Sepsis by systemic inflammation following bacterial infection is a common syndrome. There are about 1 million cases of sepsis reported in the USA annually, half of which require intensive care (1). Septic shock is associated with acute respiratory distress, cardiac dysfunction, disseminated intravascular coagulation, and a high mortality (2).
Cardiac dysfunction, characterized by compromised systolic function, reduced fractional shortening, and decreased cardiac index, is one of the most severe complications of sepsis (3). It is reported that sepsis complicated by cardiac dysfunction has a higher mortality than sepsis without cardiac dysfunction (4).
There are several reasons for cardiac dysfunction during sepsis, including cardiac inflammation, impaired cardiac energy production, β-adrenergic signaling disorder, and others (5). Of these, impaired cardiac energy production is considered one of the most important causes of cardiac dysfunction in sepsis (6). Fatty acid and glucose oxidation are the main sources of cardiac energy, both of which are strongly suppressed during sepsis (7). Restoring the cardiac energy supply may be a promising way to treat cardiac dysfunction during sepsis.
Currently, accumulated evidence shows that hydrogen gas or hydrogen-rich saline (HRS) protects against ischemic reperfusion injury, stroke, sepsis, and organ transplantation (8, 9)(8, 9). In mice with induced sepsis, treatment with hydrogen increases survival (10). In the present study, we hypothesized that HRS attenuates lipopolysaccharide (LPS)-induced cardiac dysfunction by restoring fatty acid oxidation (FAO) and thus preserving production of cardiac energy.
MATERIALS AND METHODS
Pathogen-free male Sprague-Dawley rats (body weight: 200–250 g, age: 8–10 weeks) were provided by the experimental animal center of the General Hospital of Shenyang Military Region (Shenyang, Liaoning, China). The animals were housed at 20 to 22°C, 12:12-h light–dark cycle. Animals were handled as approved by the Institutional Animal Care and Use Committee of China Medical University in accordance with the National Institutes of Health (Bethesda, MD) guidelines for the care and use of laboratory animals.
Preparation of HRS
HRS was prepared in accordance with previous studies (11). Briefly, hydrogen gas was dissolved in normal saline (NS) under 0.4 megapascal (MPa) pressure for 6 h, and stored in an aluminum bag without dead volume at 4°C under atmospheric pressure and then sterilized by γ-radiation. To ensure that the hydrogen level in the NS was at least 0.6 mmol/L, gas chromatography was performed as described by Ohsawa et al. (12).
Rats were randomly divided into four groups: a control group (CTRL), an HRS group (HRS), an LPS group (LPS), and an HRS+LPS group. Rats in the LPS group (n = 8) were injected intraperitoneally (i.p.) with 10 mg/kg LPS (Escherichia coli; Sigma, St. Louis, MO). Rats in the HRS group (n = 8) received an i.p. injection of 10 mL/kg of HRS. Rats in the HRS+LPS group (n = 8) received 10 mL/kg of HRS by i.p. injection 1 h and 4 h postadministration of 10 mg/kg of LPS. Rats in the control group (n = 8) received only an i.p. injection of 10 mL/kg of NS.
The effect of JNK inhibitor SP600125 (Sigma) on heart function was investigated in another 23 rats: eight of them received an i.v. injection of 30 mg/kg JNK inhibitor SP600125 30 min prior to an LPS challenge; the other eight rats received only a 30 mg/kg i.v. injection of JNK inhibitor SP600125. Because dimethyl sulfoxide (DMSO) was used to dissolve JNK inhibitor SP600125 (the concentration of the SP600125 DMSO stock was 30 mg/mL), five rats received an i.v. injection of the same amount of DMSO as control.
Fur was removed from the chests of all rats 1 day before the experiment. After echocardiographic examination, the rats were anesthetized by i.p. injection of 250 mg/kg of chloral hydrate and sacrificed by opening chest and drawing blood from hearts. Heart samples were collected for further pathological and molecular tests.
