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Basic Science Aspects

β2-Adrenergic Receptor Antagonist Butoxamine Partly Abolishes the Protection of 100% Oxygen Treatment Against Zymosan-Induced Generalized Inflammation in Mice

Pei, Yuping*; Bai, Xiaoguang*; Dong, Hui; Han, Han*; Zhang, Zishen*; Dong, Hailong*; Hou, Lichao; Xiong, Lize*

Author Information
doi: 10.1097/SHK.0b013e31822413a4

Abstract

INTRODUCTION

Oxygen has been used widely in clinical practice. Theoretically, hyperoxia may aggravate tissue injury, resulting from overproduction of reactive oxygen species (ROS) and nitrogen species (1). However, hyperoxia may be beneficial under certain conditions (2). It has been reported that 100% oxygen (Oxy) ventilation prevents against porcine peritonitis (3, 4) and hemorrhagic shock (5). Recently, we have demonstrated that 100% Oxy inhalation is beneficial to zymosan (ZY)-induced generalized inflammation (6, 7). So, 100% Oxy may be considered as one of the important measures for treating critical ill patients. But its underlying mechanisms remain unclear.

There is some evidence that hyperoxia protects animal models by improving tissue oxygenation (2, 3, 6). We and other authors have demonstrated that ROS scavenger protects animals with sepsis (7, 8). In the meantime, our previous study also shows that ROS scavenger pretreatment does partly abolish the protective action of 100% Oxy treatment against ZY-induced generalized inflammation in mice (7), suggesting that there may be multiple mechanisms underlying the protective effect of hyperoxia including the important role of ROS.

Inotropic agents are always necessary to maintain mean arterial pressure, cardiac output, and systemic Oxy delivery in patients with sepsis. In addition, catecholamines are reported to exert anti-inflammatory effects through binding with β2-adrenergic receptor (β2AR) expressed on the surface of various immune cells (9, 10). Stimulation of β2AR results in the formation of 3′-5′-cyclic adenosine monophosphate (cAMP) in cells and subsequently activates protein kinase A (PKA) (9, 11), thus downregulating the synthesis of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1, and upregulating synthesis of anti-inflammatory cytokines (e.g., IL-10) (9). Furthermore, β2AR agonist has been found to decrease lipopolysaccharide-induced production of inflammatory factors in monocytes (12) and protect against cecal ligation and puncture-induced sepsis (13). These observations suggest that the disturbance of the β2AR system is involved in the pathogenesis of sepsis (10).

It is reported that ROS may modulate the activity of sympathetic nervous system (14, 15), and inhibition of ROS formation decreases β2AR-mediated cAMP formation and PKA activation (16). Therefore, we hypothesized that 100% Oxy inhalation protected ZY-induced generalized inflammation by activating β2AR. Accordingly, here we examined the effects of β2AR antagonist butoxamine (BUT) on the protection of 100% Oxy inhalation against ZY-induced generalized inflammation in mice and also studied the roles of cAMP.

MATERIALS AND METHODS

Animals

We used male Imprinting Control Region mice (specific pathogen-free) 6 to 8 weeks old and weighing 20 to 25 g for the study. All animals were purchased from the Laboratory Animal Center of the Fourth Military Medical University. One week before experimental manipulation, the animals were allowed to acclimatize the experimental housing facilities. Animals were maintained in a constant 12-h light-dark cycle at 20°C to 22°C with standard food and water available ad libitum. We performed all experiments according to the National Institutes of Health guidelines. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University.

Zymosan-induced generalized inflammation model

Generalized inflammation was induced as described previously (6). 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. Mice were injected intraperitoneally with ZY at a dose of 1 g/kg. The same volume of NS was given through the same route to mice in the control group.

