蔓荆子提取物对小鼠造血系统辐射损伤的防护作用

Protective effect of Vitex trifolia L. extracts on radiation injury of hematopoietic system in mice

    摘要
  • 摘要:
    目的 探讨蔓荆子提取物对小鼠造血系统辐射损伤的防护作用。
    方法 （1）体外细胞实验：提取小鼠骨髓细胞，将骨髓细胞分为对照组、蔓荆子组、照射组和蔓荆子+照射组，蔓荆子组的给药浓度为0.01 mg/ml和0.001 mg/ml，蔓荆子+照射组的给药浓度为0.001 mg/mL，照射组和蔓荆子+照射组细胞经1 Gy照射。使用酶标仪检测细胞活力，异硫氰酸荧光素（FITC）通道检测细胞的活性氧（ROS）水平，FITC和藻红蛋白（PE）通道检测细胞的凋亡水平。（2）体内实验：采用简单随机抽样法将15只C57BL/6小鼠分为对照组（n=5）、照射组（n=5）和蔓荆子+照射组（n=5）。对照组小鼠进行假照射（0 Gy），照射组和蔓荆子+照射组小鼠进行一次性2 Gy全身照射。将蔓荆子提取物用二甲基亚砜（DMSO）配制为 500 mg/ml 的蔓荆子溶液，灌胃前用生理盐水稀释，蔓荆子+照射组小鼠于照射前给予蔓荆子提取物（400 mg/kg）0.2 ml，连续给药7 d，照射后10 d处死小鼠。使用全自动血液分析仪分别对外周血血细胞和骨髓有核细胞（BMNC）计数，使用流式细胞仪分析造血干/祖细胞的数量和百分比，检测骨髓造血细胞内ROS水平、人磷酸化组蛋白H2A变异体（γH2AX）和磷酸化的p38（pp38）的表达。符合正态分布的计量资料的2组间比较采用独立样本t检验（方差齐）。
    结果 （1）体外细胞实验：与照射组比较，0.001 mg/mL蔓荆子+照射组的骨髓细胞活力明显提高（585 485.00±37 335.80对460 384.55±53 786.37），ROS水平降低（12 260.67±232.34对17 969.67±467.24），凋亡细胞百分比显著降低（28.97±0.32）% 对 （35.33±0.35）%，且差异均有统计学意义（t=4.245、18.950、23.161，均P<0.01）。（2）体内实验：与照射组相比，蔓荆子+照射组小鼠的BMNC数量（23.34±3.01）×106个/只对（16.73±2.57）×106个/只、白细胞数量（2.80±0.35）×109个/L对（2.21±0.24）×109个/L、红细胞数量（10.54±0.51）×1012个/L对（9.68±0.26）×1012个/L、血小板数量（339.80±49.42）×109个/L对（289.40±54.08）×109个/L和血红蛋白含量（139.20±3.66） g/L对（129.20±3.87） g/L均升高，且差异均有统计学意义（t=2.582、2.824、2.999、1.376、9.739，均P<0.01）。与照射组比校，蔓荆子+照射组小鼠造血祖细胞的数量（34916.03±697.36）个/只对（26388.04±241.78）个/只和百分比（29.83±4.32）%对（22.76±2.20）%升高，造血干细胞的数量（2 074.00±23.12）个/只对（929.40±166.52）个/只升高，且差异均有统计学意义（t=5.423、9.171、3.175，均P<0.01）；造血干/祖细胞中的ROS水平下降（7 750.20±589.05）对（8 515.20±1 036.46），（9 360.20±831.97）对（10 291.40±767.57），差异均无统计学意义（t=1.435、1.839，P=0.189、0.103）；造血干/祖细胞中γH2AX的表达降低（693.20±4.82对751.60±32.72）， 且差异有统计学意义（t=3.064，P<0.01）；造血干/祖细胞中pp38表达降低（1 181.20±11.28对1 183.60±49.70，1 411.20±50.25对1 424.40±80.95），差异均无统计学意义（t=0.105、0.765， P=0.014、0.310）。
    结论 蔓荆子提取物通过降低造血细胞的氧化应激和抑制造血干细胞内的DNA损伤来达到辐射防护效果。

     

