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目的 观察调控microRNA-132 (miR-132)联合乙酰胆碱(ACh)对脂多糖(LPS)诱导的大鼠肺泡巨噬细胞炎症反应的作用.方法 分别采用FAM荧光标记的mimic/inhibitor阴性对照(NC)、miR-132 mimic/inhibitor、mimic/inhibitor NC转染大鼠肺泡巨噬细胞NR8383,通过荧光显微镜观察转染效率,实时荧光定量PCR检测miR-132的表达.转染后细胞给予LPS(1μg/mL)刺激或LPS+ ACh(10 μmoL/L)处理,采用酶联免疫吸附试验检测细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β (IL-1β)及白细胞介素-6(IL-6)浓度.两组间均数比较采用独立样本t检验,以P ＜0.05为差异具有统计学意义.结果 通过在NR8383细胞中转染mimic FAM NC确定50 nmol/L为转染mimic的最佳浓度.与mimic NC组相比,miR-132 mimic组细胞中miR-132的表达上调了(21.54±2.14)倍(P=0.001).LPS组中结果发现miR-132 mimic上清液中TNF-α(P=0.683)、IL-1β(P=0.770)、IL-6(P=0.872)水平较mimic NC差异均无统计学意义;LPS+ ACh+ mimic NC组TNF-α(P=0.008)、IL-1β(P =0.004)、IL-6 (P =0.003)水平与LPS+mimic NC组相比均下降;LPS+ ACh+ miR-132 mimic组TNF-α(P=0.001)、IL-1β (P =0.001)、IL-6 (P =0.001)水平与LPS+ ACh+ mimic NC组相比均下降.通过转染inhibitor FAM NC确定100nmol/L为转染inhibitor的最佳浓度.LPS组miR-132 inhibitor上清液中TNF-α(P=0.800)、IL-1β(P =0.449)、IL-6 (P =0.418)水平较inhibitor NC差异均无统计学意义.LPS+ ACh组结果显示miR-132 inhibitor可逆转ACh介导的TNF-α (P =0.018)、IL-1β (P =0.022)、IL-6 (P=0.032)水平的下降.结论 在体外培养环境,单独给予LPS时过表达miR-132或抑制miR-132活性不影响肺泡巨噬细胞炎症反应,但miR-132可增强ACh对LPS诱导的肺泡巨噬细胞炎症反应的抑制作用.“,”Objective To observe the effects of microRNA-132 (miR-132) combined with acetylcholine (ACh) on the lipopolysaccharide (LPS) induced inflammatory response in rat alveolar macrophages.Methods NR8383 cells were transfected with fluorescein amidite (FAM)-labeled mimic/ inhibitor negative control (NC), miR-132 mimic/inhibitor, or mimic/inhibitor NC.Transfection efficiency was observed by fluorescence microscope.The expression of miR-132 was detected by real time fluorescence quantitative PCR.The cells were treated with LPS (1 μg/mL) or LPS + ACh (10 μmol/L) after transfection.The concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the supernatant of cell culture were detected by enzyme linked immunosorbent assay (ELISA).Comparisons between two groups were done by using Student t test, and difference was statistically significant with P ＜ 0.05.Results A level of 50 nmol/L of mimic FAM NC was established as an optimal concentration for mimic transfecting NR8383 cells.The expression of miR-132 in the miR-132 mimic group was up-regulated (21.54 ± 2.14) folds compared with the NC group (P =0.001).In LPS group, there were no significant differences in the levels of TNF-α (P =0.683), IL-1β (P =0.770) and IL-6 (P =0.872) in the culture supernatant between miR-132 mimic-transfected and mimic NC-transfected alveolar macrophages.The levels of TNF-α (P =0.008), IL-1β (P =0.004) and IL-6 (P =0.003) in the LPS + ACh + mimic NC group were lower compared with the LPS + mimic NC group.The levels of TNF-α (P =0.001), IL-1β (P =0.001) and IL-6 (P =0.001) in the LPS + ACh + miR-132 mimic group were lower compared with the LPS + ACh + mimic NC group.A level of 100 nmol/L of inhibitor FAM NC was established as an optimal concentration for inhibitor transfection of cells.In LPS group, there were no significant differences in the levels of TNF-α (P =0.800), IL-1β (P =0.449) and IL-6 (P =0.418) in the supernatant of cell culture between miR-132 inhibitor-transfected cells and inhibitor NC-transfected cells.The levels of TNF-α (P =0.018), IL-1β (P=0.022) and IL-6 (P =0.032) decreased mediated by Ach could be reversed in the LPS + ACh + miR-132 inhibitor group.Conclusion In vitro culture environment, neither excessive expression of miR-132 nor inhibition of miR-132 influenced the inflammatory response of alveolar macrophages treated with LPS alone, but miR-132 can enhance ACh-mediated inhibitory effect on inflammatory response of LPS induced alveolar macrophages.