甲硫氨酸摄入限制对LPS诱导急性肺损伤小鼠巨噬细胞的影响及作用机制

Effects and mechanism of methionine restriction on macrophage for lipopolysaccharide-induced acute lung injury in mice

目的探究甲硫氨酸摄入限制(methionine restriction,MR)对脂多糖(lipopolysaccharide,LPS)诱导的急性肺损伤(acute lung injury,ALI)小鼠的巨噬细胞的影响及其作用机制。方法将36只体质量为(23±2)g的雄性C57BL/6J小鼠(6~8周龄)按随机数字表法分为3组(n=12):Sham组、LPS组和LPS+MR组。对各组小鼠的肺组织行HE染色和肺损伤病理评分。采用RT-qPCR和Western blot检测肺组织中脂多糖结合蛋白(lipopolysaccharide binding protein,LBP)和Toll样受体-4(Toll-like receptor-4,TLR4)mRNA和蛋白的表达水平;利用免疫组化技术对各组肺组织巨噬细胞的浸润情况及其相关趋化因子巨噬细胞集落刺激因子(macrophage-colony stimulating factor,M-CSF)、粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage-colony stimulating factor,GM-CSF)和C-C基序趋化因子配体3(chemokine C-C motif ligand 3,CCL3)的表达水平进行分析。结果与Sham组小鼠比较,LPS组小鼠肺组织损伤的病理评分显著升高(P<0.01)。LBP和TLR4的mRNA和蛋白表达均显著增加;CD11b和F4/80阳性细胞的数量增加,肺间质和肺泡巨噬细胞浸润明显;M-CSF、GM-CSF和CCL3的阳性细胞数量显著升高(P均<0.01)。MR可以显著改善LPS诱导的ALI,降低LPS组小鼠肺损伤病理评分(P<0.01);MR可以减少ALI小鼠肺组织LBP和TLR4 mRNA和蛋白的表达(P<0.01);与LPS组小鼠相比较,LPS+MR组小鼠肺组织CD11b、F4/80M-CSF、GM-CSF和CCL3阳性细胞数量显著降低(P<0.01)。结论MR可通过抑制巨噬细胞趋化因子的表达、进而减少巨噬细胞浸润和活化、减轻LPS诱导的肺损伤。

Objective To investigate the effects of methionine restriction(MR)on macrophages in lipopolysaccharide(LPS)-induced acute lung injury(ALI)and to explore the underlying mechanism.Methods According to the random number table method,36 male C57BL/6J mice(6~8 weeks old,23±2 g)were divided into 3 groups with 12 mice in each group:the sham group,the LPS group and the LPS+MR group.HE staining and pathological scoring of lung injury were performed in lung tissues.The expression of LPS-binding protein(LBP)and Toll-like receptor-4(TLR4)was detected by RT-qPCR and Western blotting.Macrophage-colony stimulating factor(M-CSF),granulocyte-macrophage-colony stimulating factor(GM-CSF)and chemokine C-C motif ligand 3(CCL3)which are all macrophage-associated chemokines were analyzed by immunohistochemistry.Results Compared with the sham group,the pathological score of lung injury in the LPS group was significantly increased(P<0.01);The mRNA and protein expression levels of LBP and TLR4 were significantly increased;The number of positive cells of CD11b,F4/80,M-CSF,GM-CSF and CCL3 were significantly increased(P<0.01).MR significantly improved LPS-induced ALI,and decreased the pathological score of lung injury(P<0.01);The mRNA and protein expression levels of LBP and TLR4 were decreased;Compared with the LPS group,the number of positive cells of CD11b,F4/80,M-CSF,GM-CSF and CCL3 were reduced in the LPS+MR group(P<0.01).Conclusion MR could attenuate LPS-induced ALI by inhibiting the expression of macrophage chemokines and preventing infiltration and activation of macrophage to lungs.