Oxygen treatment

We used a sealed Plexiglas chamber for Oxy treatment. At 4 and 12 h after injection of ZY, mice were put inside the chamber. Oxygen was delivered into the chamber through a tubing at a rate of 4 L/min, and carbon dioxide was removed from the chamber with Baralyme (Chemetron Medical Division, Allied Healthcare Products, Inc., St. Louis, Mo). A gas analyzer (medical gas analyzer LB-2, model 40 M; Beckman, Anaheim, Calif) was used to monitor the concentration of inspired Oxy and carbon dioxide at the outlet of the chamber. Oxygen concentration was maintained at 100% during the Oxy treatment. The room and chamber temperature was maintained at 20°C to 22°C. Control animals were exposed to room air. Food and water were available ad libitum during the treatment.

Experimental protocol

Animals were randomly assigned to the following groups (n = 30 per group): NS + air, ZY + air, ZY + Oxy, ZY + Oxy + BUT, ZY + air + BUT, and NS + air + BUT groups. In the animals of ZY + air, ZY + Oxy, ZY + Oxy + BUT, and ZY + air + BUT groups, generalized inflammation model was induced as mentioned above. In the NS + air group and NS + air + BUT group, animals were intraperitoneally injected the same volume of NS. Animals in the ZY + Oxy and ZY + Oxy + BUT groups were given Oxy treatment as described above for 3 h at 4 and 12 h after ZY injection, respectively. Animals from the other groups were exposed to room air as the control. β2-Adrenergic receptor antagonist BUT (5 mg/kg; Sigma Chemical Co) dissolved in NS was intraperitoneally injected 30 min before inhaling 100% Oxy. The survival rates were recorded on days 0.5, 1, 1.5, 2, 3, 5, and 7 after ZY or NS injection.

To further study the effects of β2AR antagonist BUT on ZY-challenged mice with Oxy treatment and its mechanisms, additional animals were used. At 24 h after ZY or NS injection, the heart, liver, lung, and kidney were harvested for histopathologic analysis (n = 8 per group) and for detecting tissue levels of inflammatory cytokines and cAMP (n = 6 per group). The blood samples were also collected for determining serum levels of lactate dehydrogenase (LDH), C-reactive protein (CRP), and inflammatory cytokines as well as cAMP (n = 6 per group). The arterial blood gas analysis was conducted at 15 and 24 h after ZY or NS injection (n = 6 per group).

Histopathologic observations

At 24 h after ZY or NS injection, mice were killed under anesthesia with 2% pentobarbital natrium. Then, the heart, liver, lung, and kidney were harvested immediately, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4- to 6-μm thickness. The sections were stained with hematoxylin-eosin after deparaffinization and rehydration. Based on the scoring standard made by Ferrer et al. (17), the histological slides were analyzed by a pathologist who was blinded to group assignment, using a scale of 1 to 4 to describe normal to severely damaged tissue (see Table, Supplemental Digital Content 1, at https://links.lww.com/SHK/A79).

Arterial blood gas analysis

Arterial blood sample was collected from the carotid artery at 15 and 24 h after ZY or NS injection. The arterial blood gas analysis was performed in all groups using a GEM Premier 3000 gas analyzer (Instrumentation Laboratory, Milan, Italy).

Serum collection

At 24 h after ZY or NS injection, blood samples were collected from the heart. Serum was separated by centrifuging at 3,000 revolutions per minute for 15 min at 4°C and was then divided into aliquots and stored at −80°C until assay.

Homogenization

At 24 h after ZY or NS injection, the heart, liver, lung, and kidney were drawn immediately. 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 (Sigma Chemical Co). Tissue homogenates were prepared in chilled phosphate buffer (0.1 M, pH 7.4) and were centrifuged at 10,000g at 4°C for 10 min. The supernatants were collected, aliquoted, and stored at −80°C until the analysis.

Assay of serum lactate and LDH

Serum levels of lactate and LDH were measured by commercially obtained assay kits (lactate and LDH; BioVision Research, Mountain View, Calif) using an automated analyzer (7600-010; Hitachi, Tokyo, Japan). All assays were done exactly as described by the manufacturer's instructions.