    Abstract:
    Objective Aims to investigate the protective effects of Vitex trifolia L. extracts on hematopoietic radiation injury in mice.
    Methods (1) For in vitro analysis, bone marrow cells were flushed and divided into control group, Vitex trifolia L. group, radiation group, and Vitex trifolia L.+radiation group. The concentrations of Vitex trifolia L. were 0.01 and 0.001 mg/mL in the V. trifolia L. group and 0.001 mg/mL in the Vitex trifolia L.+radiation group. Cells in the radiation group and Vitex trifolia L.+radiation group were irradiated with 1 Gy. Cell viability was detected by microplate reader, reactive oxygen species (ROS) level was detected by fluorescein isothiocyanate (FITC) channel, and apoptosis level was detected by FITC and phycoerythrin (PE) channels. (2) For in vivo analysis, 15 male C57BL/6 mice were divided by simple random sampling into the following groups: control group (n=5), radiation group (n=5), and Vitex trifolia L.+radiation group (ig, 400 mg/kg/day, 7 times, n=5). Mice in the control group were sham irradiated (0 Gy). Mice in the radiation group and the Vitex trifolia L.+radiation group were total body irradiated with a single dose of 2 Gy. The extract of Vitex trifolia L. was prepared with dimethyl sulfoxide to make a solution of 500 mg/mL and diluted with normal saline before gavage. Mice in the Vitex trifolia L.+radiation group were given 0.2 mL of Vitex trifolia L. extract (400 mg/kg) for 7 days before irradiation. Mice were sacrificed 10 days after irradiation. Peripheral blood and bone marrow nucleated cells (BMNC) were counted by automatic hematology analyzer. The number and percentage of hematopoietic stem progenitor cells and the levels of ROS, human phosphorylated histone H2A variants (γH2AX), and phosphorylated p38 (pp38) expression in bone marrow hematopoietic cells were analyzed by flow cytometry. Independent sample t test (homogeneity of variance) was used to compare measured data with normal distribution between two groups.
    Results The viability of bone marrow cells in the Vitex trifolia L. (0.001 mg/mL)+radiation group increased (585 485.00±37 335.80), the ROS level decreased (12 260.67±232.34), and the percentage of apoptosis significantly decreased ((28.97±0.32)%) compared with those in the radiation group ((460 384.55±53 786.37, 17 969.67±467.24, and (35.33±0.35)%, respectively)). The differences were all statistically significant (t=4.245, 18.950, 23.161, respectively; all P<0.01). The following parameters increased in comparison with the radiation group: bone marrow nucleated cells count ((23.34±3.01)×106/mouse vs. (16.73±2.57) ×106/mouse), white blood cell count ((2.80±0.35)×109/L vs. (2.21±0.24)×109/L), red blood cell count ((10.54±0.51)×1012/L vs. (9.68±0.26)×1012/L), platelet count ((339.80±49.42)×109/L vs. (289.40±54.08)×109/L), and hemoglobin content ((139.20±3.66) g/L vs. (129.20±3.87) g/L) (t=2.582, 2.824, 2.999, 1.376, 9.739, respectively; all P<0.01). The number and percentage of hematopoietic progenitor cells ((34916.03±697.36)/mouse vs. (26388.04±241.78)/mouse; (29.83±4.32)% vs. (22.76±2.20)%) and the number of hematopoietic stem cells ((2 074.00±23.12)/mouse vs.(929.40±166.52)/mouse) increased in the Vitex trifolia L.+radiation group compared with those in the radiation group, and the differences were statistically significant (t=5.423, 9.171, 3.175, respectively; all P<0.01). The levles of ROS in the hematopoietic stem cells decreased ((7 750.20±589.05) vs. (8 515.20±1 036.46), (9 360.20±831.97) vs. (10 291.40±767.57)) in the Vitex trifolia L.+radiation group compared with those in the radiation group, and the differences was not statistically significant (t=1.435, 1.839; P=0.189, 0.103). The expression of γH2AX in hematopoietic stem cells significantly decreased ((693.20±4.82) vs. (751.60±32.72), t=3.064, P<0.05). The expression of pp38 in the hematopoietic stem progenitor cells of Vitex trifolia L.+radiation group decreased compared with that in the radiation group (1181.20±11.28 vs. 1183.60±49.70, 1411.20±50.25 vs. 1424.40±80.95), but the difference was not statistically significant (t=0.105, 0.765; P=0.014, 0.310).
    Conclusion Vitex trifolia L. extracts can reduce oxidative stress and inhibit DNA damage in hematopoietic stem cells to achieve radiation protection.

     

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