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Basic Science Aspects

100% OXYGEN INHALATION PROTECTS AGAINST ZYMOSAN-INDUCED STERILE SEPSIS IN MICE

THE ROLES OF INFLAMMATORY CYTOKINES AND ANTIOXIDANT ENZYMES

Hou, Lichao*; Xie, Keliang*; Li, Nan*; Qin, Mingzhe*; Lu, Yan*; Ma, Shirong; Ji, Genlin*; Xiong, Lize*

Author Information
doi: 10.1097/SHK.0b013e31819c391a

Abstract

INTRODUCTION

Sepsis and the resultant multiple organ failure are the leading cause of death in intensive care units (1). More than 750,000 people become septic each year with a mortality rate of 30% to 40% and an approximate cost of $16.7 billion in the United States alone (1, 2). Sepsis causes high mortality worldwide, and the global market potential for sepsis treatment is estimated at more than $30 billion annually (3). A growing number of studies have found that systemic inflammatory response syndrome plays an important role in the pathogenesis of shock/sepsis/multiple-organ dysfunction syndrome (MODS) and is considered as a common pathway from sepsis/shock to MODS/multiple organ failure (4, 5). Because of the complexity of sepsis pathogenesis, it has been exceedingly difficult to develop measures that will reduce this high mortality. Thus, there is an urgent unmet medical need for an effective novel therapy for septic patients.

Many recent studies focus on early goal-directed therapy attempting to balance oxygen (Oxy) delivery and demand for severe sepsis/MODS (6). It is reported that early hyperbaric Oxy (HBO) treatment can effectively prevent the development of zymosan (ZY)-induced MODS in rats (7). The feasibility of HBO in clinical practice, however, remains limited because the HBO chamber for human is large, expensive, and unavailable universally. Compared with HBO treatment, hyperoxia treatment has been widely used in clinical practice with many advantages such as its safety, ease, and inexpensiveness. However, the application of hyperoxia treatment to the patients with critical diseases is limited because long-term hyperoxia treatment can induce Oxy toxicity associated with overproduction of reactive oxygen species (ROS), and the excessive ROS is directly related to organ injury (8, 9). Recent studies have suggested that ventilation with 100% Oxy can improve the survival rate and organ function in several shock models without affecting lung function and oxidative or nitrosative stress (10-13). A lot of work, however, is necessary to be done before its clinical application, and its detailed mechanism remains to be clarified.

Zymosan, a substance derived from the cell wall of the yeast Saccharomyces cerevisiae, has been used as a tool to induce animal model with sterile sepsis/MODS in many studies (14). The aims of the present study are 1) examining whether 100% Oxy inhalation improves organ function and survival rate in mice with ZY-induced sterile sepsis model and 2) exploring a suitable Oxy treatment protocol for treating sepsis/MODS.

MATERIALS AND METHODS

Animals

Male Imprinting Control Region mice (Specific Pathogen Free) provided by the Laboratory Animal Center of Fourth Military Medical University aged 6 to 8 weeks old and weighing 20 to 25 g were used in all experiments. The animals were housed in plastic boxes at 20°C to 22°C with a constant 12-h light-dark cycle and food and water available ad libitum. One week before experimental manipulation, the animals were allowed to acclimatize the experimental housing facilities. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals, and the experimental protocols were approved by the Institutional Animal Care and Use Committee of Fourth Military Medical University.

ZY-induced sterile sepsis model

Zymosan (Sigma Chemical Co., St. Louis, Mo) solution was prepared in normal saline (NS) to a final concentration of 25 mg/mL and was sterilized at 100°C for 80 min. All suspensions were freshly made before use. Sterile sepsis model was induced by an aseptic injection of ZY at a dose of 1 g/kg of body weight (BW, i.p.) (15). The same volume of NS was injected through the same route as the control.

Oxy treatment

The animals were placed in a sealed Plexiglas chamber. Oxy was delivered into the chamber through a tube at a rate of 4 L/min, and carbon dioxide was removed from the chamber gases with baralyme. The concentration of inspired Oxy and carbon dioxide at the outlet of the chamber was continuously monitored with a gas analyzer (medical gas analyzer LB-2, model 40 M; Beckman, Anaheim, Calif). Oxy concentration was maintained at 100% during the treatment. The room and chamber temperature was maintained at 20°C to 22°C. Air treatment was done with the animals exposed to room air. Food and water were available ad libitum during the treatment.