Enzyme-linked immunosorbent assay

The levels of TNF-α, IL-10, and cAMP in serum and tissue homogenates, as well as CRP in serum, were determined by commercially available enzyme-linked immunosorbent assay kits (TNF-α, IL-10, CRP, and cAMP; R&D Systems, Inc, Minneapolis, Minn) with a microplate reader (CA94089; Molecular Devices, Sunnyvale, Calif). All standards and samples were run in duplicate. Quantifications were performed according to the manufacturer's instructions.

Statistical analysis

The survival rates are expressed as percentage, and the histopathologic scores are expressed as median (range). The measurement data are expressed as the mean ± SEM. The intergroup differences in the levels of biochemical parameter and inflammatory cytokines were tested by one-way ANOVA followed by least significant difference test for multiple comparisons. The analysis of the survival rates was tested by Fisher exact probability method. The intergroup differences of histopathologic scores were tested by Kruskal-Wallis H method followed by Nemenyi test for multiple comparisons. The statistical analysis was performed with SPSS 13.0 software (SPSS Inc, Chicago, Ill). In all tests, P < 0.05 was considered statistically significant.

RESULTS

Oxygen treatment and BUT had no influence on breathing in ZY-challenged mice

Although Oxy is thought to be essential and beneficial to all animal life, hyperoxia has an inhibitory effect on breathing (18). In this study, to identify the impact of 100% Oxy inhalation on breathing in mice, we examined arterial pH, PO2, and PCO2 at the end of Oxy treatment immediately (15 h after ZY or NS injection).

As shown in the Figure, Supplementary Digital Content 2, at https://links.lww.com/SHK/A80, the value of arterial PO2 was 96.3 ± 2.7 mmHg in the NS + air group. The ZY-challenged mice showed the lower level of arterial PO2 (85.3 ± 5.9 mmHg). One hundred percent Oxy inhalation induced markedly higher values of arterial PO2 in the ZY + Oxy group (468.7 ± 26.5 mmHg) and ZY + Oxy + BUT group (452.9 ± 31.0 mmHg). Butoxamine has no effects on the level of arterial PO2 in ZY-challenged mice or normal mice. We observed no significant difference in the values of arterial pH and PCO2 among all groups with or without Oxy treatment. These data suggest that both Oxy treatment and BUT did not induce significant respiratory depression in all the mice.

β2AR antagonist BUT resulted in a significant reduction in survival rates of ZY-challenged mice with Oxy treatment

As was demonstrated in Figure 1, all animals survived during the observation period in the NS + air group and NS + air + BUT group. In the ZY + air group, 20% animals survived at day 7 after intraperitoneal injection of ZY at a dose of 1 g/kg body weight (P < 0.05 vs. NS + air group; n = 30 per group). With 100% Oxy treatment, the 7-day survival rate was increased to 80% in the ZY + Oxy group (P < 0.05 vs. ZY + air group; n = 30 per group), but this increase was partly reversed by administration of BUT before Oxy treatment. In the ZY + Oxy + BUT group, the 7-day survival rate was decreased to 50% (P < 0.05 vs. ZY + Oxy group; n = 30 per group). There was no significant difference in the 7-day survival rate between the ZY + air + BUT (16.7%) and ZY + air groups (20%) (P > 0.05; n = 30 per group).

F1-10
Fig. 1:
β2-Adrenergic receptor antagonist BUT partly reversed the increase in survival rate in ZY-challenged mice with 100% Oxy treatment. In the animals of ZY + air, ZY + Oxy, ZY + Oxy + BUT and ZY + air + BUT groups, generalized inflammation was induced by intraperitoneal injection of ZY (1 g/kg), and in animals of the NS + air group and NS + air + BUT group, the same volume of NS was given as a control. All animals were exposed to 100% Oxy or room air for 3 h starting at 4 and 12 h after ZY or NS injection, respectively. Animals from the ZY + Oxy + BUT group and NS + air + BUT group were intraperitoneally administered 5 mg/kg BUT dissolved in NS at 30 min before inhaling 100% Oxy. 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 group.