At 8 h post-LPS treatment, rats were fixed in a cone-shaped plastic bag for two-dimensional echocardiography with a Vevo770 system (VisualSonics Inc, Toronto, ON, Canada). The left ventricular fractional shortening and the left ventricular end-systolic dimension (LVDs) were measured at M mode. The echo examiner was blind to the treatments of the rats.
Gene expression quantification
Trizol reagent (Ambion, Carlsbad, CA) was used to isolate total ribonucleic acid (RNA) from each 100 mg rat heart sample according to the manufacturer's protocol. After isolation, concentrations of the total amount of RNA were determined by spectrometry. A SuperScript III First-Strand Synthesis kit (Invitrogen, Carlsbad, CA) was used to facilitate reverse transcription of 1 μg of the total RNA into complementary DNA (cDNA). The mRNA was quantified in an ABI 7500 fast real-time PCR machine (Applied Biosystems, New York, NY) using a SYBR Green PCR Master Mix (Invitrogen). Quantitative PCR primers used in this experiment are shown in Table 1.
Western blot analysis
Fresh rat heart tissues (150 mg per animal) were homogenized in a 1 mL cold Radio-Immunoprecipitation Assay (RIPA) buffer, consisting of 25 mM Tris–HCl pH 7.6, 1% sodium deoxycholate, 1% Nonidet P-40 (NP-40), 1% protease inhibitor cocktail, 150 mM NaCl, and 0.1% SDS. The homogenates were centrifuged at 1,500 g for 10 min at 4°C, and supernatants were collected for the Western blot analysis.
For the Western blot analysis, 20 μg of tissue homogenate protein was separated by a 4% to 15% gradient SDS-PAGE (Bioon, Shanghai, China) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Germany). The membrane was blocked in 5% fat free milk at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C. Goat anti-PGC-1α, rabbit anti-ERRα, rabbit anti-ACADM, and rabbit anti-ACADL were obtained from Sigma (St. Louis, MO). Rabbit anti-JNK and rabbit anti-phosphor-JNK were from Cell Signaling Technology (Beverly, CA). After being washed in Tris-buffered saline with Tween (TBST), the membrane was incubated with anti-goat or anti-rabbit secondary antibodies (1: 5000, Bio-Rad, Hercules, CA) at room temperature for 1 h. Chemiluminescence (Pierce, Rockford, IL) was used to investigate the signal intensities. Mouse monoclonal anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1: 5000, Sigma) was used as the loading control.
Myocardial ATP and phosphocreatine (PCr) determination
After euthanasia, 200 mg of rat heart tissue from apex was put into cold perchloric acid solution (0.4 mol/L, 2 mL) and homogenized immediately. Then samples were centrifuged at 6,000 g 4°C for 5 min. After centrifuge, 1 mL of supernatant was collected and KOH was used to adjust the pH value of the supernatant to 7.0, then the supernatant was filtered through a 0.45 μm membrane. All procedures were performed on ice and all samples were preserved on ice. The concentrations of ATP and phosphocreatine (PCr) were determined by reversed-phase high-performance liquid chromatography (HPLC, Waters Corporation, Milford, MA): the column temperature was 25°C, the mobile phase was KH2PO4/K2HPO4 buffer (220 mM; pH 7.0) containing 5% methanol and tetrabutyl ammonium hydroxide (3 mM), the UV wavelength was 205 nm and the sample injection volume was 20 μL. After different concentrations of standard solutions (St Louis, MO) were prepared, the chromatograms of standards and heart samples were acquired at a wavelength of 205 nm. Then the concentrations of ATP and PCr were obtained by measuring the areas under the curve.