Experimental design

Experiment One: Effect of 100% Oxy inhalation on ZY-induced sterile sepsis

The animals were randomly divided into 10 groups (Fig. 1A): ZY + Air, ZY + Oxy 1, 2, 3, and 4 h, NS + Air and groups. Sterile sepsis model was induced in the animals of ZY + air and ZY + Oxy 1, 2, 3, and 4h groups as previously mentioned. The animals in NS + Oxy 1, 2, 3, and 4h groups were intraperitoneally given the same volume of NS. The animals in ZY + Oxy 1, 2, 3, 4h and NS + Oxy 1, 2, 3, 4h groups were given Oxy treatment by exposure to 100% Oxy for 1, 2, 3 or 4 h at 4 and 12 h after ZY or NS injection, respectively. The animals in NS + air and ZY + air groups were exposed to air as control.

Fig. 1
Fig. 1:
The schematic diagram for grouping methods and experimental protocols. A, Effect of 100% Oxy inhalation on ZY-induced sterile sepsis. The animals were randomly divided into 10 groups: ZY + Air; ZY + Oxy 1, 2, 3, and 4h; NS + Air; and NS + Oxy 1, 2, 3, and 4h groups. Zymosan or NS were intraperitoneally given in all animals. At 4 and 12 h after ZY or NS injection, the animals were exposed to 100% Oxy or room air for 1, 2, 3, or 4 h, respectively. The survival rates were recorded on days 0.5, 1, 1.5, 2, 3, 5, and 14 after ZY or NS injection. The blood samples and organs of all survived animals 14 days after ZY or NS injection were collected for detecting serum biochemical parameters and organ histopathology. For further exploring the effect of 100% Oxy inhalation on ZY-induced sterile sepsis and its mechanisms, additional animals were used in the present study. At 24 h after ZY or NS injection, the blood samples and organs were collected for measuring the levels of serum biochemical parameters and inflammatory cytokines, as well as the activities of serum and tissue antioxidant enzymes, and organ histopathology. B, Therapeutic time window of the protective action against ZY-induced sterile sepsis by 100% Oxy inhalation. The animals were divided randomly into six groups: ZY+ Air and ZY + Oxy 4, 8, 12, 16, and 20 groups. Zymosan was intraperitoneally given, and then the animals were given Oxy treatment with the first exposure to 100% Oxy for 3 h starting at 4, 8, 12, 16, or 20 h after ZY injection, respectively, and the second exposure to 100% Oxy for another 3 h after an 8-h interval. The survival rates on days 0.5, 1, 1.5, 2, 3, 5, and 14 after ZY injection were recorded for all the animals. The arterial blood gas analysis was also performed in all groups before and after the first 100% Oxy inhalation.

The survival rates on days 0.5, 1, 1.5, 2, 3, 5, and 14 after ZY or NS injection were recorded (n = 30 per group), and all the surviving animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and then the blood samples and organs were collected for detecting serum biochemical parameters and organ histopathology.

To further study the effect of 100% Oxy inhalation on ZY-induced sterile sepsis and its mechanisms, additional animals were used for detecting the levels of serum biochemical parameters and inflammatory cytokines, as well as the activities of serum and tissue antioxidant enzymes, and organ histopathology. The grouping method and experimental protocols were the same as previously described. Based on our preliminary experiments, at 24 h after ZY or NS injection, the blood samples and organs were collected for measuring the serum levels of biochemical parameters (n = 6 per group) and inflammatory cytokines (n = 6 per group), as well as serum and tissue antioxidant enzymatic activities (n = 6 per group) and organ histopathology (n = 6 per group). The arterial blood gas analysis was also measured (n = 6 per group at each time point).

Experiment Two: Therapeutic time window of the protective action against ZY-induced sterile sepsis by 100% Oxy inhalation

To investigate the therapeutic time window of the protective action against ZY-induced sterile sepsis by 100% Oxy inhalation, the animals were divided randomly into six groups (Fig. 1B): ZY + Air and ZY + Oxy 4, 8, 12, 16, 20 groups. Sterile sepsis model was induced in all animals as previously mentioned. The animals in ZY + Oxy 4, 8, 12, 16, 20 groups were given Oxy treatment, with the first exposure to 100% Oxy for 3 h starting at 4, 8, 12, 16 or 20 h after ZY injection, respectively, and the second exposure to 100% Oxy for another 3 h after an 8-h interval. The survival rates on days 0.5, 1, 1.5, 2, 3, 5, and 14 after ZY injection were recorded (n = 30 per group). In addition, the arterial blood gas analysis was conducted in all groups before and after first 100% Oxy inhalation (n = 6 per group at each time point).