β2AR antagonist BUT blocked the improvement in organ histopathologic changes in ZY-challenged mice with Oxy treatment

Pathologic scoring system is an arbitrary mean for quantifying the visual changes of organs to allow quantitative comparisons (17). At 24 h after ZY or NS injection, organs of animals in all groups were collected for histopathologic analysis. As shown in Table 1, the pathologic scores for heart, liver, lung, and kidney in the NS + air group and the NS + air + BUT group were 1.0. In the ZY + air group, the pathologic scores for the previously mentioned organs were 3 to 3.5, respectively, which were higher than those in the NS + air group (P < 0.05; n = 8 per group). In the ZY + Oxy group, with 100% Oxy treatment, the pathologic scores for the previously mentioned organs were markedly decreased to 1 to 1.5, respectively (P < 0.05 vs. ZY + air group; n = 8 per group; Table 1), but these decreases were abolished by BUT treatment (3.0-3.5) (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 8 per group; Table 1). There were no significant differences in the pathologic scores for the previously mentioned organs between the ZY + air + BUT (3.0-4.0) and ZY + air (3.0-3.5) groups (P > 0.05; n = 8 per group; Table 1).

T1-10
Table 1:
Effects of β2AR antagonist BUT on the organ pathologic scores in ZY-challenged mice with Oxy treatment

β2AR antagonist BUT attenuated the improvement n the tissue injury indicators in ZY-challenged mice with Oxy treatment

At 24 h after ZY or NS injection, serum LDH and CRP were determined. As shown in Figure 2, in the NS + air group, the levels of serum LDH and CRP were 379.9 ± 45.9 U/L and 2.3 ± 1.7 ng/mL, respectively. In the NS + air + BUT group, the levels of serum LDH (463.8 U/L) and CRP (3.8 ± 1.9 ng/mL) had no significant differences compared with those in the NS + air group, whereas in the ZY + air group, ZY injection resulted in significant increases in the levels of serum LDH (1,449.0 ± 89.2 U/L) and CRP (235.0 ± 18.15 ng/mL) (P < 0.05 vs. NS + air group; n = 6 per group). In the ZY + Oxy group, the levels of serum LDH (992.4 ± 125.6 U/L) and CRP (88.3 ± 20.2 ng/mL) were significantly decreased compared with those in the ZY + air group (P < 0.05; n = 6 per group), but the decreases were partly abolished by administration of BUT in the ZY + Oxy + BUT group (LDH, 1,224.2 ± 199.8 U/L; CRP, 208.8 ± 19.5 ng/mL) (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 6 per group). There were no statistically significant differences in the levels of serum LDH and CRP between the ZY + air + BUT (LDH, 1,494.7 ± 96.6 U/L; CRP, 246.3 ± 23.2 ng/mL) and ZY + air (LDH, 1,449.0 ± 89.2 U/L; CRP, 235.0 ± 18.15 ng/mL) groups (P > 0.05; n = 6 per group).

F2-10
Fig. 2:
Effects of β2AR antagonist BUT on the levels of serum LDH and CRP in ZY-challenged mice with Oxy treatment. The grouping methods and experimental protocols were the same as Figure 1. The serum levels of LDH (A) and CRP (B) 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 group.

β2AR antagonist BUT prevented the decrease in serum TNF-α level and the increase in serum IL-10 level in ZY-challenged mice with Oxy treatment

