N-草酰基-D-苯丙氨酸对小鼠造血系统辐射损伤的防护作用

Protective effects of N-oxalyl-D-phenylalanine on hematopoietic system injury induced by ionizing radiation in mice

目的研究N-草酰基-D-苯丙氨酸(NOFD)对小鼠造血系统辐射损伤的防护作用。方法将18只6~8周龄的健康C57BL/6J雄性小鼠按区组随机法分为3组:对照组、4 Gyγ射线全身照射组(简称照射组)和4 Gyγ射线全身照射+5 mg/kg NOFD组(简称照射给药组),每组6只。照射给药组于照射前2、16 h及照射后3 d分别腹腔给予5 mg/kg NOFD,对照组和照射组给予等量生理盐水,给药时间和次数与照射给药组相同。采用血细胞计数仪分析各组小鼠外周血血细胞数量;采用流式细胞仪检测外周血中B细胞、T细胞、髓系细胞的百分比,骨髓细胞中造血干细胞(HSC)和造血祖细胞(HPC)的数量及百分比,骨髓细胞中磷酸化组蛋白H2AX(γ-H2AX)和线粒体活性氧(ROS)水平;采用粒细胞-巨噬细胞集落形成单位(CFU-GM)实验和脾集落形成单位(CFU-S)实验检测骨髓细胞的增殖能力。组间两两比较采用Student t检验。结果与照射组相比,照射给药组小鼠外周血中红细胞数量明显增加[(9.05±0.16)×10^(9)个/mL对(9.57±0.15)×10^(9)个/mL],T细胞的百分比升高[(11.54±0.20)%对(15.31±1.88)%],髓系细胞的百分比降低[(32.67±2.87)%对(24.90±2.19)%],HSC的数量增加[(2.24±0.54)×10^(3)个/股骨对(6.77±1.67)×10^(3)个/股骨],同时HSC和HPC在骨髓细胞中的百分比[(0.09±0.02)%对(0.59±0.13)%;(0.62±0.14)%对(1.82±0.43)%]升高,且差异均有统计学意义(t=1.998~3.633,均P<0.05)。流式细胞仪检测结果显示,与照射组相比,照射给药组小鼠的骨髓有核细胞(BMNC)和HPC中线粒体ROS自由基(MitoSOX)的平均荧光强度(MFI)明显降低[(6.66±0.56)×10^(3)对(3.19±0.25)×10^(3);(2.51±0.46)×10^(3)对(1.20±0.35)×10^(3)],且差异均有统计学意义(t=6.350、2.282,均P<0.05);同时照射给药组小鼠BMNC、HPC和HSC中γ-H2AX的MFI明显降低[(10.25±0.77)×10^(3)对(7.22±0.15)×10^(3);(18.37±2.52)×10^(3)对(12.44±0.34)×10^(3);(26.05±2.64)×10^(3)对(17.16±1.20)×10^(3)],且差异均有统计学意义(t=4.356、2.577、3.070,均P<0.05)。集落形成单位实验结果显示,与照射组相比,照射给药组CFU-GM(12.33±1.48对24.00±3.92)和CFU-S(6.00±1.07对10.83±1.01)明显增加,且差异均有统计学意义(t=2.788、3.288,均P<0.05)。结论NOFD对小鼠造血系统辐射损伤有明显的保护作用。

Objective To study the protective effects of N-oxalyl-D-phenylalanine(NOFD)on the radiation injury of hematopoietic system in mice.Methods Eighteen healthy C57BL/6J male mice aged 6-8 weeks were divided into three groups with six mice each according to randomized block design:a control group,a 4 Gyγray whole body irradiation group(TBI group),and a 4 Gyγray whole body irradiation+5 mg/kg NOFD group(TBI+NOFD group).The mice in the TBI+NOFD group were intraperitoneally given with 5 mg/kg NOFD at 2,16 h before irradiation,and 3 d after irradiation,respectively,while the control group and TBI group were intraperitoneally given with the same amount of normal saline at the same time as that in the TBI+NOFD group.The number of peripheral blood cells in each group of mice were analyzed using a blood cell counter.The percentage of B cells,T cells,and myeloid cells in peripheral blood were detected via flow cytometry.The number and percentage of hematopoietic stem cell(HSC)and hematopoietic progenitor cell(HPC)in bone marrow cells were detected via flow cytometry.Levels of phosphorylated histone H2AX(γ-H2AX)and mitochondrial reactive oxygen species in bone marrow cells were detected via flow cytometry.The proliferation ability of bone marrow cells was evaluated by counting colony-forming units-granulocyte-macrophage(CFU-GM)and colony-forming units-spleen(CFU-S).Student's t test was used for comparison between two groups.Results Compared with that in the TBI group,the number of peripheral blood erythrocytes in the TBI+NOFD group substantially increased((9.05±0.16)×10^(9)/mL vs.(9.57±0.15)×10^(9)/mL).The percentage of T cells increased((11.54±0.20)%vs.(15.31±1.88)%),whereas the percentage of myeloid cells decreased((32.67±2.87)%vs.(24.90±2.19)%).The number of HSC increased((2.24±0.54)×10^(3)/femur vs.(6.77±1.67)×10^(3)/femur),whereas the percentage of HSC and HPC in bone marrow cells significantly increased((0.09±0.02)%vs.(0.59±0.13)%,(0.62±0.14)%vs.(1.82±0.43)%;t=1.998-3.633,all P<0.05).The median fluorescence intensity(MFI)of mitochondrial reactive oxygen species(MitoSOX)in bone marrow nucleated cells(BMNC)and HPC in the TBI+NOFD group was significantly lower than that in the TBI group((6.66±0.56)×10^(3)vs.(3.19±0.25)×10^(3),(2.51±0.46)×10^(3)vs.(1.20±0.35)×10^(3);t=6.350,2.282,both P<0.05).Moreover,the MFI ofγ-H2AX in BMNC,HPC,and HSC in the TBI+NOFD group was significantly lower than that in the TBI group((10.25±0.77)×10^(3)vs.(7.22±0.15)×10^(3),(18.37±2.52)×10^(3)vs.(12.44±0.34)×10^(3),(26.05±2.64)×10^(3)vs.(17.16±1.20)×10^(3);t=4.356,2.577,3.070,all P<0.05).Compared with those in the TBI group,CFU-GM(12.33±1.48 vs.24.00±3.92)and CFU-S(6.00±1.07 vs.10.83±1.01)in the TBI+NOFD group significantly increased(t=2.788,3.288;both P<0.05).Conclusion NOFD exerts an obvious protective effect on the radiation injury of hematopoietic system in mice.