Fatty acid oxidation detection
An approximate 100 mg heart sample of each rat was used to investigate the level of fatty acid oxidation. After being incubated in a modified Krebs–Ringer buffer (10 mM HEPES (pH 7.4), 2.6 mM KCl, 115 mM NaCl, 10 mM NaHCO3, 1.2 mM KH2PO4) containing 2% BSA, 10 uCi/mL 9,10-[3H]palmitate and 0.2 mmol/mL palmitate, each heart sample was gassed with 95% O2 and 5% CO2 at 37°C for 2 h. Water was extracted from the sample by chloroform and methanol (2:1). Fatty acid oxidation was measured by the amount of 3H2O in the water.
All values are presented as means ± standard errors. Groups were compared by one-way ANOVA followed by Bonferroni correction. A P value less than 0.05 was considered statistically significant.
HRS preserved LPS-induced heart dysfunction in rats
In this study, we investigated the effect of HRS on the heart function of LPS-challenged rats. The LPS challenge decreased fractional shortening by 44% (P < 0.01, Fig. 1A) and increased LVDs by 42% (P < 0.01, Fig. 1B). However, HRS treatment increased fractional shortening by 33% (P < 0.01, Fig. 1A). HRS also decreased LVDs by 21% (P < 0.05, Fig. 1B). These data suggest that HRS significantly dampens LPS-induced heart dysfunction.
HRS restored myocardial energy metabolism aggravated by LPS challenging in rats
To determine if the improvement in heart function after administration of HRS was attributable to restored myocardial energy metabolism, we investigated the palmitate oxidation, the amount of PCr and ATP, as well as ratio of PCr to ATP of the heart tissue. LPS-challenged rats showed decreased palmitate oxidation (P < 0.01, Fig. 2A), PCr (P < 0.05, Fig. 2B), and PCr/ATP (P < 0.01, Fig. 2C), while HRS treatment significantly increased these measures by 46% (P < 0.05, Fig. 2A), 18% and 25% (P < 0.05, Fig. 2C) respectively. These results suggest that HRS treatment restores LPS-induced impaired myocardial energy metabolism.
HRS prevents LPS-induced down-regulation of PGC-1α, PPARα, and ERRα in rat heart
Between 60% and 90% of the energy needed by the healthy adult heart is produced by fatty acid oxidation (7); PGC-1α, PPARα, and ERRα are the key transcription factors for fatty acid oxidation in the heart (13, 14)(13, 14). In the next step, we investigated these genes in the mRNA and the protein level within this process of fatty acid oxidation. As shown in Figure 3A, compared with the rats in the control group, LPS-challenged rats had markedly decreased levels of PGC-1α, PPARα, and ERRα in the mRNA by 58%, 56%, and 52%, respectively (P < 0.01, Fig. 3A), which were mitigated by HRS administration (P < 0.05, Fig. 3A). In accordance with changes in their mRNA, rats in the LPS group exhibited reduction of PGC-1α, PPARα, and ERRα in their protein level, as shown by Western blot. These reductions were mitigated by HRS treatment (P < 0.05, Fig. 3A and B). These results indicate that HRS prevents LPS-induced down-regulation of PGC-1α, PPARα, and ERRα in the rat heart.
HRS reversed LPS-induced down-regulation of genes expression that was involved in fatty acid oxidation
CD36 and CPT1α, which play an important part in encoding critical proteins required for fatty acid transportation, are the target genes of PGC-1α, PPARα, and ERRα (15). Protein encoded by CD36 facilitates fatty acid transport into cardiomyocytes, while the CPT1α encoded protein plays an important part in transporting fatty acid into the mitochondria. Rats treated with LPS exhibited reductions of 63% and 58% in CD36 and CPT1α, respectively, at the mRNA level (P < 0.01, Fig. 4A). These reductions were attenuated by HRS treatment (P < 0.05, Fig. 4A and B).
