Protective Effects Of 100% Oxygen Inhalation Against Sepsis In Mice And Its Mechanisms

Sepsis, a leading cause of death in patients with critical illness, has been defined as a syndrome mainly characterized by a systemic inflammatory response induced by suspected or proven infection. Nowadays, there is no effective measures for prevention and treatment of sepsis patients. Hyperoxia inhalation as a therapeutic tool has been widely used in clinical practice. But its clinical application is limited because of oxygen toxicity. The fraction of inspired oxygen in oxygen therapy is usually less than 60 percentages. However, some recent studies have reported that early ventilation with 100% oxygen can improve survival rate and organ function. In addition, hyperoxia treatment may induce the generation of reactive oxygen species (ROS) which play an important role in the pathogenesis of sepsis. It is well known that overproduction of reactive oxygen species can lead to oxidative injury. However, some recent studies have found that moderate amounts of ROS have beneficial effect on the generation and development of diseases. Based on the above knowledge, using the zymosan-induced sepsis model, we investigate: first, the reasonable therapeutic protocol of 100% oxygen therapy for sepsis and its therapeutic time window; second, the roles of antioxidant enzymes and inflammatory cytokines in the protection of 100% oxygen therapy; and last, the effect of ROS scavenger pretreatment on the protection of 100% oxygen therapy, as well as the roles of antioxidant enzymes and inflammatory cytokines.Part I: Study on the protective effect of 100% oxygen inhalation on sepsis in mice and its therapeutic time windowMethods1. Reasonable 100% oxygen inhalation can significantly improve organ function and survival rate in a mouse model of sepsisMale ICR mice were randomly divided into 10 groups: Normal saline+Air (NS+Air), Zymosan+Air (ZY+Air), Normal Saline+Oxygen 1h, 2h, 3h, 4h (NS+Oxy 1h, 2h, 3h, 4h), and Zymosan+Oxygen 1h, 2h, 3h, 4h (ZY+Oxy 1h, 2h, 3h, 4h) groups. The sepsis model was induced by injecting intraperitoneally with zymosan at a dose of 1 g/kg of body weight, while animals in the NS control groups were intraperitoneally given the same volume of normal saline. The animals in the ZY+Oxy 1h, 2h, 3h, 4h and NS+Oxy 1h, 2h, 3h, 4h groups were given oxygen treatment by exposure to 100% oxygen for 1, 2, 3 or 4 hours at 4 and 12 hours after zymosan or normal saline injection respectively. The animals in the NS+Air and ZY+Air groups were exposed to air as negative or positive control. The observed indicators included survival rates, serum biochemical parameters (cTnI, ALT, AST, Cr, BUN), organ pathology and histopathological scores, arterial blood gas and serum lactate at different times, and then a reasonable therapeutic protocol of 100% oxygen inhalation was decided.2. Therapeutic time window of the protective action of reasonable 100% oxygen inhalation against sepsis in miceMale ICR mice were randomly divided into 6 groups: Zymosan+Air (ZY+Air) and Zymosan+Oxygen 4, 8, 12, 16, 20 (ZY+Oxy 4, 8, 12, 16, 20) groups. Sterile sepsis model was induced in all animals as mentioned above. The animals in the ZY+Oxy 4, 8, 12, 16, 20 groups were given oxygen treatment with the first exposure to 100% oxygen for 3 hours starting at 4, 8, 12, 16 or 20 hours after zymosan injection respectively, and the second exposure to 100% oxygen for another 3 hours after an 8-hour interval. The survival rates at different time points as well as arterial blood gas analysis before and after first 100% oxygen inhalation in all groups were observed, and then therapeutic time window of the protective action of reasonable 100% oxygen inhalation against sepsis was decided.Results1. Reasonable 100% oxygen inhalation can significantly improve organ function and survival rate in a mouse model of sepsisCompared with those in the NS+Air group, in the ZY+Air group, the organ pathological damage were significant, histopathological scores and levels of serum biochemical parameters (cTnI, ALT, AST, Cr, BUN) were significantly increased (P<0.05), the level of arterial oxygen partial pressure (PaO2) were significantly decreased (P<0.05), the level of serum lactate were significantly increased (P<0.05), as well as 14-day survival rate was decreased to 10% (P<0.05); Compared with those in the ZY+Air group, in the ZY+Oxy 2h, 3h groups, the organ pathological damage was significantly attenuated, histopathological scores and levels of serum biochemical parameters (cTnI, ALT, AST, Cr, BUN) were significantly decreased (P<0.05), the levels of arterial oxygen partial pressure (PaO2) were significantly increased (P<0.05), the levels of serum lactate were significantly decreased (P<0.05), as well as 14-day survival rates were increased to 60%-80% (P<0.05). However, the above indicators in the ZY+Oxy 1h, 4h groups had little significant changes (P>0.05). 