Arterial blood gas analysis

The arterial blood gas analysis was conducted in all groups at 24 h after ZY or NS injection using a GEM Premier 3000 gas analyzer (Instrumentation Laboratory, Milan, Italy).

Serum biochemical parameters assay

At the predetermined time points, animals were anesthetized, and blood samples were collected by cardiac puncture and then clotted for 30 min at 25°C. The serum was separated by centrifugation at 2,000 × g for 15 min at 4°C, aliquoted, and stored at −80°C until assayed. The samples were evaluated with a biochemistry autoanalyzer (Hitachi Autoanalyzer 7150; Hitachi, Tokyo, Japan) to measure serum levels of cardiac troponin I (cTnI; in nanograms per milliliter), alanine aminotransferase (ALT; in international units per liter), aspartate aminotransferase (AST; in international units per liter), blood urea nitrogen (BUN; in millimoles), and creatinine (Cr; in micromoles). In addition, the serum lactate was determined spectrophotometrically using a commercially available kit (lactate assay kit; BioVision Research, Mountain View, Calif).

Enzymatic activity assay

The previously obtained serum was also used for serum enzymatic activity assay.

After sampling blood, the heart, lung, liver, and kidney were removed immediately. The excised organs were immersed immediately in cold phosphate-buffered saline (pH 7.4) containing 0.16 mg/mL of heparin to remove red blood cells and clots. After the excessive fluids were cleaned, the organs were weighed and homogenized in 10 volumes of ice-cold buffer containing protease inhibitors supplied by the kits. The homogenates were centrifuged at 10,000 × g for 15 min at 4°C, and then the supernatants were collected, aliquoted, and stored at −80°C until the following analysis.

The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured using commercial kits purchased from Cayman Chemical Company (Ann Arbor, Mich). According to the manufacturer's instructions, total SOD activity was assayed by detecting superoxide radicals generated by xanthine oxidase and hypoxanthine. The reaction was monitored at 450 nm, and 1 U of SOD activity was defined as the amount of enzyme needed to exhibit 50% dismutation of superoxide radical. The CAT activity was assayed by measuring the reduction of hydrogen peroxide at 540 nm, and 1 U was defined as the amount of enzyme that would cause the formation of 1.0 nmol of formaldehyde per minute at 25°C. Glutathione peroxidase activity was assayed by measuring the rate of nicotinamide adenine dinucleotide phosphate (reduced form) oxidation by glutathione reductase-coupled reaction at 340 nm, and 1 U was defined as the amount of enzyme that would cause the oxidation of 1.0 nmol of nicotinamide adenine dinucleotide phosphate (reduced form) to NADP+ per minute at 25°C. All spectrophotometric readings were performed using a spectrophotometer (DU 640B, Beckman, Fullerton, Calif). All assays were conducted in triplicates. The protein concentration was determined using a standard commercial kit (Bio-Rad Laboratories, Hercules, Calif).

Cytokine enzyme-linked immunosorbent assay

The levels of serum TNF-α, IL-6, IL-10, and high-mobility group box 1 (HMGB1) were determined by specific enzyme-linked immunosorbent assay (ELISA) kits (TNF-α, IL-6, and IL-10; R&D Systems, Inc., Minneapolis, Minn; HMGB1, IBL, Hamburg, Germany) with a microplate reader (CA 94089, Molecular Devices, Sunnyvale, Canada). All standards and samples were run in duplicate.

Histopathological observations

After sampling blood, the heart, lung, liver, and kidney were removed immediately, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4- to 6-μm thickness. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin. Based on the scoring standard made by Ferrer et al. (16), the histological slides were blindly read and scored using a scale of 1 to 4 by two experienced pathologists (see Table, Supplemental Digital Content 1, http://links.lww.com/SHK/A13).

Statistical analysis

The histopathological scores are expressed as median (range), and the survival rates are expressed as percentage. The measurement data are expressed as the mean ± SEM. The intergroup differences of antioxidant enzymatic activities, biochemical parameter, and inflammatory cytokine levels were tested by one-way ANOVA, followed by least significant difference t test for multiple comparisons. The analysis of the survival rates was tested by Fisher exact probability method. The intergroup differences of histopathological scores were tested by the Kruskal-Wallis H method, followed by Nemenyi test for multiple comparisons. The statistical analysis was performed with SPSS 13.0 software. In all tests, a P value of less than 0.05 was considered statistically significant.

