The Therapeutic Effect Of 0.5 MAC Isoflurane In 60% Oxygen On Sepsis And Its Mechanisms

Sepsis is a major cause of death in the surgical intensive care unit(ICU) with high prevalence, high mortality, high cost of treatment, and few effective treatment strategies. Sepsis has become a critical threat to human health, but also become one of the outstanding problems in the field of critical care medicine. Therefore, it is urgent to find effective treatment strategies for sepsis. In our earlier studies, we have observed that 0.5 MAC(minimum alveolar concentration) isoflurane has a therapeutic action against experimental sepsis, inhibits abnormal inflammatory response resulting from sepsis, inhibits nuclear translocation of NF-κB after LPS stimulation, and reverses upregulation of mi RNAs m RNA induced by CLP. Based on these previous results, this project was scheduled to study the roles of nitric oxide(NO) in the mechanism underlying the protective effects of 0.5MAC isoflurane in 60% oxygen on experimental sepsis, to prove whether mi RNAs contributed to this therapeutic effect, to study the ways for combined administration of 0.5 MAC isoflurane with 60% oxygen to regulate NF-κB inflammatory pathway, to explore the roles of Nrf2/HO-1 anti-inflammatory pathway during these protective effects, and to explore whether combined administration of 0.5 MAC isoflurane/sevoflurane with 60% oxygen has a therapeutic effect on experimental sepsis inaged rats. If succeed, our project would provide more evidences for the protective action against sepsis by combined administration of 0.5 MAC isoflurane with 60% oxygen, and provide potential targets for development of new methods to treat sepsis.Objective To explore the protective effects of 0.5 MAC isoflurane in 60% oxygen on sepsis and its possible mechanism.Materials and methods Part 1. Male Imprinting Control Region/Kunming mice(ICR/Km)(25-30g) were randomly divided into seven groups: Sham+NS+Air, CLP+NS+Air, CLP+NS+100%Oxy, CLP+NS+0.5MAC ISO+60%Oxy, CLP+L-NAME+100%Oxy, CLP+L-NAME+0.5MAC ISO+60%Oxy and CLP+L-NAME+Air groups. Sepsis was induced by cecal ligation and puncture(CLP), and animals in sham group were given an operation without CLP procedure. Treatment was performed by inhalation of 100% oxygen or 0.5 MAC isoflurane in 60% oxygen for 1 h at 1 and 6 h after CLP respectively. And 20 mg/kg of NO synthase inhibitor L-NAME was administered intraperitoneally 4 h before the start of the process, and equal volume of NS was used as the control. The survival rate was observed for 7 days. Blood samples were harvested at 24 h after CLP for determining serum levels of inflammatory cytokines, femoral blood was drawn for arterial blood gas analysis, and peritoneal lavage fluid was got for bacterial culture and counting. Part 2. In our previous studies, the upregulated expression of mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p and mi R-542-3p was observed after the CLP challenge, which was reversed by treatment with 0.5 MAC isoflurane in 60% oxygen. In this study, Lentiviral vectors with m RNA of the above four mi RNAs were constructed, and were transfected into RAW264.7 cells or peripheral blood leukocytes from CLP-challenged ICR/Km mice for overexpression of these mi RNAs. Cells were exposed to 0.5 MAC isoflurane in 60% oxygen with or without LPS stimulation. Cell supernatants were harvested for determining levels of inflammatory cytokines. Part 3. Mouse macrophage cells(RAW264.7) were randomly divided into six groups: Veh,Veh+0.5 ISO+60%O, Veh+100%O, LPS, LPS +0.5 ISO+60%O and LPS+100%O groups. An in vitro sepsis was induced in RAW264.7 cells by lipopolysaccharide(LPS) stimulation, and normal medium as the vehicle. Cells were exposed to 100% oxygen or 0.5 MAC isoflurane in 60% oxygen after LPS stimulation. The expression of p-IKKα/β, p-IκBα and p-p65 protein was detected by Western blot. Part 4. In this experiment, RAW264.7 cells were grouped and treated according to the protocol described in Part 3. Then, cell supernatants were harvested for determining inflammatory cytokines. And inoculated cell slides were used for detecting the subcellular location of Nrf2. The expression of Nrf2, HO-1 m RNAs or proteins was detected by RT-PCR or Western blot. Part 5. The aged(12-14 months) and young(2-3 months) male SD rats were randomly divided into six groups: Sham+Air, Sham+0.5MAC