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摘要：
目的 评价右美托咪定对大鼠肺泡巨噬细胞缺氧/复氧损伤时表型转化的影响。 方法 大鼠肺泡巨噬细胞株NR8383培养于含20%热灭活胎牛血清的F‑12K培养基中，采用随机数字表法将细胞分为：对照组（C组），使用完全培养基，培养于常规培养箱中；缺氧/复氧组（H/R组），在三气培养箱中缺氧6 h后，置于常规培养箱中复氧6 h；右美托咪定组（D+H/R组，按右美托咪定终浓度不同分为D0.1+H/R组、D1+H/R组、D10+H/R组），在含0.1、1.0 μmol/L或10.0 μmol/L右美托咪定的培养液中孵育1 h后，更换完全培养基进行缺氧/复氧（每组设置6个复孔）。于复氧6 h后，采用细胞增殖‑毒性检测试剂盒（cell counting kit‑8, CCK‑8）法检测细胞活力，比色法检测细胞超氧化物歧化酶（superoxide dismutase, SOD）活性，分光光度计检测细胞培养上清液中乳酸脱氢酶（lactate dehydrogenase, LDH）活性，实时荧光定量聚合酶链式反应（real time quantitative polymerase chain reaction, RT‑qPCR）法检测一氧化氮合酶（nitric oxide synthase, NOS）2、干扰素（interferon, IFN）、单核细胞趋化蛋白1（monocyte chemotactic protein 1, Mcp1）、精氨酸酶1（arginase‑1, Arg1）、IL‑10、几丁质酶3样蛋白3（chitinase 3‑like protein‑3, Chi3l3）的mRNA表达，免疫荧光染色法标记诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）与Arg1。 结果 与C组比较，其余4组上清液LDH活性升高，H/R组、D0.1+H/R组和D1+H/R组细胞活力降低，H/R组、D0.1+H/R组SOD活性降低（P<0.05）；与H/R组比较，D1+H/R组和D10+H/R组细胞活力增高，D1+H/R组SOD活性增高，D1+H/R组和D10+H/R组上清液LDH活性降低（P<0.05）。与C组比较，H/R组NOS2、IFN、Mcp1、Chi3l3和IL‑10的基因表达上调，D+H/R组Mcp1、Arg1、IL‑10和Chi3l3的基因表达上调（P<0.05）；与H/R组比较，D+H/R组NOS2、Mcp1和IFN的mRNA表达下调，Arg1和IL‑10的mRNA表达上调（P<0.05）。与C组比较，H/R组和D+H/R组细胞Arg1和iNOS荧光表达均明显增强，提示Arg1和iNOS表达均增加；与H/R组比较，D+H/R组的Arg1荧光表达较H/R组更强，而iNOS荧光表达减弱，提示D+H/R组的Arg1的表达增加。 结论 右美托咪定减轻大鼠肺泡巨噬细胞缺氧/复氧损伤的机制与促进细胞向M2型极化有关。
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Abstract: Objective To evaluate the effect of dexmedetomidine on phenotypic transformation of rat alveolar macrophages during hypoxia/reoxygenation injury. Methods Rat alveolar macrophage cell line NR8383 was cultured in F‑12K media containing 20% heat‑inactivated fetal bovine serum. According to the random number table method, the cells were divided into the following groups: cells in a control group (group C) were cultured in a conventional incubator with complete media; cells in a hypoxia/reoxygenation group (group H/R) were cultured in a three‑gas incubator for hypoxia over 6 h followed by reoxygenation in a conventional incubator over 6 h. Cells in a dexmedetomidine group (group D+H/R) were sub‑divided into a D0.1+H/R group, a D1+H/R group and a D10+H/R group which were cultured with media containing 0.1, 1.0 μmol/L or 10.0 μmol/L of dexmedetomidine over 1 h, respectively. The medium was replaced for hypoxia/reoxygenation. Each group was repeated six times. After 6 h of reoxygenation, the cell viability was measured by cell counting kit‑8 (CCK‑8) assay. The activity of superoxide dismutase (SOD) was determined by colorimetric assay. The activity of lactate dehydrogenase (LDH) in the supernatant was detected by a spectrophotometer. The levels of nitric oxide synthase (NOS) 2, interferon (IFN), monocyte chemotactic protein 1 (Mcp1), arginase‑1 (Arg1), interleukin‑10 (IL‑10), and chitinase‑3‑like protein‑3 (Chi3l3) mRNA were measured by real time quantitative polymerase chain reaction (RT‑qPCR). Moreover, inducible nitric oxide synthase (iNOS) and Arg1 were stained by immunofluorescence. Results Compared with group C, LDH activity enhanced in the supernatant of the other four groups, the cell viability reduced in groups H/R, D0.1+H/R and D1+H/R, the SOD activity decreased in groups H/R and D0.1+H/R (P<0.05). Compared with group H/R, the cell viability enhanced in group D1+H/R and D10+H/R, the SOD activity increased in group D1+H/R, and the LDH activity decreased in the supernatant of groups D1+ H/R and D10+H/R decreased (P<0.05). Compared with group C, the gene expression of NOS2, IFN, Mcp1, Chi3l3 and IL‑10 was up‑regulated in group H/R, while the gene expression of Mcp1, Arg1, IL‑10 and Chi3l3 was up‑regulated in group D+H/R (P<0.05). Compared with group H/R, the expression of NOS2, Mcp1 and IFN mRNA was down‑regulated in group D+H/R, while the expression of Arg1 and IL‑10 mRNA was up‑regulated (P<0.05). Compared with group C, both Arg1 and iNOS fluorescence remarkably enhanced in groups H/R and D+H/R, which suggested that the expression of Arg1 and iNOS increased. Compared with group H/R, Arg1 fluorescence enhanced but iNOS fluorescence weakened in group D+H/R, which suggested that the expression of Arg1 in group D+H/R increased. Conclusions Dexmedetomidine alleviates the hypoxia/reoxygenation injury of alveolar macrophages in rats, which is related to promoting macrophage polarization to M2 phenotype.
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