RESULTS

Changes of arterial blood gas before and after 100% Oxy treatment in mice with ZY-induced sterile sepsis

Oxygen treatment was conducted in ZY + Oxy 1, 2, 3, 4, and NS + Oxy 1, 2, 3, and 4h groups, with the animals exposed to 100% Oxy for 1, 2, 3, or 4 h at 4 and 12 h after ZY or NS injection, respectively. The arterial blood gas was performed immediately after the first and second 100% Oxy inhalation was given. After the second 100% Oxy inhalation, the arterial partial pressure of Oxy (PO2) in ZY + Oxy 2 and 3h groups was more than 300 mmHg, whereas the arterial PO2 in ZY + Oxy 1 and 4h groups was less than 300 mmHg (data not shown).

Twice 100% Oxy inhalation for 2 or 3 h improved the survival rates in mice with ZY-induced sterile sepsis

As shown in Figure 2, 100% Oxy inhalation for 2 or 3 h starting at 4 and 12 h after ZY injection, respectively, increased the 14-day survival rate from 10% (ZY + Air group) to 60% (ZY + Oxy 2h group; P < 0.05; n = 30 per group) and 80% (ZY + Oxy 3h group; P < 0.05; n = 30 per group). The 14-day survival rates in ZY + Oxy 1 and 4h groups had no significant difference compared to that in the ZY + Air group (P > 0.05; n = 30 per group). In NS + Oxy 1, 2, 3, and 4h groups, all animals survived during the observation period (data not shown).

Fig. 2
Fig. 2:
Twice 100% Oxy inhalation for 2 or 3 h improved the survival rates in mice with ZY-induced sterile sepsis. For the ZY + Air group, the animals were treated by exposure to air after ZY injection. For the NS + Air group, the animals were treated by exposure to air after NS injection. For the ZY + Oxy 1, 2, 3, and 4h groups, the animals were treated by exposure to 100% Oxy for 1, 2, 3, 4 h at 4 and 12 h after ZY injection, respectively. The values are expressed as survival percentage (n = 30 for each group). *P < 0.05 vs. NS + Air group; P < 0.05 vs. ZY + Air group; P < 0.05 vs. ZY + Oxy 1h group; § P < 0.05 vs. ZY + Oxy 2h group; P < 0.05 vs. ZY + Oxy 3h group.

Twice 100% Oxy inhalation for 2 or 3 h prevented the abnormal changes of organ histopathology in mice with ZY-induced sterile sepsis

At 24 h after ZY or NS injection, the animals in all groups were killed for histopathological analysis. The histopathological changes in heart, lung, liver, and kidney were scored using a scale of 1 to 4 (see Table, Supplemental Digital Content 1, http://links.lww.com/SHK/A13), which is based on the scoring standard made by Ferrer et al. (16). As shown in the Table 1, the histopathological scores for heart, lung, liver, and kidney in the ZY + Air group were 3 to 3.5, much higher than those in the NS + Air group (P < 0.05; n = 6 per group).

TABLE 1
TABLE 1:
Changes of organ histopathological scores in mice with ZY-induced sterile sepsis

Exposure to 100% Oxy for 3 h starting at 4 and 12 h after ZY injection, respectively, significantly decreased the histopathological scores for these organs (Table 1; P < 0.05; ZY + Oxy 3h group vs. ZY + Air group; n = 6 per group). However, the histopathological scores for these organs in ZY + Oxy 1 and 4h groups had no statistically significant difference compared with those in the ZY + Air group (P > 0.05; n = 6 per group; Table 1).

No statistically significant difference in the histopathological scores for these organs existed between NS + Oxy 1, 2, 3, 4h, and NS + Air groups (data not shown). During the experiment for observing the survival rate, 3, 9, 18, 24, 9 animals survived more than 14 days after ZY injection in ZY + Air and ZY + Oxy 1, 2, 3, and 4h groups, respectively. In NS + Air and NS + Oxy 1, 2, 3, and 4h groups, all animals survived during the 14-day observation period. The histopathological changes in heart, lung, liver, and kidney were observed in all surviving animals after killing. We showed that the histopathological scores in ZY + Oxy 2 and 3h groups were all back to normal level (data not shown).

Twice 100% Oxy inhalation for 2 or 3 h improved tissue oxygenation in mice with ZY-induced sterile sepsis

At 24 h after ZY or NS injection, the arterial pH value, PO2, and HCO3 in the ZY + Air group were increased compared with those in the NS + Air group (P < 0.05; n = 6 per group; Fig. 3). The arterial partial pressure of carbon dioxide (PCO2) and serum lactate level were higher in the ZY + Air group than in the NS + Air group (P < 0.05; n = 6 per group; Fig. 3). The previously discussed results indicate that low tissue oxygenation occurs in ZY-induced sterile sepsis model.

