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Basic Science Aspects

EFFECTS OF REACTIVE OXYGEN SPECIES SCAVENGER ON THE PROTECTIVE ACTION OF 100% OXYGEN TREATMENT AGAINST STERILE INFLAMMATION IN MICE

Hou, Lichao*; Xie, Keliang*†; Qin, Mingzhe*‡; Peng, Daorong§; Ma, Shirong; Shang, Lei; Li, Nan*; Li, Shuzhi*; Ji, Genlin*; Lu, Yan*; Xiong, Lize*

Author Information
doi: 10.1097/SHK.0b013e3181c1b5d4

Abstract

INTRODUCTION

Sepsis/multiple organ dysfunction syndrome (MODS) is one of the most challenging clinical problems. There are a number of clinical conditions that may lead to sepsis/MODS, including shock, trauma, burn, pancreatitis, and so on (1). Although improvements in resuscitation and supportive care decrease the overall mortality among patients with sepsis/MODS, the incidence of sepsis/MODS and the total number of sepsis/MODS-related deaths are increasing because of the coming of aging society and the use of immunosuppressants (1). Therefore, it has become an imminent task to explore effective therapeutic measures for patients with sepsis.

A large number of studies have found that systemic inflammatory response syndrome plays a major role in the pathogenesis of sepsis and MODS (2). The zymosan-induced inflammation model has been widely used in many research works because zymosan, a substance derived from the cell wall of the yeast Saccharomyces cerevisiae can lead to systemic inflammation by inducing a wide range of inflammatory mediators (3).

Oxygen has been widely used in clinical practice as a medical treatment with many advantages, such as its safety, ease, and inexpensiveness. We have recently found that 100% oxygen inhalation for 2 and 3 h starting at 4 and 12 h after zymosan injection, respectively, increases the 14-day survival rate (10% to 60%-80%) in mice with zymosan-induced sterile inflammation by increasing antioxidant enzymatic activities (4). Yet, the use of hyperoxia is hindered by concerns that it could exacerbate organ injury by increasing free radical formation. It is known that oxidative stress plays an important role in the pathogenesis of sepsis/MODS, and overproduction of reactive oxygen species (ROS) can exacerbate organ injury (5, 6). Furthermore, many studies have demonstrated that a ROS scavenger can alleviate organ injury and improve survival rate of animals with sepsis (7, 8). We therefore hypothesized that ROS scavenger pretreatment may enhance the protective role of 100% oxygen treatment in mice with zymosan-induced sterile inflammation. Surprisingly, we found that ROS scavenger pretreatment blocked the protective action of 100% oxygen treatment against zymosan-induced sterile inflammation in mice, which may provide new clues for developing some strategies for the treatment of sepsis/MODS.

MATERIALS AND METHODS

Animals

Male Imprinting Control Region mice (specific pathogen free) provided by the Laboratory Animal Center of the Fourth Military Medical University, aged 6 to 8 weeks 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 with food and water available ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the National Institutes of Health guidelines for the use of experimental animals.

Zymosan-induced sterile inflammation model

Zymosan (Sigma Chemical Co, St Louis, Mo) solution was prepared in isotonic sodium chloride solution (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 inflammation was induced by an aseptic intraperitoneal injection of zymosan at a dose of 1 g/kg of body weight (BW) (3, 4). The same volume of NS was injected through the same route as the control.

Oxygen treatment

The animals were placed in a sealed Plexiglas chamber. Oxygen 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 oxygen 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, Fullerton, Calif). Oxygen concentration was maintained at 100% during the treatment. The room and chamber temperatures were maintained at 20°C to 22°C. Air treatment was done with the animals exposed to room air in the chamber. Food and water were available ad libitum during the treatment (4).

Experimental design

Part I. Effects of ROS scavenger on the survival rate in zymosan-challenged mice with 100% oxygen treatment

One hundred fifty animals were divided randomly into five groups (n = 30 per group): NS + Air + vehicle (Veh), zymosan (ZY) + Air + Veh, ZY + Air + N-acetylcysteine (NAC), ZY + oxygen (Oxy) + Veh, and ZY + Oxy + NAC groups.

Sterile inflammation was induced in all animals except the animals from the NS + Air + Veh group. The animals in the NS + Air + Veh group were intraperitoneally injected with NS.

