| Background and ObjectiveStudies have shown that, as a widely used mechanical ventilation mode, one-lung ventilation (OLV) can result in acute lung injury (ALI). Therefore, to investigate the mechanisms of ALI induced by OLV and to unravell the preventive and therapeutic effects of drugs on OLV-induced ALI are currently areas of intense investigation.Sevoflurane, a commonly used clinical anesthetic, is considered to possess protective effects on OLV-induced ALI by inhibition of inflammatory response. However, its protective mechanisms are still unclear. Studies showed that arachidonic acid (AA) and its metabolites play an important role in the development of lung injury. Thus, the purpose of this study is to investigate the effects of sevoflurane on OLV-induced ALI from arachidonic acid metabolic pathways to present novel avenues for therapeutic intervention.Methods1. Animal grouping(1) OLV-induced ALI animal modelOLV-induced ALI rabbit model was constructed by right mainstem bronchial intubation with parasternal fenestration. Totally24healthy Japanese white rabbits were randomly divided into4groups(n=6):①sham operated group(S group);②two-lung ventilation(TLV) group (T group);③TLV with parasternal fenestration group (TF group) and④OLV group (O group).(2) Exfoliation of clara cell rabbit model12healthy Japanese white rabbits were randomized into two groups (n=6): control group(C group),and naphthalene vapor (100mg/L,12h) inhaled group (NA group).(3) The protective mechanisms of sevoflurane on OLV-induced ALIHealthy Japanese white rabbits were randomized into groups as follows:①sham operated group (S group);②OLV group (O group);③OLV+sevoflurane group (OS group). The OS group was divided into4subgroups with different concentrations of sevoflurane(lvol%,2vol%,3vol%and4vol%respectively);④inhaled naphthalene vapor group (NA group);⑤inhaled naphthalene vapor before OLV group; and⑥inhaled naphthalene vapor before OLV+sevoflurane group (n=6).2. Detection indcators and methods(1) Histological scores and lung wet/dry weight (W/D) ratios were determined for lung injury assessment.(2) Expressions of lung CCSP, C-PLA2, COX2and5-LOX proteins and their homologous mRNAs were detected by western-blotting and real time-PCR, respectively.(3) PMN in the BALF were quantified by automated hematology analyzer.(4) Lung myeloperoxidase (MPO) activity, levels of lung arachidonic acid (AA), prostaglandin I2(PGI2), thromboxane A2(TXA2) and leukotrienes B4(LTB4) were quantified by ELISA.(5) Morphology and counts of clara cell were performed by electron microscope.(6) Lung histological change was observed by light microscope.Result1. One-lung ventilation (OLV) induced ALI animal modelCompared with those in S group, MPO activity and AA contents in lung tissues, PMN counts in BALF, lung W/D ratio and histopathologic score were increased in O group,T group and TF group (P<0.05).No difference was observed between T group and TF group., but all the indicators mentioned above in the two groups were lower than those in O group (P<0.05). No significant histopathologic changes were observed in S group except for mild capillary dilatation in some areas of the lung tissues. Serious hyperemia and hemorrhage in the lung tissues, thickening and exudation of alveolar wall, marked red blood cell and inflammatory cell infiltration in the alveolar space were found in O group. In both T group and TF group, pulmonary histopathologic changes alleviated significantly as compared with those in O group2. Exfoliation of clara cell rabbit modelLung W/D ratio and lung histopathologic score in NA group were the same as those in N A group, but the counts of clara cell in bronchioles and the levels of lung CCSP mRNA and protein expressions were decreased dramatically in NA group as compared with those in C group (P<0.05). No significant histopathologic changes were observed in the two groups, but the counts of clara cell in bronchioles and the secretory granules in clara cell of NA group were lower than those of C group under electron microscope.3. Effects of sevoflurane on cytosolic phospholipaseA2and clara cell secretory protein in OLV-induced injured rabbit lung tissuesIn both O group and OS group, pulmonary CCSP mRNA and protein expressions were significantly lower, while C-PLA2mRNA and protein expressions, lung W/D ratios and lung histopathologic scores were higher than those in the S group (P<0.05). Compared with O group,the OS group showed significantly increased expressions of CCSP mRNA and protein and reduced C-PLA2mRNA and protein expressions. In the4OLV+sevoflurane groups, CCSP underwent no significant changes as sevoflurane concentration