摘要目的 探讨半乳糖凝集素-9/T细胞免疫球蛋白黏蛋白-3(Galectin-9/Tim-3)信号通路在脂多糖(LPS)诱导小鼠巨噬细胞M1/M2表型极化中的意义及分子机制.方法 体外培养小鼠腹腔巨噬细胞RAW264.7,待细胞生长至80％～90％时用于实验.① 将细胞以无血清DMEM培养基培养,并分别以0(空白对照)、0.01、0.1、1、10、100 mg/L的LPS刺激24 h.采用荧光定量反转录-聚合酶链反应(RT-qPCR)或蛋白质免疫印迹试验(Western Blot)检测巨噬细胞M1型标志物白细胞介素-6(IL-6)、诱导型一氧化氮合酶(iNOS)和M2型标志物精氨酸酶-1(Arg-1)、白细胞分化抗原206(CD206)以及细胞内Tim-3、Galectin-9的表达.② 另取小鼠腹腔巨噬细胞,并分为空白对照组(用无血清DMEM培养基培养24 h)、LPS处理组(用含0.1 mg/L的LPS无血清DMEM刺激24 h)和α-乳糖预处理组(在LPS刺激前1 h用含40μmol/L Galectin-9信号拮抗剂α-乳糖的无血清DMEM进行预处理),通过封闭Galectin-9信号以验证Galectin-9在巨噬细胞M1/M2表型极化中的作用.结果 ① 低浓度LPS(0.01 mg/L、0.1 mg/L)刺激24 h后,巨噬细胞M1型标志物的表达仅轻度上调(iNOS mRNA)或无明显变化(IL-6 mRNA);而M2型标志物Arg-1 mRNA和CD206 mRNA表达则明显升高,并分别于0.1 mg/L和0.01 mg/L的LPS浓度下达到峰值〔与空白对照组比较:Arg-1 mRNA(2-ΔΔCt)为1.85±0.07比1.00±0.02,CD206 mRNA(2-ΔΔCt)为2.03±0.11比1.00±0.05,均P<0.01〕;随LPS浓度升高,IL-6 mRNA和iNOS mRNA表达持续升高,而Arg-1 mRNA和CD206 mRNA表达则逐渐降低,巨噬细胞M1/M2表型极化状态发生改变.同时,经LPS刺激后,巨噬细胞内Tim-3蛋白表达在0.01 mg/L的LPS刺激后即较空白对照组明显上调(Tim-3/GAPDH:0.84±0.04比0.69±0.02,P<0.01),并在0.1 mg/L浓度下达峰值,之后随LPS浓度升高而逐渐下降;细胞内Galectin-9及上清中分泌型Galectin-9(s-Galectin-9)蛋白表达在低浓度LPS(0.01 mg/L、0.1 mg/L)刺激后无明显变化,之后随LPS浓度升高而逐渐降低.② 与空白对照组相比,LPS处理组M1型标志物iNOS和M2型标志物Arg-1、CD206的mRNA表达均明显升高,而IL-6 mRNA表达无明显变化.使用α-乳糖预处理后,IL-6 mRNA和iNOS mRNA表达较LPS处理组进一步升高〔IL-6 mRNA(2-ΔΔCt):1.44±0.02比1.14±0.11,iNOS mRNA(2-ΔΔCt):2.45±0.04比2.01±0.08,均P<0.01〕,而Arg-1 mRNA和CD206 mRNA表达明显下降〔Arg-1 mRNA(2-ΔΔCt):0.75±0.01比1.85±0.02,CD206 mRNA(2-ΔΔCt):0.58±0.02比2.03±0.14,均P<0.01〕.同时,Galectin-9信号封闭也可减少Galectin-9胞外分泌〔与LPS处理组比较:s-Galectin-9/GAPDH为0.10±0.01比0.23±0.02,P<0.01〕,下调胞内Tim-3和Galectin-9的蛋白表达(Tim-3/GAPDH:0.28±0.01比0.43±0.01,Galectin-9/GAPDH:0.21±0.01比0.43±0.01,均P<0.01).结论 LPS通过Galectin-9/Tim-3信号通路调控巨噬细胞M1/M2表型极化,低浓度LPS可通过调节Galectin-9表达及分泌使巨噬细胞向M2表型极化,从而限制炎症发展;高浓度LPS通过下调Galectin-9的表达和分泌使巨噬细胞向M1表型极化,从而促进炎症发展.