The animals in the ZY + Oxy + Veh and ZY + Oxy + NAC groups were given oxygen treatment by exposure to 100% oxygen for 3 h starting at 4 and 12 h after zymosan injection, respectively. The animals in the NS + Air + Veh, ZY + Air + Veh, and ZY + Air + NAC groups were exposed to room air at 4 and 12 h after zymosan or NS injection, respectively.

In the ZY + Air + NAC and ZY + Oxy + NAC groups, the animals were intraperitoneally administered with 150 mg/kg NAC (Sigma, St Louis, Mo) at 30 min before oxygen inhalation, respectively (9). In the NS + Air + Veh, ZY + Air + Veh, and ZY + Oxy + Veh groups, the same volume of vehicle (NS) was given intraperitoneally at the same time points.

The survival rate was recorded on days 0.5, 1, 1.5, 2, 3, 5, and 14 after zymosan or NS injection. On day 14, all animals that survived were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and blood samples and organs were collected for detecting serum biochemical parameters and organ histopathology.

Part II. Effects of ROS scavenger on the oxygenation, biochemical parameters, and organ histopathology in zymosan-challenged mice with 100% oxygen treatment

To further confirm the effects of ROS scavenger on zymosan-challenged mice with 100% oxygen treatment, we examined tissue oxygenation, biochemical parameters, and organ histopathology. Fifty animals were used in this experiment and were assigned to five groups (n = 10 per group). The grouping method and experimental protocols were the same as previously described. At 24 h after NS or zymosan injection, all the animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and blood samples and organs were collected for detecting arterial blood gases, serum lactate, serum biochemical parameters, and organ histopathology.

Part III. Roles of inflammatory cytokines and antioxidant enzymes in the effects of ROS scavengers on the protection of 100% oxygen treatment in zymosan-challenged mice

Because sepsis/MODS is the culmination of complex interactions between the infecting microorganism and the host immune, inflammation, oxidative stress, and coagulation responses (10), we studied the roles of inflammatory cytokines and antioxidant enzymes in the effects of ROS scavengers on the protection of 100% oxygen treatment in zymosan-challenged mice.

One hundred forty-four animals were randomly divided into four groups (n = 36 per group): NS + Air + Veh, ZY + Air+ Veh, ZY + Oxy + Veh, and ZY + Oxy + NAC groups. The grouping method and experimental protocols were the same as previously described. At 2, 4, 8, 16, 24, and 32 h after NS or zymosan injection, six animals from each group were anesthetized, and blood samples were collected for measuring the levels of inflammatory cytokines as well as the activities of antioxidant enzymes in serum.

Arterial blood gas analysis

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

Serum biochemical parameter 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, nanograms per milliliter), alanine aminotransferase (ALT, international units per liter), aspartate aminotransferase (AST, international units per liter), blood urea nitrogen (BUN, millimoles per liter), and creatinine (Cr, micromoles per liter). In addition, the serum lactate was determined spectrophotometrically by using a commercially available kit (Lactate Assay Kit, BioVision Research, Mountain View, Calif).

Enzymatic activity assay

After sampling blood, the heart, lung, liver, and kidney were immediately removed. 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 excess 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 the supernatants were then 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 by 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 one unit 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 one unit was defined as the amount of enzyme that would cause the formation of 1.0 nmol of formaldehyde per minute at 25°C. The GSH-Px activity was assayed by measuring the rate of NADPH oxidation by glutathione reductase-coupled reaction at 340 nm, and one unit was defined as the amount of enzyme that would cause the oxidation of 1.0 nmol of NADPH to NADP+ per minute at 25°C. All spectrophotometric readings were performed with a spectrophotometer (DU 640B, Beckman). All assays were conducted in triplicate. The protein concentration was determined with 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 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.

Histopathologic observations

After sampling blood, the heart, lung, liver, and kidney were immediately removed, 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 in the Table Supplemental Digital Content 1 (https://links.lww.com/SHK/A22), Histological findings and scoring standard, the histological slides were blindly read and scored by two experienced pathologists.

Statistical analysis

The histopathologic scores are expressed as medians (range), and the survival rates are expressed as percentages. The measurement data are expressed as means ± SEM. The intergroup differences of antioxidant enzymes, biochemical parameters, and inflammatory cytokines were tested by one-way ANOVA followed by least significant difference t test for multiple comparisons. The analysis of survival rates was tested by Fisher exact test probability method. The intergroup differences in histopathologic scores were tested by Kruskal-Wallis H method followed by Nemenyi test for multiple comparisons. The statistical analysis was performed with SPSS 16.0 software. In all tests, a value of P < 0.05 was considered statistically significant.