Moreover, Acyl-CoA Dehydrogenase, C-2 to C-3 Short Chain (ACADS), Acyl-CoA Dehydrogenase, C-4 to C-12 Straight Chain (ACADM) and Acyl-CoA Dehydrogenase, Long Chain (ACADL) are critical genes encoding proteins required for initializing fatty acid β-oxidation, the down-regulation of which is associated with impaired fatty acid oxidation (7, 14)(7, 14). The LPS challenge decreased ACADS, ACADM, and ACADL mRNA by 65%, 57%, and 60%, respectively. Rats treated with a combination of HRS and LPS showed restored ACADS, ACADM, and ACADL mRNA expression when compared with rats treated only with LPS (P < 0.05, Fig. 4C), confirmed by Western blot analysis (Fig. 4D). These data suggest that HRS prevented LPS-induced down-regulation of genes expression involved in fatty acid oxidation.
HRS shows no effect on LPS-induced repression of glucose metabolism
Fatty acid oxidation accounts for 60% to 90% of the energy production in the healthy adult heart, and glucose metabolism accounts for the rest (7). We knew that HRS attenuated LPS-induced energy repression, but we did not know if increased fatty acid oxidation or glucose metabolism, or both, restored the energy production. To answer this question, we investigated the effect of HRS on cardiac glucose metabolism. In line with previous studies (16, 17)(16, 17), LPS showed an inhibitory effect on cardiac glucose metabolism, characterized by the down-regulation of GLUT4 in mRNA (P < 0.01, Fig. 5A), which plays an important part in transporting glucose into the cell. HRS treatment did not show an effect on the LPS-induced down-regulation of GLUT4 in mRNA.
Furthermore, rats in the LPS group showed a 50% decrease in gene expression of pyruvate dehydrogenase (PDH) (P < 0.01, Fig. 5B), an important enzyme linking glycolysis and oxidation (18), and a 7-fold increase in gene expression of pyruvate dehydrogenase kinase 4 (PDK4) (P < 0.01, Fig. 5C) that inhibits the activity of PDH. As depicted in Fig. 5, HRS treatment showed no effect on LPS-induced down-regulation of PDH and up-regulation of PDK4. To summarize, HRS treatment showed no effect on LPS-induced repression of glucose metabolism.
HRS inhibits LPS-induced activation of JNK in rat hearts
Previous studies (19) have reported that the JNK pathway is activated after an LPS challenge. In the next step, we looked at whether the JNK pathway was activated in the rat heart and if HRS treatment could inhibit the activation of the JNK pathway. Western blot analysis of the rat heart extract showed that JNK was activated by LPS administration and then dampened by HRS treatment (P < 0.05, Fig. 6).
Inhibition of JNK restores cardiac fatty acid oxidation and prevents LPS-induced heart dysfunction
Since the JNK pathway was activated in the LPS-challenged rat heart, we wanted to know if JNK activation was involved in the LPS-induced suppression of fatty acid oxidation and cardiac dysfunction. Rats challenged with the combination of LPS and the JNK inhibitor showed less activation of JNK (Fig. 7A), as well as restored fractional shortening (P < 0.01, Fig. 7B) and energy production (P < 0.05, Fig. 7C) when compared with the LPS-challenged rats.
To investigate whether fatty acid oxidation accounted for the restored energy production, palmitate oxidation of the heart samples was tested. As shown in Fig. 7D, rats treated with the combination of LPS and the JNK inhibitor exhibited a 42% increase in palmitate oxidation (P < 0.05, Fig. 7D) compared with rats treated with LPS alone. Moreover, combined administration of the JNK inhibitor and LPS increased PGC-1α, PPARα, ERRα, CD36, and CPT1α gene expression in the mRNA by 1.3-fold (P < 0.05), 1.1-fold (P < 0.05), 1.2-fold (P < 0.05), 87% (P < 0.05), and 90% (P < 0.05), respectively compared with rats treated only by LPS, which was in accordance with the change in PCr/ATP and palmitate oxidation. It is notable that treatment with the JNK inhibitor alone increased the expression of PGC-1α, PPARα, ERRα, CD36, and CPT1α in the mRNA by 3-fold (P < 0.01), 1.3-fold (P < 0.05), 1-fold (P < 0.05), 43% (P < 0.05), and 57% (P < 0.05), respectively when compared with rats in the control group. These data indicate that JNK inhibitor restored cardiac fatty acid oxidation and prevent LPS-induced heart dysfunction.
