| Acute lung injury(ALI) is an inflammatory disease with a high mortality rate, typically seen in individuals with sepsis. Sepsis and its associated lung injury is one of the main causes of death in hospitalized patients. Sevoflurane is a kind of anesthesia gas for frequently used in clinical, and sub-dose of anesthesia sevoflurane was used for sedation in ICU patients, and oxygen which the oxygen concentration was higher than the oxygen in the air, was frequently used in clinical, especially for the treatment of critically ill patients for support treatments.Additionally,studies had shown that nitric oxide(NO) was an effective bactericidal molecules, and enhanced NO-mediated bacterial clearance can improve the survival rate of septic murine. Here, we first observed that inhalation of 0.5 MAC sevoflurane in 60% oxygen protect against lethality resulting from sepsis induced by cecal ligation and puncture(CLP) or intraperitoneal injection of lipopolysccharide(LPS), and there was a time window about the protective effects of 0.5 MAC sevoflurane in 60% oxygen on LPS-induced sepsis. Treatment with 0.5 MAC sevoflurane in 60% oxygen attenuated lung injury resulting from CLP-induced sepsis and improved lung function through by inhibiting lung inflammation, and protecting alveolar endothelial and epithelial barriers. We further demonstrated that inhalation of 0.5 MAC sevoflurane in 60% oxygen protected against the lethality resulting from sepsis through NO-mediated clearance of bacteria. These results suggest that a sub-anesthetic dose of sevoflurane in 60% oxygen can effectively prevent against sepsis and its related lung injury and enhanced NO-mediated clearance of bacteria, with better clinical prospect.Research was divided into two parts: Effects of sevoflurane combined withoxygen on sepsis; Sevoflurane combined with oxygen and NO-mediated bactericidal mechanisms.Part 1: Effects of sevoflurane combined with oxygen on sepsis.Objective : To investigate the effects of sevoflurane combined with oxygen on the survival rate of septic muroideaand acute lung injury resulting from sepsis.Methods: Research was divided into three stages. Stages 1: Effects of sevoflurane combined with oxygen on survival of murine sepsis.The studay used LPS-induced mice sepsis model and CLP-induced SD rats or mice sepsis models. The control groups were given the same volume of saline intraperitoneal injection or the sham operation.100% oxygen was used as a positive treatment control.Treatment with sevoflurane combined with various concentrations of oxygen on survival of animals with LPS-induced sepsis.180 mice were randomly divided into nine groups: NS+Air, NS+100%Oxy, NS+0.5MAC Sevo +100%Oxy, LPS+Air, LPS+100%Oxy, LPS+0.5MAC Sevo +100% Oxy, LPS+0.5MAC Sevo +80%Oxy, LPS+0.5MAC Sevo +60%Oxy, LPS+0.5MAC Sevo +40%Oxy. The methods of intervention: mice were treated with 100% oxygen or 0.5 MAC sevoflurane in various concentrations of oxygen for 2 h at 6h after mice were challenged with lipopolysaccharride LPS/NS intraperitoneal injection, respectively. The control groups were given the freedom to breathe air. And the 7-day survival rate was observed. Treatment with sevoflurane combined with various concentrations of oxygen on survival of animals with CLP-induced sepsis.Using ICR/Km murine and SD rat models, 180 mice or 180 rats were randomly divided into nine groups, respectively:sham+Air, sham+100%Oxy, sham+0.5MAC Sevo +100%Oxy, CLP+Air, CLP+100%Oxy, CLP+0.5MAC Sevo +100% Oxy, CLP+0.5MAC Sevo +80%Oxy, CLP+0.5MAC Sevo +60%Oxy, CLP+0.5MAC Sevo +40%Oxy. The methods of intervention: animals were treated with 100% oxygen or 0.5 MAC sevoflurane in various concentrations of oxygen for 2 h at 6 h after CLP/Sham-operation challenge, respectively. The control groups were given the freedom to breathe the air. And the 7-day survival rate was observed.The research to the time window about the protective effects of an optimal-ratio hybrid gas of sevoflurane and oxygen on survival of animals with LPS-induced sepsis. 