abstractsObjective To investigate the meaning and molecular mechanisms of Galectin-9/ T-cell immunoglobulin mucin-3 (Tim-3) pathway on lipopolysaccharide (LPS) induced murine macrophage M1/M2 subtype polarization.Methods The murine peritoneal macrophages RAW264.7 were cultured in vitro until the cells had matured with 80％-90％ fusion rate. ① The cells were cultured in serum-free medium and treated with 0 (blank control), 0.01, 0.1, 1, 10 and 100 mg/L LPS for 24 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) or Western Blot was used to determine the expressions of M1 macrophage markers such as interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and M2 macrophage markers such as arginase-1 (Arg-1), leukocyte differentiation antigen 206 (CD206), as well as Tim-3 and Galectin-9 in the cells. ② The other mice peritoneal macrophages were divided into blank control group (cultured in serum-free DMEM medium for 24 hours), LPS treatment group (cultured in serum-free DMEM medium containing 0.1 mg/L LPS for 24 hours) and α-lactose pretreatment group (pretreated with serum-free DMEM containing 40μmol/L Galectin-9 signal antagonist 1 hour before LPS stimulation). Over closed Galectin-9 signal was used to verify the role of Galectin-9 in macrophage M1/M2 subtype polarization.Results ① After stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L) for 24 hours, the expression of M1 markers was only slightly increased such as iNOS mRNA or not significantly changed such as IL-6 mRNA in macrophages, while the expressions of M2 markers such as Arg-1 mRNA and CD206 mRNA were significantly increased and peaked at LPS concentrations of 0.1 mg/L and 0.01 mg/L [compared with blank control group:Arg-1 mRNA (2-ΔΔCt) was 1.85±0.07 vs. 1.00±0.02, CD206 mRNA (2-ΔΔCt) was 2.03±0.11 vs.1.00±0.05, both P < 0.01]. With the increase of LPS concentration, the expressions of IL-6 mRNA and iNOS mRNA continued to increase, while the expressions of Arg-1 mRNA and CD206 mRNA were gradually decreased, and the macrophage M1/M2 subtype polarization status changed. At the same time, the level of Tim-3 protein in macrophages was significantly up-regulated after stimulation with 0.01 mg/L LPS as compared with that of blank control group (Tim-3/GAPDH:0.84±0.04 vs. 0.69±0.02,P < 0.01), peaked at LPS concentrations of 0.1 mg/L, and then decreased with increasing LPS concentration. The intracellular Galectin-9 and supernatant secreted Galectin-9 (s-Galectin-9) protein levels showed no significant change after stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L), while then gradually decreased with the increase of LPS concentration. ② Compared with blank control group, the mRNA expressions of M1 marker iNOS and M2 markers Arg-1 and CD206 were significantly increased in LPS treatment group, but IL-6 mRNA level was not changed significantly. The mRNA levels of IL-6 and iNOS were further elevated after pretreatment with α-lactose as compared with that of the LPS treatment group [IL-6 mRNA (2-ΔΔCt): 1.44±0.02 vs. 1.14±0.11, iNOS mRNA (2-ΔΔCt):2.45±0.04 vs. 2.01±0.08, bothP < 0.01], while the mRNA levels of Arg-1 and CD206 were significantly decreased [Arg-1 mRNA (2-ΔΔCt): 0.75±0.01 vs. 1.85±0.02, CD206 mRNA (2-ΔΔCt): 0.58±0.02 vs. 2.03±0.14, bothP < 0.01]. Meanwhile, the blocking of Galectin-9 signaling could also reduce the extracellular s-Galectin-9 (compared with LPS treatment group: s-Galectin-9/GAPDH was 0.10±0.01 vs. 0.23±0.02,P < 0.01), down-regulated the expressions of Tim-3 and Galectin-9 (Tim-3/GAPDH: 0.28±0.01 vs. 0.43±0.01, Galectin-9/GAPDH: 0.21±0.01 vs. 0.43±0.01, bothP < 0.01).Conclusions LPS regulates macrophage M1/M2 subtype polarization via Galectin-9/ Tim-3 signaling pathway. Low-doses of LPS can limit the development of inflammation by accommodating the expression and secretion of Galectin-9 to polarize macrophages to M2. High-doses of LPS promotes the development of inflammation by down-regulating the expression and secretion of Galectin-9 to polarize macrophages to M1.