial-respiration chain complex I activity were obviously increased in the CLP + H2 group compared with the CLP group (Fig. 2, C–E; P< 0.05). However, no significant difference in mitochondrial-respiration chain complex II activity was found among the groups (Fig. 2F; P > 0.05).
Hydrogen gas reduced the degree of mitochondrial damage
In the sham and sham + H2 groups, the mitochondrial ridges were intact and showed no obvious swelling (Fig. 3, A and B), whereas the mitochondria in the CLP group underwent vacuolation and exhibited no mitochondrial ridges (Fig. 3C). In contrast, the mitochondria in the CLP + H2 group exhibited slight swelling, and some of the mitochondrial ridges disappeared (Fig. 3D).
Fig. 3:
Hydrogen gas reduced mitochondrial damage. A, B, Mitochondrial ridges were intact without swelling. C, The mitochondria became vacuolated, and mitochondrial ridges could not be observed. D, Some mitochondrial ridges disappeared, and the mitochondria exhibited slight swelling.
Hydrogen gas inhalation increased the expression of mitochondrial biogenesis-related proteins
Mitochondrial biogenesis is the process through which the production of new mitochondria plays a role in regulating mitochondrial function. The expression of PGC-1α, NRF2, and Tfam in the hippocampus was increased 6, 12, and 24 h after the induction of sepsis (Fig. 4, A–L; P< 0.05). However, hydrogen gas treatment further increased the expression of PGC-1α, NRF2, and Tfam (Fig. 4, A–L; P< 0.05).
Fig. 4:
Hydrogen gas inhalation increased the expression of mitochondrial biogenesis-related proteins. A–D, The expression of PGC-1α, NRF2, and Tfam 6 h after the operation was detected by Western blotting. The results from quantitative analyses of PGC-1α, NRF2, and Tfam are shown. E–H, The expression of PGC-1α, NRF2, and Tfam expression 12 h after the operation was detected by Western blotting. The results from quantitative analyses of PGC-1α, NRF2, and Tfam are shown. I–L, The expression of PGC-1α, NRF2, and Tfam expression 24 h after the operation was detected by Western blotting. The results from quantitative analyses of PGC-1α, NRF2, and Tfam are shown. The data are expressed as the means ± SDs (n = 6, per time, one-way analysis). ∗ P< 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. CLP indicates cecal ligation and puncture; NRF2, nuclear respiratory factor 2; PGC-1α, peroxisome proliferator-activated receptor gamma co-activator 1α; SD, standard deviation; Tfam, mitochondrial transcription factor A.
The effects of hydrogen gas on survival rate and pathological changes were reversed by SR-18292, an inhibitor of PGC-1α
All the mice in the CLP + SR-18292 group died within 4 days after CLP, and all the mice in the CLP + SR-18292 + H2 group were dead 5 days after CLP (Fig. 5A). The survival rate of both of these groups was markedly decreased compared with that of the CLP + H2 group (Fig. 5A). The pyramidal neurons of the CLP + SR-18292 + H2 group showed a sparse arrangement, and the number of normal neurons in this group was significantly decreased compared with that in the CLP + H2 group (Fig. 5, B and C; P< 0.05).
Fig. 5:
The effects of hydrogen gas on the survival rate and pathological changes were deteriorated by SR-18292, an inhibitor of PGC-1α. A, The 7-day survival rates of the septic mice in each group are shown. B, The Nissl bodies in neurons and pathological changes were observed by Nissl staining. C, The number of normal neurons was calculated through Nissl staining. The values in (A) are expressed as the survival rates (n = 20, log-rank test). # P< 0.05 vs. the CLP group; & P< 0.05 vs. the CLP + H2 group. The data in (C) are expressed as the means ± SDs (n = 6, one-way analysis). # P< 0.05 vs. the CLP group; & P< 0.05 vs. the CLP + H2 group. CLP indicates cecal ligation and puncture; PGC-1α, peroxisome proliferator-activated receptor gamma co-activator 1α; SD, standard deviation.