2. Therapeutic time window of the protective action of reasonable 100% oxygen inhalation against sepsis in miceCompared with those in the ZY+Air group, in the ZY+Oxy 4, 8, 12 groups, the survival rates were significantly increased (P<0.05), and the PaO2 levels were also increased (P<0.05, more than 300mmHg). However, the survival rates in the ZY+Oxy 16, 20 groups had no significant changes (P>0.05).ConclusionsReasonable 100% oxygen inhalation can significantly improve organ damage and function, tissue oxygenation, and thus increase the survival rate in a mouse model of sepsis, and its therapeutic time window is 12 hours. Part II: Study on the mechanisms of the protective effects of 100% oxygen inhalation on sepsis in miceMethods1. The roles of inflammatory cytokines and antioxidant enzymes during the therapeutic effect of early reasonable 100% oxygen inhalation on sepsis in miceMale ICR mice were randomly divided into 10 groups: The grouping methods were the same as Methods 1, Part I . At 24 hours after zymosan or normal saline injection, the levels of serum pro-inflammatory cytokines TNF-α, IL-6, HMGB1 and anti-inflammatory cytokine IL-10, as well as the activities of serum and organ antioxidant enzymes SOD, CAT, GSH-Px were observed in all groups.Results1. The roles of inflammatory cytokines and antioxidant enzymes during the therapeutic effect of early reasonable 100% oxygen inhalation on sepsis in miceCompared with those in the NS+Air group, in the ZY+Air group, the levels of serum pro-inflammatory cytokines TNF-α, IL-6, HMGB1 were significantly increased (P<0.05), and the levels of anti-inflammatory cytokine IL-10 and the activities of serum and organ antioxidant enzymes SOD, CAT, GSH-Px were also significantly decreased (P<0.05). Compared with those in the ZY+Air group, in the ZY+Oxy 2h, 3h groups, the levels of serum pro-inflammatory cytokines TNF-α, IL-6, HMGB1 were significantly decreased (P<0.05), and the levels of anti-inflammatory cytokine IL-10 and the activities of serum and organ antioxidant enzymes SOD, CAT, GSH-Px were also significantly increased (P<0.05). However, the above indicators in the ZY+Oxy 1h, 4h groups had no significant changes (P>0.05).ConclusionsEarly reasonable 100% oxygen inhalation can decrease the levels of serum pro-inflammatory cytokines, increase the level of serum anti-inflammaotry cytokine, and raise the activities of serum and organ antioxidant enzymes, suggesting the protection of 100% oxygen against sepsis is via regulating the levels of inflammatory cytokines and the activities of antioxidant enzymes.Part III: ROS scavenger pretreatment can block the protection of 100% oxygen therapy against sepsis and its mechanismMethods1. Effects of ROS scavenger on the protection of 100% oxygen therapy against sepsis in miceMale ICR mice were randomly divided into 11 groups: Normal saline+Air (NS+Air), Zymosan+Air (ZY+Air), Zymosan+Air+Vehicle (ZY+Air+Veh), Zymosan+Air+ROS scavenger (N-acetylcysteine (NAC) or Vitamin C (Vit C) or dimethylthiourea (DMTU)), Zymosan+Oxygen (ZY+Oxy), Zymosan+Oxygen+Vehicle (ZY+Oxy+Veh) and Zymosan+Oxy+ROS scavenger (NAC or Vit C or DMTU) groups. The animals in the NS+Air group were intraperitoneally given normal saline as the control. Sterile sepsis was induced in the animals from the other 10 groups. The animals in the ZY+Oxy, ZY+Oxy+Veh and ZY+Oxy+ROS scavenger groups were given oxygen treatment by exposure to 100% oxygen for 3 hours at 4 and 12 hours after zymosan injection respectively. The animals in the NS+Air, ZY+Air, ZY+Air+Veh and ZY+Air+ROS scavenger groups were exposed to room air as the control. In the ZY+Air+ROS scavenger and ZY+Oxy+ROS scavenger groups, the animals were intraperitoneally pretreated with 150 mg/kg NAC or 150 mg/kg Vit C or 50 mg/kg DMTU at 30 minutes before oxygen treatment respectively. In the ZY+Air+Veh and ZY+Oxy+Veh group, the same volume of normal saline was given intraperitoneally at the same time points. The survival rates, serum biochemical parameters (cTnI, ALT, AST, Cr, BUN), organ histopathology and histopathological scores, as well as arterial blood gas and serum lactate at different time points in all group were observed, and then the effect of ROS scavenger pretreatment on the protection of 