increased,but C-PLAt mRNA and protein expressions, lung W/D ratios and lung histopathologic scores all decreased gradually as the concentrations of sevoflurane increased (P<0.05).No significant pathological changes were observed in the S group except for mild capillary dilatation in some areas of lung tissues.In the0group,the lung tissues presented with severe hyperemia and hemorrhage, alveolar wall thickening and exudation, with markedly red blood cell and inflammatory cell infiltration in alveolar space. These pathological changes above mentioned in lung tissues were alleviated gradually as the concentration of sevoflurane increased.4. The role of clara cell secretory protein in the protective effects of sevoflurane against OLV-induced ALICompared with those in the S group, lung CCSP mRNA and protein expressions were decreased in all the other groups(P<0.05), while lung C-PLA2mRNA and protein expressions,the contents of lung AA, lung W/D ratio and histopathologic score were increased (P<0.05), except that those in the NA group; Lung CCSP mRNA and protein expressions in NA group were the same as those in the NAO Group and NAOS group, but were significant lower than those in the O group and OS group (P<0.05), while the other indicators were significant higher than those in the latter four groups(P<0.05). Compared with those in the O group, lung CCSP mRNA and protein expressions decreased significantly in the NAO group and NAOS groups(P<0.05),but increased significantly in the OS group(P<0.05),while the rest indicators were increased significantly in the OS and NAOS groups(P<0.05),but decreased significantly in NAO group(P<0.05). Except for the lung CCSP mRNA and protein expressions were significant lower(P<0.05), the rest indicators were significant higher in the NAO group than those in the OS group and NAOS groups (P <0.05). The levels of lung CCSP mRNA and protein expressions increased significantly,but the other indicators decreased significantly in the OS group as compared with those in the NOS group(P<0.05). No apparent pathologic changes were found in both S group and NA group. A large area of hyperemia and hemorrhage in lung tissues, thickening and exudation in alveolar wall, marked red blood cell and inflammatory cell infiltration in alveolar space were found in O group and the pathologic changes above-mentioned were further deterioration in the NAO group. In the OS group, scattered regions of hyperemia and hemorrhage spots, mild red blood cell and inflammatory cell infiltration in alveolar space and apparent incrassation and exudation in alveolar wall were found. Pathologic changes in NAOS group were aggravated apparently as compared with those in the OS group.5. The effects of sevoflurane on AA metabolism COX2and5-LOX pathways in OLV-induced ALICOX2and5-LOX mRNA and protein expressions and the contents of LTB4, TXA2and PGI2in the lungs, lung wet/dry(W/D) ratio and histopathologic score were significantly higher, while PGI2/TXA2ratio was significantly lower in O group and OS group than those in S group (P<0.05). Compared with those in O group, all the indicators above-mentioned were decreased significantly but PGI2/TXA2ratio was elevated markedly in OS group(P<0.05). No significant histopathologic changes were observed in S group except for mild capillary dilatation in some areas of the lung tissues. Serious hyperemia and hemorrhage in the lung tissues, thickening and exudation of alveolar wall, marked red blood cell and inflammatory cell infiltration in the alveolar space were found in O group. These pulmonary histopathologic changes were alleviated significantly in OS group.Conclusion1. Combined the right mainstem bronchial intubation with parasternal fenestration can be used to construct rabbit model of OLV-induced ALI.2. OLV induced ALI may be associated with the increased expressions of C-PLA2by downregulating the expression of CCSP, and the activations of COX2and5-LOX pathways to result in increased generation of arachidonic acid (AA) and its metabolities in injured rabbit lung tissues.3. The protective effects of sevoflurane on OLV-induced ALI may be through the inhibition of C-PLA2expression by up-regulating the expression of CCSP or through other mechanisms to down-regulate the expression of C-PLA2.4. Sevoflurane can produce protective effects against on OLV-induced ALI possibly by reducing AA metabolities and regulating PGI2/TXA2ratio via inhibition of COX2and5-LOX pathways.5. Arachidonic acid generation and metabolism pathways in lung may be a novel avenues for therapeutic potential intervention of sevoflurane on OLV-induced ALI. |
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