SR-18292 inhibited the improvement in cognition and mitochondrial function
The Y-maze test showed that SR-18292 reduced the hydrogen gas-mediated increase in the percentage of alternation (Fig. 6A; P< 0.05). However, no significant difference in the number of line crossings was found among the four groups (Fig. 6B; P> 0.05). The MMP, ATP content, and mitochondrial-respiration chain complex I activity obtained in the CLP + SR-18292 + H2 group were clearly decreased compared with those found in the CLP + H2 group (Fig. 6, C–E; P< 0.05).
Fig. 6:
SR-18292 inhibited improvements in cognition function and mitochondrial function. A, B, The cognition function 3 days after the operation was assessed using the Y-maze test. The percentage of alternation was determined with the spontaneous alternation test, and the number of line crossings was unchanged. C, The JC-1 aggregate/monomer ratio was used to calculate the MMP. D, The ATP content was detected based on fluorescein. E, The mitochondrial-respiration chain complex I activity was detected 24 h after the operation. The data are expressed as the means ± SDs (n = 8 in (A, B), n = 6 in (C–E), one-way analysis). # P< 0.05 vs. the CLP group; & P< 0.05 vs. the CLP + H2 group. ATP indicates adenosine triphosphate; CLP, cecal ligation and puncture; MMP, mitochondrial membrane potential; SD, standard deviation.
SR-18292 inhibited the activation of PGC-1α and decreased the expression of mitochondrial biogenesis-related proteins
The injection of SR-18292 significantly increased the expression of acetyl-PGC-1α compared with that found in the CLP + H2 group (Fig. 7, A and B; P< 0.05). Moreover, the expression levels of NRF2 and Tfam were decreased in the CLP + SR-18292 + H2 group (Fig. 7, A, C, D; P< 0.05).
Fig. 7:
SR-18292 decreased the expression of mitochondrial biogenesis-related proteins. A–D, The expression of acetyl-PGC-1α, NRF2, and Tfam 24 h after the operation was detected by Western blotting. The results from quantitative analyses of acetyl-PGC-1α, NRF2, and Tfam are shown. The data are expressed as the means ± SDs (n = 6, one-way analysis). # P< 0.05 vs. the CLP group; & P< 0.05 vs. the CLP + H2 group. CLP indicates cecal ligation and puncture; NRF2, nuclear respiratory factor 2; PGC-1α, peroxisome proliferator-activated receptor gamma co-activator 1α; SD, standard deviation; Tfam, mitochondrial transcription factor A.
DISCUSSION
As a worldwide disease, sepsis has an unacceptably high incidence and is associated with a high clinical cost and high mortality. In 2017, an estimated 48.9 million incident cases of sepsis were recorded worldwide and 11.0 million sepsis-related deaths were reported, representing 19.7% of all global deaths (20). The comprehension of this problem by clinicians and lay persons has increased over the past 10 years, and improved outcomes have been observed. In 2017, the World Health Assembly and WHO made sepsis a global health priority and adopted a resolution to improve the prevention, diagnosis, and management of sepsis (21). The brain damage induced by SAE is one of the earliest and most frequent components of organ injury in sepsis, and our previous studies revealed that the inhalation of 2% H2 can be a therapeutic strategy to protect against septic brain injury (22, 23). In this study, sepsis model was developed successfully, and brain injury was observed in septic mice 24 h after sepsis. The survival rate was clearly decreased in sepsis. The Y-maze test was used to evaluate the working memory, which reflects the short-term memory, of mice (23), and the results showed that the working memory of mice in sepsis was significantly decreased compared with that of sham group. In addition, sepsis presented a lower number of normal neurons and a higher number of apoptotic cells compared with the sham group. However, 2% H2 treatment improved the survival rate, working memory, pathological changes, and neuronal apoptosis rate in sepsis mice.