In the present study, we found that: HRS i.p. administration restored cardiac function impaired by an LPS challenge. Preserved heart function by HRS treatment was mainly due to rescued cardiac energy metabolism through the restoration of cardiac fatty acid oxidation in rats.
Sepsis is one of the major causes of death in ICUs (1). Patients with sepsis-induced cardiac dysfunction have a higher mortality than those without such cardiac dysfunction (4). Suppressed fatty acid oxidation contributing to impaired cardiac energy metabolism is associated with cardiac dysfunction in sepsis (5). Given that fatty acid oxidation produces 60% to 90% of the energy needed by the adult heart, while glucose metabolism produces the other 10% to 30% (7), accumulated evidence shows that both fatty acid oxidation and glucose metabolism are dramatically suppressed in sepsis-induced heart dysfunction (20). This is different from pressure-overload heart failure in which glucose metabolism is up-regulated to compensate for the decreased fatty acid oxidation (7). Consistent with previous studies (20), our work showed that cardiac fatty acid oxidation was markedly suppressed after an LPS challenge and was not compensated by glucose metabolism.
Since the first report by Ohsawa et al. (12) in 2007 that hydrogen shows benefits against oxidative stress, growing evidence has shown that hydrogen or HRS treatment has a protective effect on sepsis-induced organ dysfunction such as acute lung injury, renal failure, liver injury, and so on (8, 9, 21)(8, 9, 21)(8, 9, 21). There are several possible mechanisms underlying the benefits of molecular hydrogen: first, it was reported that hydrogen can accumulate in the lipid phase of biomembranes, especially in unsaturated lipid regions that are the main targets of initial oxidative damage, and neutralizes the effect of hydroxyl radicals (OH) (22). It is known that OH is the major trigger of the chain reaction of free radicals, activating of which can cause serious cell damage (23). Thus, molecular hydrogen may suppress the chain reaction and protects cells from oxidative damage. Second, molecular hydrogen shows protective effects by directly reducing peroxynitrite (ONOO−) and regulating gene expression (24); and ONOO− is an oxidant that can damage a wide array of molecules in cells, including DNA and proteins (25). Third, hydrogen shows protective effects by inducing the gene expression of antioxidants. It was reported that hydrogen could induce the expression of superoxide dismutase (SOD), Heme oxygenase (HO-1), catalase, and myeloperoxidase (26) that all are antioxidants with protective effects against stress and inflammation. Also many studies have shown that hydrogen influences some signaling pathways, Xie et al. reported that hydrogen ameliorates LPS-induced lung injury by inactivating NF-κB cell signaling (27), another study showed that hydrogen acts as therapeutic gas by inactivating the cell signaling pathway of apoptosis signal-regulated kinase (ASK1), p38 MAP kinase, JNK, and IκBα (28).
The heart acts as an engine, beating about 100,000 times a day to maintain the 10 tons of blood pumped through the whole body every day. An insufficient energy supply results in mechanical heart failure (7). PPARα and ERRα are nuclear-receptor transcription factors controlling the expression of the enzymes related to fatty acid oxidation and energy production (13, 14, 29)(13, 14, 29)(13, 14, 29). PGC-1α, a coactivator that binds to nuclear receptors, such as PPARα, ERRα, and nuclear respiratory factors (NRFs), boosts the transcriptional level of critical genes related to energy metabolism to meet the energy demands of different environments (13, 29)(13, 29). Previous studies have reported that compromised cardiac energy metabolism is associated with the down-regulated expression of PGC-1α, PPARα, ERRα, and downstream genes in animal models of sepsis (20, 30)(20, 30). In line with previous studies, we found that the ratio of PCr to ATP used to evaluate the energy level in the heart was significantly decreased after an LPS challenge. Along with the decreased PCr/ATP, the transcriptional levels of PGC-1α, PPARα, and ERRα were down-regulated by an LPS challenge in the rat's heart. Moreover, the downstream genes involved in fatty acid metabolism such as CD36, which transports fatty acid into the cell; CPT1α, which transports free fatty acids into mitochondria; ACADM, which breaks down medium chain fatty acids ACADL, which breaks down long chain fatty acids; and citrate synthase, a pace-making enzyme in the first step of the citric acid cycle, were all down-regulated. HRS restored these processes.
