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目的探讨甘氨酸抑制脂多糖介导的鼠枯否细胞激活效应相关机制和甘氨酸的最佳用药时机。方法将40只BALB/c小鼠分为内毒素组、预防组、早期治疗组和后期治疗组,每组10只。分离培养枯否细胞后,内毒素组加入脂多糖(10mg/L),预防组、早期治疗组和后期治疗组分别在加入脂多糖前24h、加入后0和4h加入甘氨酸(1mmol/L),分别在加入脂多糖后0、1、2、6和12h,采用逆转录聚合酶链反应及蛋白印迹法测定枯否细胞的白细胞介素1受体相关激酶4(IRAK4)mRNA和蛋白表达水平,用酶联免疫吸附法检测枯否细胞的核因子κB(NFκB)活性和培养上清液的肿瘤坏死因子α(TNFα)含量。结果脂多糖刺激后,预防组的IRAK4mRNA和蛋白表达、NFκB活性的相对峰值分别为3.64±1.13、34.54±10.31、0.47±0.10,TNFα峰值为(1780.70±210.17)pg/ml,与早期治疗组比较,差异均无统计学意义,但与内毒素组和后期治疗组比较,各峰值均明显降低,差异有统计学意义。结论提前或者在脂多糖刺激的同时应用甘氨酸,能有效抑制脂多糖介导的枯否细胞激活效应,其作用机制之一可能为抑制IRAK4的表达。
Objective To investigate the mechanism of glycine in inhibiting lipopolysaccharide-induced murine cell activation and the optimal timing of glycine. Methods 40 BALB / c mice were divided into endotoxin group, prophylaxis group, early treatment group and post-treatment group, 10 in each group. Lipopolysaccharide (10mg / L) was added to the endotoxin group after the isolation and cultivation of Kupffer cells. Glycine (1mmol / L) was added to the prophylaxis group, the early treatment group and the later treatment group respectively 24h before adding lipopolysaccharide and 0 and 4h after the addition. The mRNA and protein expression of interleukin-1 receptor-related kinase 4 (IRAK4) in Kupffer cells were determined by reverse transcription polymerase chain reaction and Western blotting at 0, 1, 2, 6 and 12 h after adding lipopolysaccharide, The activity of nuclear factor kappa B (NFκB) and the content of tumor necrosis factor α (TNFα) in supernatant of Kupffer cell were detected by enzyme-linked immunosorbent assay. Results The relative peak values of IRAK4 mRNA and protein expression and NFκB activity of the prophylaxis group were 3.64 ± 1.13,34.54 ± 10.31 and 0.47 ± 0.10, respectively, and the peak value of TNFα was (1780.70 ± 210.17) pg / ml after stimulation with LPS. Compared with the early treatment group , The differences were not statistically significant, but compared with the endotoxin group and the late treatment group, the peak values were significantly lower, the difference was statistically significant. Conclusions The application of glycine in advance or in the stimulation of lipopolysaccharide can effectively inhibit LPS-mediated activation of Kupffer cells. One of the mechanisms may be that it inhibits the expression of IRAK4.