Mitochondria, as the “powerhouses” of eukaryotic cells, provide the energy needed for cellular metabolism through oxidative phosphorylation. Mitochondria not only generate ATP but also participate in cell physiology and pathology, such as ion homeostasis, cell redox, transport of metabolites, and cell death (7). The close interaction between mitochondria and cell function explains the many important roles played by mitochondria and mitochondrial dysregulation in diverse diseases, such as neurological disease, cancer, and metabolic disease (24). Mitochondrial dysfunction has been regarded as a critical event in sepsis-induced multiple organ dysfunction syndrome (25). To evaluate the effects of H2 on sepsis-induced mitochondrial dysfunction, we detected the generation of ATP, the change in the MMP and the activities of mitochondrial-respiration chain complexes I and II using commercial kits.
As a mitochondrial electrochemical gradient, the MMP is generated by the oxidative phosphorylation system, which includes the electron transport chain (ETC) and ATP synthase (26). The transport of H+ across the inner mitochondrial membrane is key to the formation of the MMP (7). The energy used to pump H+ across the inner mitochondrial membrane is provided by the ETC, which comprises a group of enzymes that are arranged in an orderly manner on the inner mitochondrial membrane and transport electrons. In the ECT, mitochondrial-respiration chain complexes I (NADH dehydrogenase) and II (succinate dehydrogenase) are major sites for electron entry. Electrons are transferred to complex III (bc1 complex) from complex I or II and then to complex IV (cytochrome c oxidase) via cytochrome c and eventually are donated to O2(26). After the above process, the ETC extracts the energy from electrons to H+ for the formation of the MMP. Therefore, the MMP constitutes an intermediate form of energy storage that provides energy for the synthesis of ATP by ATP synthase (7, 27). Therefore, stability of both intracellular ATP and the MMP is necessary for normal cell function. It has been reported that hydrogen gas protects against sepsis-induced acute lung injury via regulation of mitochondrial function (7). Our results showed that the MMP, ATP content, and complex I activity are decreased in septic mice, whereas H2 treatment clearly increased the MMP, ATP content, and complex I activity. However, no significant difference in complex II activity was found among the four groups. These results indicated that H2 treatment can improve mitochondrial function and that the mechanism might be related to the activation of complex I but not complex II.
Mitochondria are highly dynamic organelles that maintain homeostasis through balanced mitochondrial biogenesis, mitochondrial fission, and fusion and mitophagy (28). As a process involving the generation of new mitochondria, mitochondrial biogenesis is crucial for maintaining both the quantity and the quality of mitochondria. Peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1alpha (PGC-1α) is a master regulator and crucial metabolic node of mitochondrial biogenesis (29). PGC-1α can be activated by phosphorylation or deacetylation, and activated PGC-1α activates NRF2 and Tfam and thus promotes the synthesis of mitochondrial DNA (11). Both clinical and animal trials have shown that mitochondrial biogenesis is associated with the prognosis of sepsis (13, 30). In this study, the expression levels of PGC-1α, NRF2, and Tfam were increased in the septic mice compared with the sham group, and H2 treatment further increased the levels of those proteins. This finding indicated that H2 increases mitochondrial biogenesis in septic mice.
PGC-1α plays an important role in regulating the process of mitochondrial biogenesis, and the inhibitory effect of SR-18292 on PGC-1α has been confirmed (15). The deacetylation of PGC-1α serves as foundation for the function of PGC-α (11). However, SR-18292 effectively inhibits the deacetylation of PGC-1α (14). We injected SR-18292 into the abdominal cavity of mice and found that SR-18292 deteriorated the effect of hydrogen gas on sepsis-induced brain injury and mitochondrial dysfunction. The survival rate, number of normal neurons, percentage of alternation, MMP, ATP content, and mitochondrial-respiration chain complex I activity were decreased in the CLP + SR-18292 + H2 group compared with the CLP + H2 group. SR-18292 increased the expression of acetyl-PGC-1α and inhibited the activation of PGC-1α. Moreover, the expression levels of NRF2 and Tfam were also reduced by the intra-abdominal injection of SR-18292.
In conclusion, our research indicates that H2 might be an effective therapeutic strategy for sepsis-induced brain injury. This study also verifies that the protective effect of H2 on sepsis-induced brain injury involves the improvement of mitochondrial biogenesis through the activation of PGC-1α.
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