二甲双胍对脂多糖激活小胶质细胞炎症因子释放的影响及机制探讨

Effect of metformin on release of inflammatory factor from LPS-stimulated microglia and its possible mechanism

目的观察二甲双胍对脂多糖(LPS)刺激的小胶质细胞炎症因子释放的影响,并探讨其机制。方法取对数生长期的小胶质细胞系BV2细胞,以台盼蓝拒染法观察二甲双胍是否具有细胞毒性作用。将BV2细胞随机分为两组,LPS组予LPS 50 ng/m L刺激24 h;观察组分别以0.02、2、8、16 mmol/L二甲双胍预处理24 h,再以LPS50 ng/m L刺激24 h。采用ELISA法检测两组细胞上清液中的TNF-α水平,以细胞计数法计算增殖细胞数,以MTT法检测细胞增殖活性,以碘化丙啶染色+流式细胞术检测细胞周期,以改良的碘化丙啶单染法+流式细胞术检测细胞凋亡。结果不同浓度二甲双胍处理的小胶质细胞均台盼蓝拒染;与LPS组相比,观察组随着二甲双胍浓度增加细胞上清液中TNF-α逐渐减少,增殖细胞数及增殖活性的吸光度值逐渐降低,细胞凋亡率逐渐增加,2、8、16mmol/L时差异有统计学意义(P<0.05、0.01、0.01)。观察组随着二甲双胍浓度增加,G1期细胞比例逐渐增加,8、16 mmol/L时与LPS组相比差异有统计学意义(P<0.05、0.01)。结论二甲双胍能减少LPS激活的小胶质细胞炎症因子的释放,其机制可能与抑制小胶质细胞增殖、促进小胶质细胞凋亡有关。

Objective To observe the effect of metformin on the release of inflammatory factor from lipopolysaccharide （LPS）-stimulated microglia and to investigate the possible mechanism.Methods The cytotoxicity of metformin to BV2 cells in logarithmic growth phase was tested by Trypan blue exclusion.Then BV2 cells were randomly divided into LPS stimulation group （LPS group,cells were cultured with LPS in 50 ng/mL for 24 hours）and LPS plus Metformin group （ob-servation group,cells were cultured with metformin in 0.02,2,8,and 16 mmol/L for 24 hours first,then cultured with LPS in 50 ng/mL for 24 hours）.TNF-αwas detected by ELISA.The proliferation was calculated by Cell counting.The cell viability was tested by MTT.The cell cycle was evaluated by propidium iodide staining ＋flow cytometry.The apoptot-ic rate was analyzed by improved propidium iodide staining ＋flow cytometry.Results The microglia treated with differ-ent concentrations of metformin refused staining of the trypan blue exclusion.Compared with LPS group,metformin de-creased the level of TNF-α,inhibited the proliferation of microglia and increased the apoptotic rate in a dose-dependent manner （P 〈0.05,0.01,0.01 at 2,8,and 16 mmol/L respectively）.With the increased metformin concentration,the percentage of microglia in the G1 phase was increased.Significant difference was found in the percentage of microglia in the G1 phase between the LPS group and observation group with 8 and 16 mmol/L （P 〈0.05,P 〈0.01）.Conclusion Met-formin decreases the release of inflammatory factor from LPS-stimulated microglia,and the mechanism may be related to the inhibition of microglia proliferation and promotion of microglia apoptosis.