Fig. 3
Fig. 3:
Twice 100% Oxy inhalation for 2 or 3 h improved tissue oxygenation in mice with ZY-induced sterile sepsis. The grouping methods and experimental protocols were the same as in Figure 2. The arterial pH value (A), PCO2 (B), PO2 (C), and HCO3 (D) as well as serum lactate (E) were measured at 24 h after ZY or NS injection. The values are expressed as mean ± SEM (n = 6 for each group). *P < 0.05 vs. NS + Air group; P < 0.05 vs. ZY + Air group; P < 0.05 vs. ZY + Oxy 1h group; § P < 0.05 vs. ZY + Oxy 2h group; P < 0.05 vs. ZY + Oxy 3h group.

Twice 100% Oxy inhalation for 2 or 3 h improved the tissue oxygenation, whereas twice 100% Oxy inhalation for 1 or 4 h did not. In ZY + Oxy 2 and 3h groups, the arterial pH value, PO2 and HCO3 were higher than those in the ZY + Air group (P < 0.05; n = 6 per group; Fig. 3). The arterial PCO2 and serum lactate level were lower in ZY + Oxy 2 and 3h groups than in the ZY + Air group (P < 0.05; n = 6 per group; Fig. 3). However, compared with the ZY + Air group, the arterial pH value, PO2, HCO3, and PCO2 had no significant changes in ZY + Oxy 1 and 4h groups. The serum levels of lactate were higher in ZY + Oxy 1 and 4h groups than in ZY + Oxy 2 and 3h groups (P < 0.05; n = 6 per group; Fig. 3).

Twice 100% Oxy inhalation for 2 or 3 h prevented the abnormal changes of serum biochemical parameters in mice with ZY-induced sterile sepsis

At 24 h after ZY or NS injection, the animals in all groups were killed to measure serum biochemical parameters. As shown in Figure 4, the levels of serum cTnI, ALT, AST, Cr, and BUN at 24 h after ZY injection increased significantly in the ZY + Air group (P < 0.05 vs. NS + Air group; n = 6 per group).

Fig. 4
Fig. 4:
Twice 100% Oxy inhalation for 2 or 3 h prevented the abnormal changes of serum biochemical parameters in mice with ZY-induced sterile sepsis. The grouping methods and experimental protocols were the same as in Figure 2. The values are expressed as mean ± SEM (n = 6 for each group). *P < 0.05 vs. NS + Air group; P < 0.05 vs. ZY + Air group; P < 0.05 vs. ZY + Oxy 1h group; § P < 0.05 vs. ZY + Oxy 2h group; P < 0.05 vs. ZY + Oxy 3h group.

Exposure to 100% Oxy for 2 or 3 h at 4 and 12 h after ZY injection, respectively, attenuated the abnormal changes in serum biochemical parameters. In contrast, shorter or longer Oxy exposures did not have the same benefit. The levels of serum cTnI, ALT, AST, Cr, and BUN in ZY + Oxy 2 and 3h groups were lower than those in the ZY + Air group (P < 0.05; n = 6 per group). However, the serum levels of cTnI, ALT in ZY + Oxy 1h group and cTnI, Cr, BUN in ZY + Oxy 4h group had no statistically significant differences compared with those in the ZY + Air group.

No statistically significant difference in the serum levels of cTnI, ALT, AST, Cr, and BUN existed between the NS + Oxy 1, 2, 3, 4h and NS + Air groups (data not shown). During the experiment for observing the survival rate, 3, 9, 18, 24, and 9 animals survived more than 14 days after ZY injection in ZY + Air and ZY + Oxy 1, 2, 3, and 4h groups, respectively. In NS + Air and NS + Oxy 1, 2, 3, and 4h groups, all animals survived during the 14-day observation period. In all survived animals, the serum biochemical parameters were determined after they were killed. We showed that the serum levels of cTnI, ALT, AST, Cr, and BUN in ZY + Oxy 2 and 3h groups were all back to normal level (data not shown).

Twice 100% Oxy inhalation for 2 or 3 h attenuated the abnormal changes in the levels of serum inflammatory cytokines in mice with ZY-induced sterile sepsis

Based on the results of our preliminary experiment, the changes of serum inflammatory cytokine levels were observed at 24 h after ZY or NS injection in mice with or without Oxy treatment. The levels of serum TNF-α, IL-6, IL-10, and HMGB1 significantly increased in the ZY + Air group compared with those in the NS + Air group (Fig. 5; P < 0.05; n = 6 per group).