Systemic inflammation is also thought to be another contributing factor to cardiac dysfunction in septic patients (5). Under the challenge of sepsis, increased circulating inflammatory cytokines, such as tumor necrosis factor α (TNFα) and interleukin (IL)-1, are observed in septic patients and animals with induced sepsis. Neutralizing the circulatory TNFα in animals with induced sepsiss increased survival rates and thus seems to be an effective therapeutic strategy (31). This same strategy, however, was proved to be ineffective in improving mortality in septic patients (32). Likewise, although inhibition of IL-1 improved survival rates in experimental animals with sepsis induced by LPS or cecal ligation and puncture (CLP) (33), this same treatment strategy shows little effect on attenuating mortality in septic patients. In recent years, growing evidence shows that hydrogen gas mitigates sepsis by decreasing systemic inflammatory cytokines such as TNFα, IL-1, and IL-6 (27). Consistent with previous studies, we found that HRS decreased the expression of TNFα and IL-1 in the mRNA (see Figure, Supplemental Digital Content 1, at http://links.lww.com/SHK/A327) in the rat heart, but it was not possible to determine that decreased TNFα and IL-1 after HRS treatment contributes to the restored heart function, after impairment by an LPS challenge. Further work is needed to address this question.
Previous studies (19, 30)(19, 30) have shown that the JNK pathway is activated during sepsis, and the administration of hydrogen gas or HRS inhibits this activation. Moreover, the JNK pathway is found to be involved in tissue energy homeostasis (34). In order to prove that the JNK pathway activation is involved in compromised cardiac energy production, we treated rats with JNK inhibitor SP600125 prior to an LPS challenge and found that JNK inhibitor attenuated LPS-induced suppression of fatty acid oxidation and restored heart function, as shown by increased pamitate oxidation and restored gene expressions associated with fatty acid oxidation, including PGC-1α, PPARα, ERRα, CD36, and CPT1α.
It is known that the NF-κB signaling pathway, which is associated with regulating the expression of cytokines and chemokines, is activated after an LPS challenge (27). An activated NF-κB signaling pathway is involved in a high expression of cytokines, chemokines, and organ injury (5). Reported by Xie et al. (27), hydrogen treatment inhibits NF-κB p65 nuclear translocation and decreases the expression of TNFα and IL-6, thus preventing acute lung injury in rats with induced sepsis. An activated NF-κB signaling pathway is also related to the down-regulation of PGC-1α in rats with induced cardiac hypertrophy (35). In the present study, HRS treatment impeded the LPS-induced down-regulation of the expression of genes related to cardiac fatty acid oxidation, thus restoring heart function. The mechanism of the protective effect of HRS may be multifactorial, so the possibility that an inactivated NF-κB signaling pathway may play a part in this protective effect cannot be excluded. Further work is needed to investigate whether the NF-κB signaling pathway is involved in heart dysfunction during sepsis.
In conclusion, our study revealed that HRS attenuated LPS-induced heart dysfunction through restoring fatty acid oxidation by mitigating c-Jun N-terminal kinase activation in rats.
The experiments in the current study were carried out in the Experimental Center of the Shengjing Hospital, China Medical University, and General Hospital of Shenyang Military Region.
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