七氟烷预处理对大鼠肢体远端缺血再灌注诱导肺损伤的保护作用

目的 探讨七氟烷预处理对大鼠肢体远端缺血再灌注（IR）所诱发肺损伤的保护作用及其机制。方法 选用28只成年健康雄性SD大鼠,按数字表法随机分为4组,每组7只。假手术组（Sham组）：仅分离出大鼠双侧股动静脉,不夹闭,暴露5 h后取材;七氟烷预处理+假手术组（S-S组）：大鼠吸入2.5%七氟烷30 min后,同Sham组操作;肢体远端缺血再灌注组（IR组）：建立大鼠下肢IR模型,分离出大鼠双侧股静脉和股动脉,夹闭双侧股动脉3 h,恢复血供再灌注2 h后取材;S-IR组：先进行七氟烷预处理,方法同S-S组,七氟烷预处理后操作同IR组。实验结束后,收集各组大鼠左心室血液和双侧肺组织,观察以下项目：（1）测定各组大鼠肺组织的湿重与干重的比值（W/D）、总肺含水量（TLW）;（2）光镜下观察各组大鼠肺组织病理变化;（3）采用相应试剂盒检测各组大鼠肺组织和血清中氧化应激因子脂质氧化（MDA）、超氧化物歧化酶（SOD）含量及炎症因子肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6水平;（4）蛋白质印迹法（Western blot）检测各组大鼠肺组织中磷酸化p38a丝裂原活化蛋白激酶（p-p38）、磷酸化腺苷酸活化蛋白激酶（p-AMPK）及沉默信息调节因子-1（SIRT1）蛋白的表达水平。结果 （1）各组大鼠肺组织W/D、TLW水平比较：IR组、S-IR组均高于Sham组、S-S组,S-IR组均低于IR组,差异均有统计学意义（F=144.75、133.21,P值均<0.001）。（2）各组大鼠肺组织病理改变：Sham组肺泡形态结构正常,肺泡内无炎性细胞浸润,肺泡间隔无液体积聚;S-S组肺泡结构大致正常,肺泡壁无明显增厚,肺泡间隔亦未发现炎性细胞浸润,提示无明显损伤;IR组肺泡形态结构破坏严重,广泛的肺泡间隔增厚,肺泡间质充满大量水肿液,其间浸润着大量的炎性细胞;与IR组相比,S-IR组炎性细胞浸润和水肿程度有明显减轻。（3）肺组织和血清中：IL-1β、IL-6、TNF-α、MDA含量比较,IR组、S-IR组均高于Sham组、S-S组,S-IR组均低于IR组,差异均有统计学意义（P值均<0.05）;SOD含量比较,IR组、S-IR组均低于Sham组、S-S组,S-IR组均高于IR组,差异均有统计学意义（P值均<0.05）;S-S组与Sham组间IL-1β、IL-6、TNF-α、MDA及SOD含量差异均无统计学意义（P值均>0.05）。（4）肺组织p-p38蛋白相对表达量比较：IR组、S-IR组均高于Sham组、S-S组,S-IR组均低于IR组,差异均有统计学意义（P值均<0.05）;p-AMPK、SIRT1蛋白相对表达量比较：IR组、S-IR组均低于Sham组、S-S组,S-IR组均高于IR组,差异均有统计学意义（P值均<0.05）;S-S组与Sham组肺组织p-p38、p-AMPK、SIRT1蛋白相对表达量差异均无统计学意义（P值均>0.05）。结论 对于肢体远端IR所诱发的肺损伤,七氟烷预处理可起到一定的保护作用,其机制可能与促进p-AMPK、SIRT1蛋白表达及抑制p-p38蛋白表达,进而减轻肺部炎症反应有关。

Protective effect of sevoflurane preconditioning on lung injury induced by distal limb ischemia-reperfusion