Fig. 5
Fig. 5:
Twice 100% Oxy inhalation for 2 or 3 h prevented the abnormal changes of serum inflammatory cytokines in mice with ZY-induced sterile sepsis. The grouping methods and experimental protocols were the same as in Figure 2. The values are expressed as mean ± SEM (n = 6 for each group). *P< 0.05 vs. NS + Air group; P < 0.05 vs. ZY + Air group; P < 0.05 vs. ZY + Oxy 1h group; § P < 0.05 vs. ZY + Oxy 2h group; P < 0.05 vs. ZY + Oxy 3h group.

As shown in Figure 5, in ZY + Oxy 2 and 3h groups, the levels of serum TNF-α, IL-6, and HMGB1 were significantly lower than those in ZY + Air and ZY + Oxy 1 and 4h groups (P < 0.05; n = 6 per group). The serum IL-6 levels in ZY + Oxy 1 and 4h groups had no statistically significant difference compared with that in the ZY + Air group.

The serum IL-10 level in the ZY + Oxy 3h group increased with no statistically significant difference compared with that in the ZY + Air group (P > 0.05; n = 6 per group). The serum IL-10 levels in ZY + Oxy 1 and 4h groups were lower than that in the ZY + Air group (P < 0.05; n = 6 per group). No significant difference in the levels of serum TNF-α, IL-6, IL-10, and HMGB1 existed between the NS + Oxy 1, 2, 3, 4h, and NS + Air groups (data not shown).

Twice 100% Oxy inhalation for 2 or 3 h prevented the abnormal changes of antioxidant enzymatic activities in mice with ZY-induced sterile sepsis

Based on the results of our preliminary experiment, we also observed the changes of serum and tissue antioxidant enzymatic activities at 24 h after ZY or NS injection in mice with or without Oxy treatment. In the ZY + Air group, the activities of SOD, CAT, and GSH-Px in serum, heart, lung, liver, and kidney were significantly downregulated (Figs. 6 and 7; P < 0.05 vs. NS + Air group; n = 6 per group).

Fig. 6
Fig. 6:
Twice 100% Oxy inhalation for 2 or 3 h prevented the downregulation of serum antioxidant enzymatic activities in mice with ZY-induced sterile sepsis. A, Superoxide dismutase activity; B, CAT activity; C, GSH-Px activity. The grouping methods and experimental protocols were the same as in Figure 2. The values are expressed as mean ± SEM (n = 6 for each group). *P < 0.05 vs. NS + Air group; P < 0.05 vs. ZY + Air group; P < 0.05 vs. ZY + Oxy 1h group; § P < 0.05 vs. ZY + Oxy 2h group; P < 0.05 vs. ZY + Oxy 3h group.
Fig. 7
Fig. 7:
Twice 100% Oxy inhalation for 2 or 3 h prevented the downregulation of tissue antioxidant enzymatic activities in mice with ZY-induced sterile sepsis. A, Superoxide dismutase activity; B, CAT activity; C, GSH-Px activity. The grouping methods and experimental protocols were the same as in Figure 2. The values are expressed as mean ± SEM (n = 6 for each group). *P < 0.05 vs. NS + Air group; P < 0.05 vs. ZY + Air group; P < 0.05 vs. ZY + Oxy 1h group; § P < 0.05 vs. ZY + Oxy 2h group; P < 0.05 vs. ZY + Oxy 3h group.

In ZY + Oxy 2 and 3h groups, the activities of SOD, CAT and GSH-Px in serum, and these organs were significantly higher than those in the ZY + Air group (P < 0.05; n = 6 per group). However, the activities of SOD, CAT, and GSH-Px in serum and these organs in the ZY + Oxy 1h group, as well as the activities of SOD and CAT in serum and these organs in the ZY + Oxy 4h group, had no significant difference compared with those in the ZY + Air group (Figs. 6 and 7).

No statistically significant difference in the activities of SOD, CAT, and GSH-Px in serum and these organs existed between the NS + Oxy 1, 2, 3, 4h, and NS + Air groups (data not shown).

In summary, twice 100% Oxy inhalation for 2 or 3 h improved tissue oxygenation, organ function, and survival rates by regulating the inflammatory cytokines and antioxidant enzymes in mice with ZY-induced sterile sepsis. To explore the suitable treatment protocol, the therapeutic time window of Oxy treatment was further investigated.

