Zhang M, Li Z, Pei W, Li X, Yang H, Zhu X, Lü K
Key Laboratory of Non- coding RNA Transformation Research of Anhui Higher Education Institution, Wannan Medical College, Wuhu 241001, China.
Central Laboratory, Yijishan Hospital, Wannan Medical College, Wuhu 241001, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Mar 20;43(3):393-399. doi: 10.12122/j.issn.1673-4254.2023.03.08.
To explore the effect of M2 macrophage-derived exosomal lncRNA NR_028113.1 on macrophage polarization and its possible mechanism.
Bone marrow-derived macrophages (BMDMs) from BALB/c mice were isolated and cultured . After IL-4 treatment to induce M2 macrophage polarization, exosomes (M2-exo) were extracted from the supernatant of M2 macrophages and identified. The expression of lncRNA in M2-exo was detected by qRT-PCR. BMDMs were co-cultured with M2-exo (100 μg/mL) or PBS for 48 h, and the changes in cellular expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF- were detected using qRT-PCR and Western blotting. The percentage of CD206 cells was analyzed using flow cytometry, and the phosphorylation levels of JAK2/STAT3 proteins were detected using Western blotting. A lncRNA smart silencer was designed to specifically inhibit the expression of lncRNA NR_028113.1 in the M2 macrophages, from which exosomes were extracted and co-cultured with BMDMs for 48 h. The mRNA expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-, CD206 cell percentage and the phosphorylation levels of JAK2/STAT3 proteins were detected using qRT-PCR, flow cytometry and Western blotting.
LncRNA NR_028113.1 was highly expressed in the exosomes of M2 macrophages ( < 0.05). Co-culture with M2-exo significantly increased mRNA expressions of M2 macrophage marker genes Arg1, YM-1 and FIZZ1 ( < 0.05), lowered the expressions of iNOS and TNF- ( < 0.05), and increased CD206 cell percentage and JAK2/STAT3 protein phosphorylation level in BMDMs ( < 0.05). After inhibiting the expression of lncRNA NR_028113.1 in M2 macrophages, the extracted M2-exo caused significant down-regulation of the mRNA expressions of Arg1, YM-1 and FIZZ1 and up-regulation of iNOS and TNF- mRNA ( < 0.05), resulting also in signi-ficantly reduced CD206 cell percentage and lowered phosphorylation levels of JAK2/STAT3 proteins in co-cultured BMDM ( < 0.05).
M2 macrophage-derived exosomal lncRNA NR_028113.1 can significantly promote M2 polarization of macrophages possibly by activating the JAK2/STAT3 signaling pathway.
探讨M2巨噬细胞来源的外泌体长链非编码RNA（lncRNA）NR_028113.1对巨噬细胞极化的影响及其可能机制。
分离培养BALB/c小鼠骨髓来源的巨噬细胞（BMDMs）。经白细胞介素-4（IL-4）处理诱导M2巨噬细胞极化后，从M2巨噬细胞的上清液中提取外泌体（M2-exo）并进行鉴定。采用实时定量聚合酶链反应（qRT-PCR）检测M2-exo中lncRNA的表达。将BMDMs与M2-exo（100μg/mL）或磷酸盐缓冲液（PBS）共培养48小时，采用qRT-PCR和蛋白质印迹法检测精氨酸酶1（Arg1）、半乳糖凝集素-3（YM-1）、抵抗素样分子α（FIZZ1）、诱导型一氧化氮合酶（iNOS）和肿瘤坏死因子-α（TNF-α）细胞表达水平的变化。采用流式细胞术分析CD206细胞的百分比，采用蛋白质印迹法检测Janus激酶2（JAK2）/信号转导和转录激活因子3（STAT3）蛋白的磷酸化水平。设计lncRNA智能沉默剂特异性抑制M2巨噬细胞中lncRNA NR_028113.1的表达，从中提取外泌体并与BMDMs共培养48小时。采用qRT-PCR、流式细胞术和蛋白质印迹法检测Arg1、YM-1、FIZZ1、iNOS和TNF-α的信使核糖核酸（mRNA）表达水平、CD206细胞百分比以及JAK2/STAT3蛋白的磷酸化水平。
lncRNA NR_028113.1在M2巨噬细胞的外泌体中高表达（P<0.05）。与M2-exo共培养显著增加了M2巨噬细胞标志物基因Arg1、YM-1和FIZZ1的mRNA表达（P<0.05），降低了iNOS和TNF-α的表达（P<0.05），并增加了BMDMs中CD206细胞的百分比和JAK2/STAT3蛋白的磷酸化水平（P<0.05）。抑制M2巨噬细胞中lncRNA NR_028113.1的表达后，提取的M2-exo导致Arg1、YM-1和FIZZ1的mRNA表达显著下调，iNOS和TNF-α的mRNA上调（P<0.05），同时共培养的BMDM中CD206细胞百分比显著降低，JAK2/STAT3蛋白的磷酸化水平降低（P<0.05）。
M2巨噬细胞来源的外泌体长链非编码RNA NR_028113.1可能通过激活JAK2/STAT3信号通路显著促进巨噬细胞向M2极化。