RESULTS

Changes in survival rates

Oxygen treatment was given by exposure to 100% oxygen for 3 h starting at 4 and 12 h after zymosan and NS injections, respectively. With oxygen treatment, the 14-day survival rate was increased from 10% (ZY + Air + Veh group) to 80% (ZY + Oxy + Veh group, P < 0.05; n = 30 per group; Fig. 1). However, ROS scavenger NAC pretreatment prevented this increase of survival rates induced by oxygen treatment (P < 0.05; ZY+ Oxy + NAC group vs. ZY + Oxy + Veh group respectively, n = 30 per group; Fig. 1).

Fig. 1
Fig. 1:
ROS scavenger NAC pretreatment attenuated the beneficial effect of 100% oxygen treatment on survival rates in mice challenged with zymosan. The survival rates were recorded on days 0.5, 1, 1.5, 2, 3, 5, and 14 after zymosan or NS injection. The values represent percentages (n = 30 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group. d-days.

When only ROS scavenger NAC was used in zymosan-stimulated mice, the survival rates were increased significantly (P < 0.05; ZY + Air + NAC group vs. ZY + Air + Veh group, respectively, n = 30 per group; Fig. 1).

In addition, we investigated the influences of two other ROS scavengers, vitamin C (Vit C; Sigma Chemicals Co, Poole, UK) and dimethylthiourea (DMTU; Sigma-Aldrich, St Louis, Mo), on the beneficial effect of 100% oxygen treatment in mice challenged with zymosan. The results of survival rates were similar with that of NAC; detailed data were shown in the Figure Supplemental Digital Content 2 (https://links.lww.com/SHK/A23), ROS scavenger Vit C or DMTU pretreatment attenuated the beneficial effect of 100% oxygen treatment on survival rates in mice challenged with zymosan. The animals of ZY + Air + Vit C and ZY + Oxy + DMTU groups were intraperitoneally administered 150 mg/kg Vit C and 50 mg/kg DMTU, respectively (11-13), at 30 min before oxygen inhalation. The survival rates were recorded on days 0.5, 1, 1.5, 2, 3, 5, and 14 after zymosan or NS injection. The values represent percentages (n = 30 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

The previous results indicated that zymosan injection significantly reduced survival rates in mice, and oxygen treatment increased survival rates of mice with zymosan stimulation, which was abolished by ROS scavenger pretreatment. Nevertheless, when ROS scavenger NAC, Vit C, or DMTU was used in zymosan-stimulated mice receiving room air, any of them increased the survival rates.

Organ histopathologic changes

At 24 h after zymosan or NS injection, the histopathologic scores for heart, lung, liver, and kidney were 3 to 3.5 in the ZY + Air + Veh group, much higher than those in the NS + Air + Veh group (P < 0.05; n = 10 per group; Table 1).

TABLE 1
TABLE 1:
Effect of ROS scavenger on organ histopathologic scores in zymosan-challenged mice with 100% oxygen treatment

With oxygen treatment, the histopathologic scores for heart, lung, liver, and kidney were significantly decreased (P < 0.05; ZY + Oxy + Veh group vs. ZY + Air + Veh group; n = 10 per group). However, ROS scavenger NAC pretreatment attenuated this protective role (P < 0.05; ZY + Oxy + NAC group vs. ZY + Oxy + Veh group, respectively; n = 10 per group).

With only ROS scavenger NAC being used in zymosan-stimulated mice, the histopathologic scores for heart, lung, liver, and kidney were decreased significantly (P < 0.05; ZY + Air + NAC group vs. ZY + Air + Veh group, respectively; n = 10 per group).

During the experiment for observing survival rate in the ZY + Air + Veh, ZY + Air + NAC, ZY + Oxy + Veh, and ZY + Oxy + NAC groups, there were 3, 21, 24, 6, animals that survived for 14 days after zymosan injection, respectively. All animals in the NS + Air + Veh group survived during the 14-day observation period. Histopathologic changes in heart, lung, liver, and kidney were observed in all survived animals after sacrifice. There were no differences among groups in mice that survived 14 days (data not shown).