Activation of the β2-mediated pathway downregulates the production of TNF-α and upregulates the level of IL-10 (9). In this study, TNF-α and IL-10 in serum and organs were tested. In the NS + air group, the levels of serum TNF-α and IL-10 were 12.6 ± 6.9 pg/mL and 24.0 ± 11.8 pg/mL, respectively. In the ZY + air group, the levels of serum TNF-α (210.3 ± 21.5 pg/mL) and IL-10 (169.6 ± 58.2 pg/mL) were significantly increased compared with those in the NS + air group (P < 0.05; n = 6 per group; Fig. 3). With 100% Oxy treatment, serum TNF-α level was markedly decreased, and serum IL-10 level was further increased (TNF-α, 89.2 ± 17.2 pg/mL; IL-10, 278.3 ± 39.4 pg/mL) (P < 0.05, ZY + Oxy group vs. ZY + air group; n = 6 per group; Fig. 3), but these effects on the above inflammatory cytokines were partly abolished by intraperitoneal injection of BUT before Oxy inhalation (TNF-α, 132.7 ± 21.2 pg/mL; IL-10, 221.3 ± 40.2 pg/mL) (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 6 per group; Fig. 3). No statistically significant differences in the levels of serum TNF-α and IL-10 were present between the ZY + air + BUT (TNF-α, 220.4 ± 23.4 pg/mL; IL-10, 156.6 ± 56.7 pg/mL) and ZY + air groups (P > 0.05; n = 6 per group; Fig. 3), and there were no significant differences in the levels of serum TNF-α and IL-10 between the NS + air + BUT (TNF-α, 15.5 ± 5.2 pg/mL; IL-10, 24.5 ± 6.5 pg/mL) and NS + air groups (P > 0.05; n = 6 per group; Fig. 3), which demonstrated that BUT had no significant influences on the levels of serum TNF-α and IL-10 in ZY-challenged mice and in normal mice.

F3-10
Fig. 3:
Effects of β2AR antagonist BUT on TNF-α and IL-10 productions in ZY-challenged mice with Oxy treatment. The grouping methods and experimental protocols were the same as Figure 1. TNF-α (A, C) and IL-10 (B, D) 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 group.

Moreover, in the ZY + air group, the levels of TNF-α and IL-10 in the heart, liver, lung, and kidney were significantly increased compared with those in the NS + air group (P < 0.05; n = 6 per group; Fig. 3). In the ZY + Oxy group, the levels of TNF-α in the heart, liver, lung, and kidney were markedly decreased compared with those in the ZY + air group (P < 0.05; n = 6 per group), whereas the levels of IL-10 in the heart, liver, lung, and kidney were significantly increased when compared with those in the ZY + air group (P < 0.05; n = 6 per group; Fig. 3). These effects of 100% Oxy treatment on the inflammatory cytokines in the previously mentioned organs were partly reversed by BUT treatment (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 6 per group; Fig. 3). Butoxamine had no significant effects on the levels of tissue TNF-α and IL-10 in ZY-challenged mice (P > 0.05, ZY + air + BUT group vs. ZY + air group; n = 6 per group) and in normal mice (P > 0.05, NS + air + BUT group vs. NS + air group; n = 6 per group).

β2AR antagonist BUT partly abolished the improvement of tissue oxygenation in ZY-challenged mice with Oxy treatment

As shown in Figure 4, at 24 h after ZY or NS injection, the levels of arterial pH value (7.26 ± 0.05), PO2 (72.5 ± 9.0 mmHg), and HCO3 (18.4 ± 1.5 mmol/L) in the ZY + air group were decreased markedly compared with those in the NS + air group (pH value, 7.38 ± 0.03; PO2, 108.0 ± 7.3 mmHg; HCO3, 24.6 ± 0.9 mmol/L) (P < 0.05; n = 6 per group). With Oxy treatment, the levels of arterial pH value, PO2, and HCO3 were significantly increased in the ZY + Oxy group (pH, 7.36 ± 0.02; PO2, 94.0 ± 3.1 mmHg; HCO3, 22.2 ± 1.4 mmol/L) (P < 0.05, ZY + Oxy group vs. ZY + air group; n = 6 per group), and these increases were reversed by BUT treatment in the ZY + Oxy + BUT group (pH, 7.31 ± 0.04; PO2, 82.7 ± 7.3 mmHg; HCO3, 20.2 ± 1.3 mmol/L) (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 6 per group).

F4-10
Fig. 4:
Effects of β2AR antagonist BUT on tissue oxygenation in ZY-challenged mice with Oxy treatment. The arterial pH value (A), PO2 (B), PCO2 (C), HCO3 (D), and serum lactate (E) were measured at 24 h after ZY or NS injection. The grouping methods and experimental protocols were the same as Figure 1. 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 group.

