MicroRNA-132通过调控胆碱能通路减轻肺泡巨噬细胞炎症反应  

MicroRNA-132 inhibits inflammation of alveolar macrophages by regulating cholinergic anti-inflammatory pathway

作　　者：刘芬[1] 李勇[2] 赵宁[1] 李东海[3] 江榕[1] 曾振国[1] 邵强[1] 彭菲菲[1] 王燕[1] 钱克俭[1] 

机构地区：[1]南昌大学第一附属医院重症医学科,江西南昌330006 [2]南昌大学第一附属医院肿瘤科,江西南昌330006 [3]南昌大学第一附属医院神经外科,江西南昌330006

出　　处：《中国病理生理杂志》2016年第2期261-266,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81101410;No.81460292);江西省自然科学基金资助项目(No.20122BAB205002)

摘　　要：目的:探讨microRNA-132(miR-132)通过调控胆碱能通路减轻肺泡巨噬细胞炎症反应的作用机制。方法:向大鼠肺泡巨噬细胞NR8383中转染miR-132 mimic、mimic阴性对照(NC)、miR-132 inhibitor或inhibitor NC,转染后用脂多糖(LPS)和(或)乙酰胆碱(ACh)处理细胞;采用real-time PCR检测乙酰胆碱酯酶(AChE)的m RNA表达;采用Western blot检测细胞中AChE、信号转导及转录激活因子3(STAT3)和磷酸化STAT3(p-STAT3),以及细胞浆和细胞核中核因子κB(NF-κB)蛋白的改变;采用AChE活性测试盒检测细胞上清液中AChE活性的改变;采用免疫荧光检测NF-κB的核移位变化。结果:上调或下调miR-132不影响AChE的mRNA相对表达水平;上调miR-132可使AChE蛋白及活性的水平均显著降低(P<0.05),下调miR-132可使AChE蛋白及活性的水平均显著升高(P<0.05)。当给予LPS+ACh作用时,miR-132 mimic组对NF-κB p65核移位的抑制作用较mimic NC组更强(P<0.05),miR-132 inhibitor组较inhibitor NC组更弱(P<0.05);miR-132 mimic组对STAT3及p-STAT3蛋白的抑制作用较mimic NC组更强(P<0.05)。结论:miR-132通过调控胆碱能通路减轻肺泡巨噬细胞炎症反应的作用机制可能是miR-132靶向作用AChE,抑制AChE对ACh的水解作用,从而增强ACh的抗炎作用,抑制NF-κB及STAT3的活化。AIM： To investigate the role of microRNA-132 （miR-132） on alveolar macrophage inflammation. METHODS： Rat alveolar macrophage cell line NR8383 was transfected with miR-132 mimic, mimic negative control （NC）, miR-132 inhibitor, or inhibitor NC. The cells were divided into transfection group, transfection ＋ lipopolysaccha- ride （LPS）group, and transfection ＋ LPS ＋ acetylcholine （ACh）group. The mRNA expression of acetylcholinesterase （ACHE） was detected by real-time PCR. The protein levels of ACHE, signal transducer and activator of transcription 3 （STAT3） and phosphorylated STAT3 （p-STAT3） in the cells, and nuclear factor-KB （NF-KB） in the cytoplasm and nu- cleus were analyzed by Western blot. The activity of AChE in the culture supernatant was measured by AChE activity assay kit. The nuclear translocation of NF-KB was detected by immunofluorescence assay. RESULTS： Up-regulation or down- regulation of miR-132 had no effect on the mRNA expression of ACHE. However, up-regulation of miR-132 decreased the protein level of AChE compared with mimic NC group （P 〈 0. 05 ）. Transfection with miR-132 inhibitor increased the pro- tein expression of AChE compared with inhibitor NC group （ P 〈 0. 05 ）. In the alveolar macrophages treated with LPS ＋ ACh, the inhibition of nuclear translocation of NF-κB p65 in miR-132 mimic group was more effective than that in mimic NC group （P 〈0. 05）. The inhibitory effect in miR-132 inhibitor group was weaker than that in inhibitor NC group （P 〈0. 05 ）. The inhibitory effect of miR-132 mimic on the protein levels of STAT3 and p-STAT3 was stronger than that of mimic NC （P 〈 0. 05 ）. CONCLUSION： miR-132 in LPS-stimulated alveolar macrophages reinforced ACh-mediated anti-inflam- matory reaction by targeting AChE to suppress ACh hydrolyzation, which was related to the suppression of NF-κB and STAT3 activation.

关 键 词：MicroRNA-132 乙酰胆碱酯酶 乙酰胆碱 

分 类 号：R392.12[医药卫生—免疫学]