ISO+60%Oxy, Sham+0.5MAC SEV+60%Oxy, CLP+Air, CLP+0.5MAC ISO+60%Oxy and CLP+0.5MAC SEV+60%Oxy groups. Sepsis was induced by CLP, and Sham group were given an operation without CLP procedure. Treatment was performed by inhalation of 0.5 MAC isoflurane in 60% oxygen for 1 h at 1 and 6 h after CLP respectively,or 0.5 MAC sevoflurane in 60% oxygen for 2 h at 6 h after CLP. The survival rate was observed for 7 days. Blood, organs and bronchoalveolar lavage fluid(BALF) were collected at 24 h after CLP for determining serum levels of biochemical parameters and inflammatory cytokines, arterial blood gas analysis, histopathologic observation, wet-to-dry weight ratio of lung and total protein in BALF.Results Part 1. We observed that inhalation of 100% oxygen and 0.5 MAC isoflurane in 60% oxygen increased the survival rate of septic mice, inhibited abnormal changes of inflammatory cytokines, improved tissue oxygenation, and enhanced peritoneal bacteria clearance, which were reversed by NO synthase inhibitor L-NAME. Therefore, these results suggest that NO participates in the mechanism underlying protective action against sepsis by 100% oxygen and 0.5 MAC isoflurane in 60% oxygen.Part 2. Overexpression of mi R-542-3p, mi R-3074-2-3p and mi R-133a-3p in RAW264.7 cells, partially reversed the inhibition on increased proinflammatory factors after LPS stimulation by treatment of 0.5 MAC isoflurane in 60% oxygen. Overexpression of mi R-133a-3p, mi R-3074-2-3p and mi R-542-3p in peripheral blood leukocytes from CLP-challenged mice, also partially reversed the inhibitive effects on increased proinflammatory factors after CLP by treatment of 0.5 MAC isoflurane in 60% oxygen. These results suggest that mi RNAs may contribute to this therapeutic effect on sepsis by treatment of 0.5 MAC isoflurane in 60% oxygen. Part 3. Treatment of 100% oxygen and 0.5 MAC isoflurane in 60% oxygen inhibited the levels of p-IKK α/β, p-IκB α, p-p65 protein expression in cells after LPS stimulation. It was suggested that 100% oxygen and 0.5 MAC isoflurane in 60% oxygen exerted their protective action against sepsis by inhibition of NF-κB activation through downregulation of phosphorylation of IKK α/β, IκB α and p65 protein. Part 4. With LPS stimulation, the levels of inflammatory cytokines in cell supernatants were significantly increased when compared to those in Veh group. With treatment of 100% oxygen or 0.5 MAC isoflurane in 60% oxygen, the levels of inflammatory cytokines in cell supernatants were significantly decreased, and the m RNA levels of Nrf2 and its target gene of HO-1 were increased significantly when compared with those in LPS group(P<0.05). Treatment of 100% oxygen or 0.5 MAC isoflurane 60% oxygen increased Nrf2 protein expression level in cell nucleus and HO-1 protein expression level in cell with LPS-stimulation. These results indicated that Nrf2/HO-1 pathway is involved in the mechanism underlying protective action against sepsis by 100% oxygen and 0.5 MAC isoflurane in 60% oxygen. Part 5. For the aged and young SD rats, the 7-day survival rates were lower in CLP group than in Sham group. Inhalation of 0.5 MAC isoflurane/sevoflurane in 60% oxygen improved the 7-day survival rates of aged and young SD rats after CLP. In addition, inhalation of 0.5 MAC isoflurane/sevoflurane in 60% oxygen improved the abnormal changes of lung injury indicators such as lung tissue protein leakage and wet to dry weight ratio, serum biochemical indicators, inflammatory cytokines and tissueoxygenation in aged rats. These results indicated that combined administration of 0.5 MAC isoflurane/sevoflurane in 60% oxygen protected against experimental sepsis in aged rats.Conclusion According to the above results, it was demonstrated that, NO participates in the mechanism of protective action against sepsis by 0.5 MAC isoflurane in 60% oxygen; and mi RNAs may also contribute to this therapeutic effect; 0.5 MAC isoflurane in 60% oxygen exerts its protective action against sepsis by inhibiting NF-κB activation through downregulation of p-IKKα/β, p-IκBα, p-p65 protein expression; Nrf2/HO-1 mediated anti-inflammatory pathway may be involved in this protective effect on in-vitro sepsis by 0.5 MAC isoflurane in 60% oxygen. In addition, we also demonstrated that 0.5 MAC isoflurane/sevoflurane in 60% oxygen has a therapeutic effect on CLP-induced sepsis in aged rats.