The levels of arterial PCO2 and serum lactate are 37.5 ± 1.9 mmHg and 1.3 ± 0.2 mmol/L, respectively. Meanwhile, ZY injection significantly increased the levels of arterial PCO2 (50.17 ± 3.3 mmHg) and serum lactate (9.1 ± 1.8 mmol/L) (P < 0.05, ZY + air group vs. NS + air group; n = 6 per group). With Oxy treatment, the levels of arterial PCO2 and serum lactate were decreased markedly in the ZY + Oxy group (PCO2,40.3 ± 3.3 mmHg; serum lactate, 5.4 ± 0.9 mmol/L) (P < 0.05, ZY + Oxy group vs. ZY + air group; n = 6 per group); these decreases were reversed by BUT treatment (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 6 per group).

Butoxamine had no statistically significant influences on the levels of arterial pH value, PO2, HCO3, PCO2, and serum lactate in ZY-challenged mice (P > 0.05, ZY + air + BUT group vs. ZY + air group; n = 6 per group) and in normal mice (P > 0.05, NS + air + BUT group vs. NS + air group; n = 6 per group; Fig. 4).

cAMP contributed to the effects of β2AR antagonist BUT on ZY-challenged mice with Oxy treatment

To define the downstream signaling events of the β2AR pathway, we examined the possible involvement of cAMP (9). In NS + air group, the level of serum cAMP is 13.6 ± 3.1 pmol/L. We observed that ZY increased serum cAMP level (44.2 ± 11.3 pmol/L) at 24 h after ZY injection (P < 0.05, ZY + air group vs. NS + air group; n = 6 per group; Fig. 5). Oxygen treatment further elevated the level of serum cAMP (138.5 ± 24.3 pmol/L) (P < 0.05, ZY + Oxy group vs. ZY + air group; n = 6 per group; Fig. 5), and this increase was partly reversed by intraperitoneally injected BUT in the ZY + Oxy + BUT group (64.3 ± 14.3 pmol/L) (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 6 per group; Fig. 5).

F5-10
Fig. 5:
Effects of β2AR antagonist BUT on the cAMP production in ZY-challenged mice with Oxy treatment. The grouping methods and experimental protocols were the same as Figure 1. The levels of cAMP in serum (A) and tissues (B) 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 group.

In contrast, the levels of cAMP in the heart, liver, lung, and kidney in the ZY + air group were significantly decreased compared with those in the NS + air group (P < 0.05 vs. NS + air group; n = 6 per group). However, Oxy treatment increased the levels of cAMP in the heart, liver, lung, and kidney (P < 0.05, ZY + Oxy group vs. ZY + air group; n = 6 per group; Fig. 5), and the increases were partly reversed by BUT (P < 0.05, ZY + Oxy + BUT group vs. ZY + Oxy group; n = 6 per group).

Butoxamine had no marked influences on the levels of serum and tissue cAMP in ZY-challenged mice (P > 0.05, ZY + air + BUT group vs. ZY + air group; n = 6 per group) and in normal mice (P > 0.05, NS + air + BUT group vs. NS + air group; n = 6 per group).

DISCUSSION

In the present study, we found that β2AR antagonist BUT partly abolished the protection of 100% Oxy against ZY-induced generalized inflammation in mice. We also showed that ZY led to the increased serum cAMP level and the decreased tissue cAMP level. In addition, Oxy treatment increased both serum and tissue cAMP, which were partly abolished by BUT treatment.

Sepsis remains a major cause of high mortality in the intensive care unit, with an annual increase in incidence of about 9% and a mortality of about 25% (19). Systemic inflammatory response syndrome is frequently observed after surgery or trauma, leading to the possibility of developing sepsis and subsequent multiple organ dysfunction syndrome (MODS) (20). Our experiments were performed on a widely accepted model of generalized inflammation that has been used in many previous studies, and the changes of organ function, severity of tissue injury, inflammatory response, and survival rates were used as monitoring criteria (21-24).