In addition, we investigated the influences of two other ROS scavengers, Vit C and DMTU, on the beneficial effect of 100% oxygen treatment in mice challenged with zymosan. The results of histopathologic scores were similar to those of NAC; detailed data were shown in the Table Supplemental Digital Content 3 (https://links.lww.com/SHK/A24), ROS scavenger Vit C or DMTU pretreatment blocked the beneficial effect of 100% oxygen treatment on organ histopathologic scores in mice challenged with zymosan. At 24 h after NS or zymosan injection, all animals were anesthetized, and the organ samples were collected for measuring the histopathologic scores. The values of histopathologic scores represent medians (range) (n = 10 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

These results indicated that zymosan injection led to the histopathologic injury for heart, lung, liver, and kidney in mice, and 100% oxygen treatment reduced this organ injury, which was blocked by ROS scavenger pretreatment. However, when ROS scavenger NAC, Vit C, or DMTU was used in zymosan-stimulated mice receiving room air, it reduced this organ injury.

Changes in biochemical parameters for heart, liver, and kidney functions

As seen in Figure 2, the levels of serum cTnI, ALT, AST, Cr, and BUN in the ZY + Air + Veh group were increased significantly at 24 h after zymosan injection (P < 0.05; ZY + Air + Veh group vs. NS + Air + Veh group; n = 6 per group).

Fig. 2
Fig. 2:
ROS scavenger NAC pretreatment blocked the beneficial effect of 100% oxygen treatment on serum biochemical parameters in mice challenged with zymosan. At 24 h after NS or zymosan injection, all the animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and blood samples were collected for detecting serum biochemical parameters. The values represent mean ± SEM (n = 6 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

With oxygen treatment, the levels of serum cTnI, ALT, AST, Cr, and BUN were reduced (P < 0.05; ZY + Oxy + Veh group vs. ZY + Air + Veh group; n = 6 per group). However, ROS scavenger NAC pretreatment attenuated the decrease in the levels of serum cTnI, ALT, AST, Cr, and BUN induced by oxygen treatment (P < 0.05; ZY + Oxy + NAC group vs. ZY + Oxy + Veh group, respectively; n = 6 per group).

With only ROS scavenger NAC being used in zymosan-stimulated mice, the levels of serum cTnI, ALT, AST, Cr, and BUN were significantly decreased (P < 0.05; ZY + Air + NAC group vs. ZY + Air + Veh group, respectively; n = 6 per group).

At 14 days after zymosan or NS injection, the changes in the levels of serum cTnI, ALT, AST, Cr, and BUN were also examined in all animals that survived. There were no differences among groups in mice that survived 14 days (data not shown).

In addition, we investigated the influences of two other ROS scavengers, Vit C and DMTU, on the beneficial effect of 100% oxygen treatment in mice challenged with zymosan. The results of serum cTnI, ALT, AST, Cr, and BUN were similar with that of NAC; detailed data were shown in the Figure Supplemental Digital Content 4 (https://links.lww.com/SHK/A25), ROS scavenger NAC pretreatment blocked the beneficial effect of 100% oxygen treatment on serum biochemical parameters in mice challenged with zymosan. At 24 h after NS or zymosan injection, all the animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and blood samples were collected for detecting serum biochemical parameters. The values represent mean ± SEM (n = 6 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

Based on these results, we found that zymosan injection resulted in organ dysfunction at 24 h after zymosan challenge in mice, and oxygen treatment improved this organ dysfunction, which was abolished by ROS scavenger pretreatment. However, when ROS scavenger NAC, Vit C, or DMTU was used in zymosan-stimulated mice receiving room air, it improved this organ dysfunction.

Changes in tissue oxygenation

The tissue oxygenation was measured at 24 h after zymosan challenge by determining the levels of arterial blood gases and blood lactate. We found that zymosan injection increased serum lactate level (9.72 ± 0.23 mmol/L in the ZY + Air + Veh group vs. 1.51 ± 0.10 mmol/L in the NS + Air + Veh group) and decreased arterial PO2 (55.93 ± 1.37 mmHg in the ZY + Air + Veh group vs. 97.88 ± 2.44 mmHg in the NS + Air + Veh group) (P < 0.05; n = 6 per group; Fig. 3). The results showed that zymosan injection led to lower tissue oxygenation in mice.