270 mice were randomly divided into nine groups: NS+Air, LPS+Air, LPS+0.5MAC Sevo +60% Oxy(12h), LPS+0.5MAC Sevo +60%Oxy(10h), LPS+0.5MAC Sevo +60%Oxy(8h), LPS+0.5MAC Sevo +60%Oxy(6h), LPS+0.5MAC Sevo +60%Oxy(4h), LPS+0.5MAC Sevo +60%Oxy(2h), LPS+0.5MAC Sevo +60%Oxy(0h). After ICR/Km mice challenged by intraperitoneal injection of LPS 0h, 2h, 4h, 6h, 8h, 10 h, 12 h, 100% oxygen or 0.5 MAC sevoflurane in 60% oxygen was inhaled for 2 h, respectively. The control groups were given the freedom to breathe the air. And the 7-day survival rate was observed.Stages 2: Effects of an optimal-ratio hybrid gas of sevoflurane and oxygen on inflammatory response of experimental rats with sepsis. Male SD rats were randomly divided into 6 groups: sham+Air, sham+100% Oxy, sham+0.5MAC Sevo +60%Oxy, CLP+Air, CLP+100%Oxy, CLP +0.5MAC Sevo +60% Oxy. The methods of intervention: animals were treated with 100% oxygen or 0.5 MAC sevoflurane in 60% oxygen for 2 h at 6 h after CLP/Sham-operation challenge, respectively. The control groups were given the freedom to breathe air. 24 h after the operation, the levels of inflammatory cytokines(IL-1β, TNF-α, IL-6, HMGB, IL-10) in serum and peritoneal fluid were evaluated.Stages 3: Effects of an optimal-ratio hybrid gas of sevoflurane and oxygen on lung injury and lung function of sepsis. Male SD rats were randomly divided into 6 groups: sham+Air, sham+100% Oxy, sham+0.5MAC Sevo +60%Oxy, CLP+Air,CLP+100%Oxy, CLP +0.5MAC Sevo +60% Oxy. The methods of intervention was the same as previous. 24 h after the operation, Sampals were assessed for pathological changes in the lung tissue, the number of neutrophils gathered in the lung tissue sections, lung injury indicators(Lung wet and dry ratio, MPO activity in lung tissue, contents of total protein and albumin in BAFL) and lung function parameters( arterial blood gas analysis:levels of p H, Pa O2, Pa O2/Fi O2, Pa CO2, and lactate).Results: Theprotective effect of 0.5MAC Sevoflurane in 60% oxygen improved CLP or LPS induced sepsis was the best, and the protective effect of 0.5MAC sevoflurane in 60% oxygen on LPS-induced sepsis had a therapeutic time window which was within 8h for LPS-induction, but the best intervention was performed at 6h after induction. 0.5MAC Sevoflurane in 60% oxygen significantly inhibited inflammatory response of sepsis induced by CLP. And 0.5MAC sevoflurane in 60% oxygen improved CLP-induced septic lung injury and lung function in septic rats.Part 2: Sevoflurane combined with oxygen improved survival of sepsis and NO-mediated bactericidal mechanisms.Objective : To explore sevoflurane combined with oxygen improved survival of sepsis through by NO-mediated bactericidal mechanisms.Methods: Experiment was divided into three parts.Part one, whether sevoflurane combined with oxygen enhanced bacterial clearance of CLP-induced sepsis rats. Male SD rats were randomly divided into 6 groups: sham+Air, sham+100% Oxy, sham+0.5MAC Sevo +60%Oxy, CLP+Air, CLP+100%Oxy, CLP +0.5MAC Sevo +60% Oxy. Using CLP-induced sepsis model, the control groups were given sham operation.. The intervention of 100% oxygen or 0.5MAC sevoflurane in 60% oxygen was the same as previous.24 h after operation, the peritoneal lavage fluid was obtained and cultured over night on blood-agar base plates at 37 ℃, and the CFUs were counted.Parttwo, whether inhibition to NOS will reverse the protective effect of sevoflurane combined with oxygen about improvement of sepsis survival.Mice were randomly divided into ten groups: sham+Air,CLP+NS+100% Oxy, CLP+NS+0.5MAC Sevo +60%Oxy, CLP+NS+Air, CLP+ L-NAME1 +Air, CLP+ L-NAME1+100%Oxy,CLP + L-NAME1 +0.5MAC Sevo +60% Oxy, CLP+ L-NAME2 +Air, CLP+ L-NAME2+100%Oxy, CLP + L-NAME2 +0.5MAC Sevo +60% Oxy. The intervention of 100% oxygen or 0.5MAC Sevoflurane in 60% oxygen was the same as previous. Toinhibit NOS, treatment with N G-nitro L-arginine methylester(L-NAME)(10 mg/kg or 20 mg/kg) was administered intraperitoneally 4 h before treatment of 100% oxygen or 0.5 MAC sevoflurane in 60% oxygen. The same volume of NS was given for the control groups. And the 7-day survival rate was observed.Part three,whether inhibition to NOS will reverse the effects of sevoflurane combined with oxygen improved bacterial clearance. Mice were randomly divided into sevone groups:sham+Air, CLP+NS+100% Oxy, CLP+NS+0.5MAC Sevo +60%Oxy, CLP+NS+Air, CLP+ L-NAME +Air, CLP+ L-NAME+100%Oxy, CLP + L-NAME +0.5MAC Sevo +60% Oxy. The intervention of 100% oxygen or 0.5MAC Sevoflurane in 60% oxygen and inhibition to NOS were the same as previous( L-NAME, intraperitoneal injection 20mg/kg).Results: 0.5MACsevoflurane combined with 60% oxygen enhanced bacterial clearance of CLP-induced sepsis rats.Inhibition to NO synthase prevents an improvement of survival rate of CLP-challenged mice by inhalation of 0.5 MAC sevoflurane in 60% oxygen. Inhibition to NO synthase blocks an improvement of bacterial clearance of CLP-challenged mice by inhalation of 0.5 MAC sevoflurane in 60% oxygen.Conclusion: Sub-anesthetic dose of sevoflurane in 60% oxygen effectively improve sepsis and its related lung injury and enhance sterilization mechanism through NO-mediated clearance of bacteria. |
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