Oxygen is an atmospheric gas essential for survival of all living creatures. Because of its low costs and easy availability, Oxy therapy has been considered as one of the most critical consideration in the management of various medical and surgical disorders (2). However, the data on hyperoxia-related oxidative stress are controversial. Several experimental studies demonstrate that 100% Oxy inhalation or ventilation can improve the organ function and survival rates of animals with sepsis or septic shock (3, 5-7). On the other hand, it is well known that ROS accounts for Oxy toxicity (1). Reactive oxygen species scavenger can alleviate organ injury and improve survival rates of animals with sepsis (8). It is interesting that hyperoxia may trigger repair mechanisms and increase antioxidative capacity through overproduction of ROS (1). Bigdeli et al. (25) report that normobaric hyperoxia can produce free radical Oxy and increase antioxidant enzyme activity to prevent against ischemia brain injury. We have found that ROS scavenger pretreatment partly abolishes the protective effects of 100% Oxy (7), suggesting that ROS may be associated with the protection of 100% Oxy. In addition, some data show that ROS may modulate the sympathetic nervous system activity (14, 15), although the relationship between them has not been fully elucidated. Furthermore, inhibition of ROS formation decreases β2AR-mediated cAMP formation and PKA activation (16). Therefore, we hypothesized that 100% Oxy inhalation protected against sepsis through activation of β2AR signaling pathway, although the detailed mechanisms of ROS and β2AR pathway during Oxy treatment need further studies.

The pathogenesis of sepsis is characterized with uncontrolled systemic inflammatory response (19). TNF-α, produced by activated monocytes and macrophages, is a major proinflammatory cytokine during the inflammatory response induced by ZY, correlating with the severity of sepsis (20). IL-10 is a potent anti-inflammatory cytokine acting primarily on antigen-presenting cells such as macrophages and dendritic cells (17). Activation of β2AR-mediated pathway brings about downregulation of TNF-α and upregulation of IL-10 (9). Volman and colleagues (19) report that TNF-α mRNA and IL-10 mRNA are strongly upregulated in the lung, liver, and kidney in ZY-induced murine model for the MODS. In this study, we observed the production of TNF-α and IL-10 in the heart, liver, lung, kidney, and serum in all animals, and the results are consistent with those of the above studies.

The sympathetic nervous system, a major component of the autonomic nervous system, innervates all lymphoid organs (9). Evidences support the inflammatory modulation of β-adrenergic receptor, and β2 activation as well as β1 blockade seems to downregulate proinflammatory response by modulating the cytokine production profile (11). β2-Adrenergic receptor, a member of the guanine nucleotide regulatory protein-coupled receptor, is widely expressed on various tissues and cells involved in the host immune response (22). Recent studies show that activation of β2AR exerts the anti-inflammatory effects, increasing the release of IL-10 and decreasing the level of TNF-α (9, 11, 16). Stimulation of β2AR results in activation of adenylyl cyclase via stimulatory G proteins, which leads to the subsequent increase in cytoplasmic cAMP, a second messenger in the signal transduction system (11). 3′-5′-Cyclic adenosine monophosphate activates cAMP-dependent protein kinase, modulating many cellular processes mainly through activating PKA (9, 11).

It has been demonstrated that research data consistent with the previous data. β2-Adrenergic receptor mediates immunomodulatory action, and activation of β2AR provides beneficial effects to the host suffering from sepsis (8). A selective β2AR agonist, terbutaline, protects against cecal ligation and puncture-induced sepsis through inhibition of proinflammatory mediators and attenuation of oxidant production (13). Overexpression of renal β2AR has an anti-inflammatory effect and maintains renal function following lipopolysaccharide-induced endotoxemia through the cAMP-PKA pathway (26, 27).