Fig. 3
Fig. 3:
ROS scavenger NAC pretreatment blocked the beneficial effect of 100% oxygen treatment on serum lactate and arterial blood gases in mice challenged with zymosan. At 24 h after NS or zymosan injection, all the animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and blood samples were collected for detecting arterial blood gases and serum lactate. The values represent mean ± SEM (n = 6 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

Oxygen treatment significantly decreased serum lactate level (4.02 ± 0.11 mmol/L in the ZY + Oxy + Veh group vs. 9.72 ± 0.23 mmol/L in the ZY + Air + Veh group) and increased arterial PO2 (96.43 ± 1.80 mmHg in the ZY + Oxy + Veh group vs. 55.93 ± 1.37 mmHg in the ZY + Air + Veh group) (P < 0.05; n = 6 per group; Fig. 3), which suggested that oxygen treatment improved the tissue oxygenation in zymosan-stimulated mice. However, ROS scavenger NAC pretreatment abolished the improvement of tissue oxygenation induced by oxygen treatment (P < 0.05; ZY + Oxy + NAC group vs. ZY + Oxy + Veh group, respectively; n = 6 per group; Fig. 3).

With only ROS scavenger NAC being used in zymosan-stimulated mice, the serum lactate level was decreased and the arterial PO2 was increased (P < 0.05; ZY + Air + NAC group vs. ZY + Air + Veh group, respectively; n = 6 per group; Fig. 3), which demonstrated that when ROS scavenger NAC was used in zymosan-stimulated mice receiving room air, it improved this tissue oxygenation.

In addition, we investigated the influences of two other ROS scavengers, Vit C and DMTU, on the beneficial effect of 100% oxygen treatment in mice challenged with zymosan. The results of tissue oxygenation were similar with that of NAC, the detailed data were shown in the Figure Supplemental Digital Content 5 (https://links.lww.com/SHK/A26), ROS scavenger Vit C or DMTU pretreatment blocked the beneficial effect of 100% oxygen treatment on serum lactate and arterial blood gases in mice challenged with zymosan. At 24 h after NS or zymosan injection, all the animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and blood samples were collected for detecting arterial blood gases and serum lactate. The values represent mean ± SEM (n = 6 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

Changes in serum inflammatory cytokines

We detected the levels of serum TNF-α, HMGB1, and IL-10 at 2, 4, 8, 16, 24, and 32 h after zymosan or NS injection. The results showed that the levels of serum TNF-α, IL-10, and HMGB1 were significantly increased from 2 to 32 h after zymosan injection (P < 0.05; ZY + Air + Veh group vs. NS + Air + Veh group; n = 6 per group at each time point; Fig. 4). With oxygen treatment, the increase in the levels of serum TNF-α and HMGB1 from 8 to 32 h after zymosan injection was attenuated (P < 0.05; ZY + Oxy + Veh group vs. ZY + Air + Veh group, n = 6 per group at each time point; Fig. 4), whereas serum IL-10 level was further increased (P < 0.05; ZY + Oxy + Veh group vs. ZY + Air + Veh group; n = 6 per group at each time point; Fig. 4). However, with ROS scavenger NAC pretreatment, the levels of serum TNF-α and HMGB1 from 8 to 32 h after zymosan injection were higher than those in the ZY + Oxy + Veh group (P < 0.05; n = 6 per group at each time point; Fig. 4), whereas serum IL-10 levels were significantly decreased in the ZY + Oxy + NAC group (P < 0.05 vs. ZY + Oxy + Veh group; n = 6 per group at each time point; Fig. 4).

Fig. 4
Fig. 4:
ROS scavenger NAC pretreatment blocked the beneficial effect of 100% oxygen treatment on serum inflammatory cytokines in mice challenged with zymosan. At 2, 4, 8, 16, 24, and 32 h after NS or zymosan injection, six animals from each group were anesthetized, and blood samples were collected for measuring the levels of inflammatory cytokines. The values represent mean ± SEM (n = 6 per group at each time point). *P < 0.05 vs. NS+ Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

Based on the previous results, we did another experiment and detected the levels of serum TNF-α, IL-10, and HMGB1 at 24 h after zymosan injection in the ZY + Oxy + Vit C and ZY + Oxy + DMTU groups. The ROS scavenger Vit C or DMTU pretreatment had similar results with those of NAC pretreatment, as shown in the Figure Supplemental Digital Content 6 (https://links.lww.com/SHK/A27), ROS scavenger Vit C or DMTU pretreatment blocked the beneficial effect of 100% oxygen treatment on serum inflammatory cytokines in mice challenged with zymosan. At 24 h after NS or zymosan injection, all animals were anesthetized, and blood samples were collected for measuring the levels of inflammatory cytokines. The values represent mean ± SEM (n = 6 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

With only ROS scavenger NAC, Vit C, or DMTU being used in zymosan-stimulated mice, the levels of serum TNF-α and HMGB1 were decreased significantly and the level of serum IL-10 was increased significantly (data not shown).