Objective To investigate the protective effect and mechanism of sevoflurane preconditioning on lung injury induced by the distal ischemia reperfusion (IR) of rat limbs. Methods Twenty-eight healthy adult male SD rats were selected. The rats were randomly divided into 4 groups (7 rats per group) by using a random number table. In the sham operation group (sham group), only the bilateral femoral arteries and veins were isolated without clamping, and the materials were taken after 5 h of exposure. The sevoflurane preconditioning+sham operation group (S-S group) inhaled 2.5% sevoflurane for 30 min. Then, the operation was the same as that of the sham group. In the distal limb IR group (IR group), the rat model of the lower limb IR was established by isolating the bilateral femoral veins and the femoral arteries, clamping the bilateral femoral arteries for 3 h, restoring blood supply for 2 h after reperfusion, and then extracting the materials. The sevoflurane preconditioning+distal limb IR group (S-IR group) was pretreated with sevoflurane in advance by the same method as that of the S-S group. The operation after sevoflurane pretreatment was the same as that of the IR group. At the end of the experiment, the left ventricular blood and bilateral lung tissues in each group were collected. (1) The wet weight/dry weight (W/D) ratio and total lung water content (TLW) were measured. (2) Pathological changes of the lung tissues were observed under a light microscope. (3) The content of oxidative stress factors, including lipid oxidation (MDA) and superoxide dismutase (SOD), and the levels of inflammatory factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in the lung tissues and sera were measured by corresponding kits. (4) Protein expression levels of phosphorylated p38α mitogen-activated protein kinase (p-p38), phosphorylated adenosine monophosphate activated protein kinase (p-AMPK), and silent information regulator factor-1 (SIRT1) in the lung tissues were determined by Western blot. Results (1) The W/D and TLW of the IR and S-IR groups were higher than those of the sham and S-S groups, and corresponding values of the S-IR group were lower than those of the IR group; all with statistically significant differences (F=144.75, 133.21; all P values<0.001). (2) Histopathological sections of the lung tissues in the sham group showed normal alveolar morphological structure, no inflammatory cell infiltration in the alveoli, and no fluid accumulation in the alveolar septa. Histopathological sections of the lungs in the S-S group showed grossly normal alveolar architecture, no obvious thickening of the alveolar walls, and no inflammatory cell infiltration in the alveolar septa. These results suggested no significant damage. Compared with the sham group, the histopathological sections of lungs in the IR group showed severe destruction of the alveolar morphological structure, extensive alveolar septal thickening, alveolar interstitium filled with a large amount of edema fluid, and infiltration with a large number of inflammatory cells. Inflammatory cell infiltration and edema were remarkably reduced in the S-IR group compared with the IR group. (3) The levels of IL-1β, IL-6, TNF-α, and MDA in the lung tissues and sera of the IR and S-IR groups were higher than those of the sham and S-S groups, while those of the S-IR group were lower than those of the IR group; all with statistically significant differences (all P values <0.05). The SOD contents in the IR and S-IR groups were lower than those in the sham and S-S groups, and that in the S-IR group was higher than that in the IR group; all with statistically significant differences (all P values <0.05). No significant differences were found in the amounts of IL-1β, IL-6, TNF-α, MDA, and SOD in the lung tissues between the S-S and sham groups (all P values >0.05). The p-p38 protein levels in the IR and S-IR groups were higher than those in the sham and the S-S groups, and that in the S-IR group was lower than that in the IR group; all with statistically significant differences (all P values <0.05). (4) The relative protein expression levels of p-AMPK and SIRT1 in the lung tissues in the IR and S-IR groups were lower than those in the sham and S-S groups, and that in the S-IR group was higher than that in the IR group; all with statistically significant differences (all P values <0.05). No significant differences were found in the relative protein expression levels of p-p38, p-AMPK, and SIRT1 between the lung tissues of the S-S and sham groups (all P values >0.05). Conclusion For the lung injury induced by distal limb IR, sevoflurane preconditioning may play a protective role. The mechanism may be related to the promotion of p-AMPK, SIRT1 protein expression, and inhibition of p-p38 protein expression, which may then attenuate lung inflammation.