Therapeutic time window of twice 100% Oxy inhalation for 3 h in mice with ZY-induced sterile sepsis

As shown in Figure 8, we found that the therapeutic time window of Oxy treatment with twice 100% Oxy inhalation for 3 h was less than 12 h after ZY injection in mice. Oxy treatment with the first 100% Oxy inhalation for 3 h starting at 4, 8, or 12 h after ZY injection, respectively, increased the 14-day survival rate from 10% (ZY + Air group) to 90% (ZY + Oxy 4h group; P < 0.05; n = 30 per group) and 70% (ZY + Oxy 8 and ZY + Oxy 12 groups; P < 0.05; n = 30 per group). Oxy treatment with the first 100% Oxy inhalation starting at later than 12 h after ZY injection had little protective or even detrimental effect (Fig. 8). We also showed that the arterial PO2 in the animals after the first exposure to 100% Oxy for 3 h beginning at less than 12 h after ZY injection was more than 300 mmHg (data not shown). However, when 100% Oxy inhalation was given at 16 or 20 h after ZY injection, the arterial PO2 was less than 250 mmHg (data not shown).

Fig. 8
Fig. 8:
Twice 100% Oxy inhalation for 3 h starting at less than 12 h after ZY injection improved the survival rates in mice with ZY-induced sterile sepsis. Sterile sepsis model was induced in all the animals as previously mentioned. The animals in ZY + Oxy 4, 8, 12, 16, and 20h groups were given Oxy treatment, with the first exposure to 100% Oxy for 3 h starting at 4, 8, 12, 16, or 20 h after ZY injection, respectively, and the second exposure to 100% Oxy for another 3 h after an 8-h interval. The values are expressed as survival percentage (n = 30 for each group). *P < 0.05 vs. NS + Air group; P < 0.05 vs. ZY + Air group; P < 0.05 vs. ZY + Oxy 1h group; § P < 0.05 vs. ZY + Oxy 2h group; P < 0.05 vs. ZY + Oxy 3h group.

DISCUSSION

In the present study, we found that 100% Oxy inhalation for 2 or 3 h starting at 4 and 12 h after ZY injection, respectively, improved tissue oxygenation, organ function, and survival rate in mice with ZY-induced sterile sepsis via regulating the serum levels of inflammatory cytokines and the activities of serum and tissue antioxidant enzymes, but twice 100% Oxy inhalation for 1 or 4 h did not.

Zymosan is a substance derived from the cell wall of the yeast S. cerevisiae. Intraperitoneal injection of a high dose of ZY (0.8-1.0 g/kg BW) can induce a sepsis/MODS model in rats or mice (14). In the present study, ZY (1.0 g/kg BW, i.p.) resulted in increased clinical scores, progressive weight loss, lower tissue oxygenation, the abnormal increase of serum biochemical parameters levels, and organ histopathological scores at 24 h after ZY injection, suggesting that ZY injection induces a sterile sepsis model.

Decreased Oxy delivery and cellular hypoxia are important factors in the pathophysiology of sepsis/MODS (17). Oxy therapy has been widely used in clinical practice with many advantages. It is simple to administer, safe, noninvasive, inexpensive, and can be started promptly after sepsis onset. Moreover, Oxy has multiple beneficial biochemical, molecular, and hemodynamic effects. Oxy inhalation is often required for the treatment of patients with critical conditions such as ischemia and/or hypoxia (18), but clinical application of hyperoxia treatment is limited because of its toxicity associated with overproduction of ROS induced by hyperoxia (8). The ROS production is directly related to Oxy tension (19). Sepsis/MODS is associated with oxidative stress resulting from enhanced formation of ROS (20). Meanwhile, it has been found that hyperoxia treatment can impede bacterial phagocytosis (21). Therefore, hyperoxia may theoretically aggravate tissue injury in patients with sepsis/MODS. However, some studies have reported that hyperoxia has antibiotic activity (22), and perioperative hyperoxia can reduce wound infections after abdominal surgery (23). Furthermore, it is suggested that ventilation with 100% Oxy can improve survival rate and organ function as well as reduce systemic inflammatory response in several shock models (10, 12, 24-26). Recently, in a porcine model of early hyperdynamic septic shock, ventilation with 100% Oxy has been shown to improve organ function without affecting lung function and oxidative or nitrosative stress (10). Interestingly, Cuzzocrea et al. (7) has found that HBO treatment at 4 and 11 h after ZY injection could significantly improve the 72-h survival rate and organ function in ZY-induced MODS model. Our previous study has demonstrated that hyperoxia plays a key role in HBO pretreatment (27). Another study has shown that HBO treatment administered twice daily doubles the survival rate during a 4-day observation period in a rat model with peritonitis, whereas the HBO treatment with only once daily fails to influence the outcome (28). The present study demonstrated that 100% Oxy inhalation significantly improved tissue oxygenation, organ function, and survival rate in mice with ZY-induced sterile sepsis. We further observed that exposure to 100% Oxy for 2 or 3 h starting at 4 and 12 h after ZY injection, respectively, prevented the abnormal changes of tissue oxygenation, serum biochemical parameters, and organ histopathology induced by ZY and significantly improved the 14-day survival rate. However, exposure to 100% Oxy for 1 or 4 h starting at same time points had little preventive effects, suggesting that suitable 100% Oxy inhalation duration was necessary for Oxy treatment against sepsis.