The previous results showed that zymosan injection resulted in sterile inflammation by significantly increasing the levels of serum TNF-α, IL-10, and HMGB1 in mice. Oxygen treatment exerted a protective action on this inflammation model by decreasing the levels of serum TNF-α and HMGB1 and by increasing the level of serum IL-10 in these zymosan-stimulated mice, which was abolished by the pretreatment with NAC, Vit C, or DMTU. However, when ROS scavenger NAC, Vit C, or DMTU was used in mice receiving room air, any of them had a beneficial effect on this sterile inflammation model by regulating serum levels of TNF-α, IL-10, and HMGB1.

Changes in serum and tissue antioxidant enzymatic activities

We first detected the activities of serum SOD, CAT, and GSH-Px at 2, 4, 8, 16, 24, and 32 h after zymosan or NS injection. The results showed that the activities of serum SOD, CAT, and GSH-Px were decreased significantly from 16 to 32 h after zymosan injection (P < 0.05; ZY + Air + Veh group vs. NS + Air + Veh group; n = 6 per group; Fig. 5). Oxygen treatment significantly increased the activities of serum SOD, CAT, and GSH-Px from 8 to 32 h after zymosan injection (P < 0.05 vs. ZY + Air + Veh group; n = 6 per group; Fig. 5). However, with ROS scavenger NAC pretreatment, the activities of serum SOD, CAT, and GSH-Px were significantly decreased from 8 to 32 h after zymosan injection (P < 0.05 vs. ZY + Oxy + Veh group; n = 6 per group; Fig. 5).

Fig. 5
Fig. 5:
ROS scavenger NAC pretreatment blocked the beneficial effect of 100% oxygen treatment by preventing the increase in serum antioxidant enzymatic activities in mice challenged with zymosan. At 2, 4, 8, 16, 24, and 32 h after NS or zymosan injection, six animals from each group were anesthetized, and blood samples were collected for measuring the activities of serum antioxidant enzymes. The values represent mean ± SEM (n= 6 per group at each time point). *P < 0.05 vs. NS + Air + Veh group; P<0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

Based on the previous results, we did another experiment and examined the activities of serum SOD, CAT, and GSH-Px at 24 h after zymosan injection in mice with ROS scavenger Vit C or DMTU pretreatment, and there were similar results with those of NAC pretreatment (see the Figure Supplemental Digital Content 7 [https://links.lww.com/SHK/A28], ROS scavenger Vit C or DMTU pretreatment blocked the beneficial effect of 100% oxygen treatment by preventing the increase in serum antioxidant enzymatic activities in mice challenged with zymosan). At 24 h after NS or zymosan injection, all animals were anesthetized, and blood samples were collected for measuring the activities of serum antioxidant enzymes. The values represent mean ± SEM (n = 6 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 0.05 vs. ZY + Oxy + Veh group.

With only ROS scavenger NAC, Vit C, or DMTU being used in zymosan-stimulated mice, the activities of serum SOD, CAT, and GSH-Px were increased significantly (data not shown).

Besides, we detected the activities of SOD, CAT, and GSH-Px in heart, lung, liver, and kidney for all 6 groups at 24 h after zymosan or NS injection. Looking at changes in the activities of SOD, CAT, and GSH-Px in serum, results were similar to activities of SOD, CAT, and GSH-Px in the heart, lung, liver, and kidney (see the Figure Supplemental Digital Content 8 [https://links.lww.com/SHK/A29] ROS scavenger NAC, Vit C or DMTU pretreatment blocked the beneficial effect of 100% oxygen treatment by preventing the increase in tissue antioxidant enzymatic activities in mice challenged with zymosan). At 24 h after NS or zymosan injection, all animals were anesthetized, and then heart, lung, liver, and kidney were collected for measuring the activities of tissue antioxidant enzymes. The values represent mean ± SEM (n = 6 per group). *P < 0.05 vs. NS + Air + Veh group; P < 0.05 vs. ZY + Air + Veh group; P < 05 vs. ZY + Oxy + Veh group.

The previous results demonstrated that zymosan injection led to sterile inflammation in mice by significantly decreasing the activities of SOD, CAT, and GSH-Px in serum and tissue. Oxygen treatment had a protective effect on this sterile inflammation model by markedly increasing the activities of serum and tissue SOD, CAT, and GSH-Px in these zymosan-stimulated mice, which was abolished by the pretreatment with NAC, Vit C, or DMTU. However, when ROS scavenger NAC, Vit C, or DMTU was used in mice receiving room air, any of them was beneficial to this sterile inflammation model by regulating the activities of SOD, CAT, and GSH-Px in serum and tissue.