100% oxygen therapy against sepsis was decided.2. ROS scavenger pretreatment can block the protection of 100% oxygen therapy against sepsis in mice: the roles of inflammatory cytokines and antioxidant enzymesMale ICR mice were randomly divided into 11 groups: The grouping methods were the same as Methods 1, Part III. At different time points after zymosan or normal saline injection, the levels of serum pro-inflammatory cytokines TNF-α, IL-6, HMGB1 and anti-inflammatory cytokine IL-10, as well as the activities of serum and organ antioxidant enzymes SOD, CAT, GSH-Px were observed, and then the roles of inflammatory cytokines and antioxidant enzymes were determined. Results1. Effects of ROS scavenger on the protection of 100% oxygen therapy against sepsis in miceCompared with those in the ZY+Air group, in the ZY+Oxy group, the levels of serum biochemical parameters (cTnI, ALT, AST, Cr, BUN) and organ histopathological scores were significantly decreased (P<0.05), PaO2 was significantly increased (P<0.05), the level of serum lactate was significantly decreased (P<0.05), and 14-day survival rates were significantly increased (P<0.05). When compared with those in the ZY+Oxy group, in the ZY+Oxy+ROS scavenger (NAC or Vit C or DMTU) groups, the levels of serum biochemical parameters (cTnI, ALT, AST, Cr, BUN) and organ histopathological scores were significantly increased (P<0.05), PaO2 was significantly decreased (P<0.05), the level of serum lactate was significantly increased (P<0.05), and 14-day survival rates were significantly decreased (P<0.05). However, when compared with those in the ZY+Air group, in the ZY+Air+ROS scavenger (NAC or Vit C or DMTU) groups, the levels of serum biochemical parameters (cTnI, ALT, AST, Cr, BUN) and organ histopathological scores were significantly decreased (P<0.05), PaO2 was significantly increased (P<0.05), the level of serum lactate was significantly decreased (P<0.05), and 14-day survival rates were significantly increased (P<0.05).2. ROS scavenger pretreatment can block the protection of 100% oxygen therapy against sepsis in mice: the roles of inflammatory cytokines and antioxidant enzymesCompared with those in the ZY+Air group, in the ZY+Oxy group, the levels of serum pro-inflammatory cytokines TNF-α, IL-6, HMGB1 were significantly decreased (P<0.05), and the level of anti-inflammatory cytokine IL-10 were significantly increased (P<0.05), as well as the activities of serum and organ antioxidant enzymes SOD, CAT, GSH-Px were significantly increased (P<0.05). When compared with those in the ZY+Oxy group, in the ZY+Oxy+ROS scavenger (NAC or Vit C or DMTU) groups, the levels of serum pro-inflammatory cytokines TNF-α, IL-6, HMGB1 were significantly increased (P<0.05), and the level of anti-inflammatory cytokine IL-10 were significantly decreased (P<0.05), as well as the activities of serum and organ antioxidant enzymes SOD, CAT, GSH-Px were significantly decreased (P<0.05). However, when compared with those in the ZY+OxyAir group, in the ZY+Air+ROS scavenger (NAC or Vit C or DMTU) groups, the levels of serum pro-inflammatory cytokines TNF-α, IL-6, HMGB1 were significantly decreased (P<0.05), and the level of anti-inflammatory cytokine IL-10 were significantly increased (P<0.05), as well as the activities of serum and organ antioxidant enzymes SOD, CAT, GSH-Px were significantly increased (P<0.05).ConclusionsROS scavenger pretreatment can block the protection of 100% oxygen therapy against sepsis in mice, suggesting that the protection of 100% oxygen therapy against sepsis is associated with the production of ROS induced by exposure to 100% oxygen, and its mechanism is partly through attenuating the regulating effects of 100% oxygen therapy on the levels of inflammatory cytokines and activities of antioxidant enzymes. Summary1. Early reasonable 100% oxygen inhalation can significantly improve organ function, tissue oxygenation, and thus increase the survival rates in septic mice.2. The therapeutic time window of early reasonable 100% oxygen inhalation in septic mice is 12 hours.3. The mechanism underlying the protection of 100% oxygen therapy against sepsis is partly through decreasing the levels of serum pro-inflammatory cytokines, increasing the level of serum anti-inflammatory cytokine, and increasing the activities of serum and tissue antioxidant enzymes.4. ROS scanveger pretreatment does not enhance but block the pretection of 100% oxygen therapy against sepsis in mice partly via blocking the regulating effects of 100% oxygen therapy on inflammatory cytokines levels and antioxidant enzymatic activities.