To define the role of β2AR in the protective effect of Oxy treatment, a selective β2AR antagonist, BUT, was applied in this study. We observed that an intraperitoneal injection of BUT (5 mg/kg) before Oxy inhalation partly reversed the protection of 100% Oxy, which demonstrated the important roles of β2AR in this protective effect of hyperoxia. This antagonistic effect of BUT on 100% Oxy treatment was, most likely, not due to its effect of β2AR antagonist on airway and oxygenation. The doses of 2 mg/kg to 25 mg/kg of BUT have been used in several previous studies, and there was no evidence that this dose range of BUT affects the oxygenation of animals (28-30). Kato and colleagues (31) report that BUT (20 mg/kg) does not affect the levels of arterial pH, PO2, and PCO2 in rats. In this study, if BUT (5 mg/kg) induced bronchospasm, the oxygenation of mice treated with BUT would be worse than the mice treated with NS. However, our arterial blood gas analysis showed that BUT at 5 mg/kg had no significant influence on the levels of pH, PO2, and PCO2 in normal mice and ZY-challenged mice with or without Oxy treatment. Moreover, we observed that BUT did abolish the protection of 100% Oxy treatment against sepsis. The β1AR is mainly expressed in the heart, and β2AR is mainly expressed in the airway. It is demonstrated that the adrenergic modulation of the cellular immune system is independent of an altered cardiac function or arterial blood pressure, and β2AR blockade does not affect arterial blood pressure during systemic inflammation or following hemorrhagic shock in humans and animals (22, 32).

3′-5′-Cyclic adenosine monophosphate is considered as a key intracellular second messenger and has been shown to have anti-inflammatory and tissue-protective effects at increased levels (10). It has been reported that the anti-inflammatory effect of β2AR activation is dependent on an increase in intracellular cAMP levels (9, 11). In this study, we evaluated cAMP concentrations in the heart, liver, lung, kidney, and serum. The tissue levels represent cAMP in parenchymal cells, and the serum level represents cAMP level in nonparenchymal cells, including endothelial cells and inflammatory cells (33). We demonstrated that the serum cAMP level was higher, and the tissue cAMP levels were lower in ZY-challenged mice. We also showed that Oxy treatment increased both serum cAMP level and tissue cAMP levels, which were partly abolished by BUT pretreatment. Based on the previous literatures, the CD4+ T-helper type 1 (TH1) and TH2 balance is involved in the pattern of cytokine production in sepsis (34), and activation of adenylate cyclase leads to a shift toward TH2-type responses, which are anti-inflammatory, whereas downregulation of intracellular cAMP stimulates the TH1-type response, resulting in inflammation (10). The above statements suggest that Oxy treatment may modulate the TH1/TH2 balance to protection of ZY-induced generalized inflammation. Further studies are needed on the possible mechanisms.

There is some evidence that Oxy treatment may be an adjunct measure during the first 12 to 24 h of septic shock (2). Our results also suggest that moderate 100% Oxy inhalation in the early phase is a potential measurement for sepsis/MODS. However, all these arguments favoring the O2 treatment exclusively originate from experimental animals. Moreover, neither the window of opportunity nor the exposure period necessary to cause O2 toxicity is known in clinical studies. Therefore, preclinical studies and controlled clinical trials are needed to test the safety and efficacy of Oxy treatment for sepsis/MODS.

In summary, we conclude that BUT pretreatment partly abolished the protective action of 100% Oxy treatment in the ZY-challenged mice, which provides further evidence for the mechanism of Oxy treatment preventing against ZY-induced generalized inflammation. Treatments targeting β2AR may be developed as an effective therapy for sepsis/MODS. Furthermore, this protective effect appears due to elevation of cAMP.

ACKNOWLEDGMENTS

The authors thank Prof Wenyong Wang and Prof Yiling Zhao from the Department of Pathology, Fourth Military Medical University, for assisting in histopathologic analysis; Prof Lei Shang from the Department of Health Statistics, Fourth Military Medical University, for help in the statistics analysis; and Prof Shanlu Liu from the Department of Molecular Microbiology and Immunology, Bond Life Science Center, University of Missouri, and Dr Kening Wang in the Medical Virology Section, Laboratory of Infectious Diseases, NIAID, National Institutes of Health, for their insightful comments.

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Keywords:

Zymosan; inflammatory cytokine; adrenergic receptor; 100% oxygen treatment; cyclic adenosine monophosphate

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