DISCUSSION

The present study demonstrated that 100% oxygen treatment improved tissue oxygenation, attenuated organ dysfunction, reduced histopathologic injury, decreased the levels of serum proinflammatory cytokines, increased the level of serum anti-inflammatory cytokine and the activities of serum and tissue antioxidant enzymes, and increased the survival rates in mice challenged with zymosan. Pretreatment with ROS scavenger Vit C, NAC, or DMTU abolished this protective action of 100% oxygen treatment against sterile inflammation by preventing its regulatory action on the proinflammatory and anti-inflammatory cytokines as well as the antioxidant system. However, when ROS scavenger Vit C, NAC, or DMTU was used in mice receiving room air, it had a beneficial effect on this sterile inflammation model.

Based on our previous study and other studies, intraperitoneal injection of a high dose of zymosan (0.8-1.0 g/kg BW) can induce a generalized inflammation model in rats or mice (3, 4). In the present study, zymosan (1.0 g/kg BW, intraperitoneal injection) successfully induced sterile inflammation model in mice characterized by the decrease of survival rates of mice, histopathologic injury, organ dysfunction, abnormally decreased tissue oxygenation, as well as the increased levels of serum proinflammatory cytokines and anti-inflammatory cytokines, and the decreased activities of antioxidant enzymes in serum and tissue.

The concept of oxygen as a therapeutic agent was introduced in the 1920s by Alvin Barach (14). Since then, oxygen therapy has been widely used in clinical practice as a mainstay of supportive treatment for patients experiencing hypoxemia and critical illness. There are about 800,000 patients receiving long-term oxygen therapy in the United States at a yearly cost of $1.8 billion (15). It has been reported that hyperoxia ventilation improves organ function and survival rate in several models of shock (16-20). Our recent study has also found that 100% oxygen inhalation for 2 and 3 h starting at 4 and 12 h, respectively, after zymosan injection benefits the outcome of mice with sterile sepsis. In the present study, we further showed that 100% oxygen inhalation for 3 h starting at 4 and 12 h after zymosan injection had a protective action in zymosan-challenged mice.

It is believed that improved tissue oxygenation and decreased systemic inflammatory response play an important role in the protection of hyperoxia treatment (16, 18, 20). Our recent study has found that 100% oxygen inhalation for 2 and 3 h starting at 4 and 12 h, respectively, after zymosan injection has beneficial effects in mice with sterile sepsis, whereas twice 100% oxygen inhalation for 1 or 4 h has little preventive effects (4). These results suggest that there might be more important mechanisms underlying the protective effects of hyperoxia treatment on sepsis.

Evidence of massive oxidative stress is well established in critical illnesses characterized by tissue I/R injury and by an intense systemic inflammatory response such as during sepsis (9). Oxidative stress could exacerbate organ injury and thus overall clinical outcome (9). An excessive production of ROS contributes to an overwhelming inflammatory response and tissue injury (9). A growing number of studies have demonstrated that various ROS scavengers have been shown to ameliorate multiple organ dysfunction, blunt proinflammatory cytokines, and thus improve survival rate in various animal models of sepsis or endotoxemia (7-9). In the present study, we also found that when a ROS scavenger (NAC, Vit C, or DMTU) is being used alone, any of them ameliorated tissue oxygenation, reduced organ injury, improved organ dysfunction, increased antioxidant enzymatic activities, reduced proinflammatory cytokines, and thus improved survival rates in mice challenged with zymosan. Our previous study has shown that 100% oxygen treatment exerts its protection by improving the activities of antioxidant enzymes (4). The present study was scheduled to investigate whether ROS scavenger pretreatment enhanced the protective action of oxygen treatment against sepsis. Surprisingly, we found that ROS scavenger pretreatment partially abolished the protective action of 100% oxygen treatment against zymosan-induced sterile inflammation in mice.