The uncontrolled and exaggerated inflammatory responses as well as the disbalance of proinflammatory and anti-inflammatory cytokines play a major role in the pathogenesis of sepsis/MODS (29, 30). The inflammatory cytokines include early inflammatory cytokines such as proinflammatory cytokines TNF-α, IL-6, and anti-inflammatory cytokine IL-10, and the late inflammatory cytokine HMGB1 (31). The interaction of early and late inflammatory cytokines can facilitate organ dysfunction and injury in sepsis (32, 33). In the present study, we found that both early inflammatory cytokines and late inflammatory cytokine HMGB1 were markedly increased after ZY injection, suggesting that the late inflammatory cytokine HMGB1 might play a critical role in the development of ZY-induced sterile sepsis. We further observed that 100% Oxy inhalation for 1 or 4 h at 4 and 12 h after ZY injection, respectively, decreased serum anti-inflammatory cytokine more markedly than proinflammatory cytokines in mice with ZY-induced sterile sepsis. Twice 100% Oxy inhalation for 2 or 3 h at the same time points decreased the levels of serum proinflammatory cytokines and increased the level of serum anti-inflammatory cytokine.

Recent studies have reported that the decreased antioxidant enzymatic activities are related to the development of sepsis/MODS (7, 34-36). We observed that 100% Oxy inhalation for 2 or 3 h at 4 and 12 h after ZY injection, respectively, could prevent the decrease of serum and tissue antioxidant enzymatic activities, whereas 100% Oxy inhalation for 1 or 4 h did not. Another study also reported that the protection of hyperoxia treatment is associated with the generation of antioxidant enzymes (37).

We further showed that the therapeutic time window of twice 100% Oxy inhalation for 3 h in mice with ZY-induced sterile sepsis was less than 12 h after ZY injection. When 100% Oxy inhalation began later than 12 h after ZY injection, however, there was little protective or even detrimental effect. It is known that hyperoxia inhalation can increase arterial PO2, then restore cellular energy metabolism and improve hemodynamics, and so on (9, 11). Our results showed that the arterial PO2 in the animals with exposure to 100% Oxy inhalation starting at less than 12 h after ZY injection was more than 300 mmHg.

It is generally believed that hyperoxia treatment can improve the organ function, but long-term hyperoxia treatment can induce Oxy toxicity (8, 9). Therefore, one of the main aims of this study is to explore a suitable Oxy treatment protocol for treating sepsis/MODS. The present results indicated that 2 or 3 h of 100% Oxy exposure is beneficial, but shorter or longer 100% Oxy exposure is not. We also found that the 2- or 3-h Oxy treatment protocol improves tissue oxygenation, increases the antioxidant enzymatic activities, decreases the proinflammatory cytokines, and increases the anti-inflammatory cytokine, but the shorter or longer Oxy treatment protocol has little beneficial effect. We speculate that shorter Oxy treatment may not provide enough Oxy to improve tissue oxygenation, but longer Oxy treatment may induce Oxy toxicity. The Oxy toxicity may attenuate the beneficial effect of Oxy treatment.

We conclude that twice 100% Oxy inhalation for 2 or 3 h has protective action on ZY-induced sterile sepsis in mice, whereas longer or shorter 100% Oxy inhalation does not. The present results may provide a potential cue for developing effective therapeutic strategies for the patients with sepsis/MODS.

ACKNOWLEDGMENTS

The authors thank Professor Qing Li and Professor Yanping Hui from the Department of Pathology, Fourth Military Medical University, for assisting in histopathological analysis, Professor Lei Shang from the Department of Health Statistics, Fourth Military Medical University, for help in the statistics analysis, and Professor Shanlu Liu from the Department of Microbiology and Immunology, McGill University, Montreal, Canada, for insightful comments.

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Keywords:

Sepsis; sterile; multiple organ dysfunction syndrome; inflammatory cytokine; antioxidant enzyme; 100% oxygen

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