The Effect Of The Cholinergic Anti-inflammatory Pathway On Acute Lung Injury Induced By Oleic Acid And Lipopolysaccharide In Rats

Part One Two-hit Animal Model of Acute Lung Injury Induced by Oleic Acid and LipopolysaccharideObjective: To compare the characteristics of the animal models of acute lung injury induced by oleic acid(OA) and/or lipopolysaccharide (LPS) in rats.Methods: Twenty four male Wistar rats were randomly divided into four groups(n=6). These groups included a control group (no interventions), a group administered LPS only (10 mg·kg-1, intraperitoneally), a group administered OA only (0.15 ml·kg-1, intravenously), and a group administered LPS+OA (OA given 30 minutes after LPS). The mean arterial blood pressure (MAP) and heart rate (HR) were observed continuously. Blood and tissue samples were collected 120 minutes after the first drug injection.The TLR2 and TLR4 mRNA, NF-κB p65, MPO of lung tissue and serum IL-6 were measured respectively. The blood gas analysis, wet to dry ratio (W/D) of right lung, and pathological changes were also observed.Results: As compared with the control group, the expression of TLR4 mRNA and NF-κBp65 and the activity of MPO in lung tissue and serum IL-6 levels were increased in LPS group, but the W/D ratio and gas exchange were no statistical significance. In contrast, OA caused severe alveolar damage with associated abnormalities in gas exchange and the W/D ratio while the expression of TLR2 mRNA of lung tissue was increased, but the expression of NF-κBp65 and the activity of MPO in lung tissue and serum IL-6 levels had no change. When given together, LPS and OA acted synergistically to increase the expression of TLR4 mRNA, TLR2 mRNA, NF-κBp65 and MPO activity in lung tissue and serum IL-6 levels, the W/D ratio and PaCO2 significantly increased and pH and PaO2 markedly decreased. The histopathologic changes of lung tissue showed alveolar hemorrhage, necrosis and proteinaceous alveolar edema.Conclusion:"Two-hit"model of acute lung injury induced by oleic acid combined with lipopolysaccharide is an ideal model for ALI, which is better mimic clinical manifestation. Part Two The Effect of the Cholinergic Anti-inflammatory Pathway on Acute Lung Injury Induced by Oleic Acid and Lipopolysaccharide in RatsObjective: To investigate the pathogenesis of the acute lung injury(ALI) induced by oleic acid and lipopolysaccharide in rats with the method of gene chip and the effect of the cholinergic anti-inflammatory pathway(CAP) on this kind of animal modal, observe the role of the spleen in the cholinergic anti-inflammatory pathway.Methods: Forty-two male Wistar rats were randomly divided into seven groups(n=6).①Control group (saline, 10ml·kg-1, intraperitoneally),②ALI group (0.15ml·kg-1 OA administrated intravenously 30min after intraperitoneally injection of 10mg·kg-1 LPS),③ST group: electrical stimulation(5V, 2ms, 1Hz) of the right cervical vagus nerve trunk for 10min before LPS and OA administration, and continous for another 10min after LPS was given,④THA group: 1.5mg·kg-1 THA was given intravenously before LPS and OA administration,⑤SP group: LPS and OA were administrated 10min after splenectomy,⑥SP+ST group: performed the same as ST group 10min after splenectomy,⑦SP+THA group: performed the same as THA group 10min after splenectomy. The mean arterial blood pressure(MAP) and heart rate(HR) were observed continuousiy. Blood and tissue samples were collected 120 minutes 120 minutes after the first drug injection in all groups. The TLR signaling pathway microarray, TLR2 and TLR4 mRNA, NF-κBp65, MPO of lung tissue and serum IL-6 were measured respectively. The blood gas analysis, wet to dry ratio (W/D) of right lung, and pathological changes were also observed. Genes with ratio average > 2 was identified as variance genes.Results: As compared with group C, The MAP, HR, pH and PaCO2 markedly decreased from 60min after the operation completed in group ALI while the histopathologic changes of lung tissue showed alveolar hemorrhage, necrosis and proteinaceous alveolar edema. W/D ratio, the activity of MPO, NF-κB expression and the level of serum IL-6 significantly increased. As compared with group C, there were 32 genes which interacted with TLR were up-regulated and the expressions of the TLR2 and TLR4 mRNA were markedly increased in other six groups, but no statistical changed between these six groups.As compared with group ALI, The MAP, HR, pH and PaO2 markedly increased in group ST and group THA as well as PaCO2 decreased. The lung tissue pathological changed slightly. W/D ratio and the activity of MPO significantly reduced. The expression of NF-κB p65 protein and the amount of serum IL-6 markedly decreased. Among the NF-κB pathway, only 6 genes were down-regulated.As compared with the ALI group, the MAP, HR, pH and PaO2 markedly increased in the SP group after the operation completed. The lung tissue pathological changes alleviated. W/D ratio and the activity of MPO significantly reduced. The expression of NF-κB p65 protein and the amount of serum IL-6 markedly decreased. As compared with the SP group, The changes of MAP, HR, W/D ratio, the expression of NF-κB p65 protein and the amount of serum IL-6 showed no statistcs difference in the SP+ST group and SP+THA group except increased PaO2 and decreased MPO.MAP, HR, pH and PaO2 markedly decreased in group SP+ST as compared with group ST and in group SP+THA as compared with group THA. W/D ratio, the activity of MPO, the expression of NF-κB p65 protein and the amount of serum IL-6 significantly increased in the comparison of the two pairs. The up-regulated genes were all related with NF-κB pathway.Conclusions: 1. The TLR2 and TLR4 gene which were present in the lung tissue of rats were activated by the"two-hit"of LPS and OA, and it can activate multi-strip intra-cellular inflammatory signal pathway and induced acute lung injury ultimately.2. Electrical stimulation of the vagus nerve or administered THA intravenously can protect the lung against ALI induced by LPS and OA via activating cholinergic anti-inflammatory pathway. 3. The CAP can inhibit the activation of NF-κB and reduce the production of proinflammatory cytokine but no effect to the expression of TLRs.4. Spleen play an important role in the CAP, Splenectomy significantly attenuate the protective effect of CAP on ALI.