It is well known that hyperoxia treatment can induce the production of ROS, and oxygen toxicity is considered to be associated with overproduction of ROS (5, 6). In addition, the production of ROS induced by hyperoxia is also considered to play a critical role in its antibacterial effect (21). Some recent reports suggest that ROS mediates useful actions in relation to cellular functions (22); the ROS induced by hyperoxia inhalation has the ability to act as an important signaling molecule (22). It is now well established that moderate amounts of ROS have all the prerequisites to serve as secondary messengers affecting gene expression, as long as their production does not overrun a toxic threshold (23). In the present study, pretreatment with a ROS scavenger Vit C, NAC, or DMTU partly abolished the beneficial effects of 100% oxygen treatment on zymosan-induced sterile inflammation, suggesting an important role of ROS in the mechanism of hyperoxia treatment against sepsis. According to our studies and a related article (22), we presume that moderate levels of ROS were produced during the 100% oxygen treatment. Then, the ROS, as a trigger, increased the activities of antioxidant enzymes in serum and tissue. The increased activities of antioxidant enzymes can lead to the removal of excessive ROS subsequently induced by zymosan, which results in decreased oxidation injury and produces a protective effect on sepsis/MODS. However, the detailed mechanism is unclear. Some previous studies have found that moderate levels of ROS, especially superoxide anions and hydrogen peroxide, have been shown to regulate signaling cascades mediating the responses to vasoactive peptides, growth factors, cytokines, hormones, and coagulation factors, as well as to physical and chemical stress (23), which may pave the new way to study for the mechanism. Nevertheless, this study is an intriguing phenomenon that may provide the basis for mechanistic studies.

Many studies suggest that oxidative stress and inflammation can interact in many conditions (24, 25). It is believed that 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 (2, 26). The inflammatory cytokines include early inflammatory cytokines such as proinflammatory cytokines TNF-α and IL-6 and anti-inflammatory cytokine IL-10, as well as the late inflammatory cytokine HMGB1 (27, 28). The early and late inflammatory cytokines can interact and facilitate the organ dysfunction and injury in sepsis (29). Some recent studies have demonstrated that depletion of antioxidant enzymes, such as SOD, CAT, and GSH-Px, and the oxidative damage of lipids and proteins by ROS are involved in the pathogenesis of sepsis/MODS (30-32). Our previous study has found that 100% oxygen treatment exerts its protection against sterile sepsis via increasing the activities of antioxidant enzymes (SOD, CAT, and GSH-Px) and the level of serum anti-inflammatory cytokine (IL-10), as well as decreasing the levels of serum proinflammatory cytokines (TNF-α, IL-6, and HMGB1) (4). In the present study, we showed that when ROS scavenger Vit C, NAC, or DMTU was used in the zymosan-stimulated mice not receiving oxygen treatment, any of them could increase the activities of antioxidant enzymes and the level of anti-inflammatory cytokine (IL-10) and decrease the levels of early proinflammatory cytokine (TNF-α) and late proinflammatory cytokine (HMGB1). However, we found that ROS scavenger (Vit C or NAC or DMTU) pretreatment blocked the regulatory action of 100% oxygen treatment on proinfammatory and anti-inflammatory cytokines, as well as the antioxidant system in zymosan-challenged mice, further supporting that ROS may be associated with the protection of 100% oxygen treatment against sterile inflammation. Recently, another study has found that SOD1-deficient mice with more ROS production have less serum cytokines and are less susceptible to LPS-induced septic shock (33), which also support our findings.

Although we got our results from a zymosan-induced systemic inflammation mouse model, we think that the scientific and clinical significance of this finding may be very important. The zymosan-induced inflammation model has been widely used in many research works because zymosan, a substance derived from the cell wall of the yeast S. cerevisiae, can lead to systemic inflammation by inducing a wide range of inflammatory mediators (3). In the present study, we also found that zymosan (1.0 g/kg BW, intraperitoneal injection) successfully induced sterile inflammation model in mice. It is well known that systemic inflammatory response syndrome plays a major role in the pathogenesis of sepsis and is the common pathway from sepsis to MODS (2). However, the detail protocol of 100% oxygen inhalation for treating the disease needs to be further studied before its clinical application. This finding may be very important for treating sepsis and MODS.

We conclude that ROS scavenger pretreatment can block the protective role of 100% oxygen treatment in mice challenged with zymosan, which suggests that ROS may be associated with the protection of 100% oxygen against sterile inflammation. Our results also provide further evidence that when ROS scavenger was used in zymosan-challenged mice not receiving oxygen treatment, it is beneficial by way of its regulatory action on the levels of inflammatory cytokines and the activities of antioxidant enzymes. These data may help develop new strategies for treating patients with sepsis/MODS.

ACKNOWLEDGMENTS

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

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Keywords:

Inflammation; sterile; sepsis; multiple organ dysfunction syndrome; reactive oxygen species; inflammatory cytokine; antioxidant enzyme; 100% oxygen treatment

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