目的：研究吲哚-3-甲醇（I3C）对小鼠骨髓造血细胞辐射损伤的保护作用及机制。方法（1）密度梯度离心法获得CD45.1亚型C57BL/6J小鼠骨髓有核细胞，经0 mol/L、10-8 mol/L~10-3 mol/L I3C处理后，接受不同剂量（0 Gy、1 Gy、4 Gy）的137Csγ-射线照射；继续培养18 h后采用生物发光法检测细胞活力。（2）设空白对照组和10-6 mol/L I3C组，经上述3种剂量射线照射后，接种于甲基纤维素半固体培养基中培养7 d，观察骨髓粒-单核巨噬细胞集落（CFU-GM）形成情况。（3）取24只CD45.2亚型小鼠接受8 Gy 137Csγ-射线照射作为受体，CD45.1亚型小鼠骨髓有核细胞（供体）设空白对照组、4 Gy照射组和4 Gy照射+10-6 mol/L I3C组。将供体与竞争者（CD45.2亚型）骨髓细胞混和后，接种于受体小鼠体内（每组8只），流式细胞术检测受体小鼠外周血细胞中供体来源的细胞比例。（4）细胞设空白对照组、10-6 mol/L I3C组、1 Gy照射组和1 Gy照射+10-6 mol/L I3C组。培养24 h后收集细胞，提取蛋白后Western blot法检测各组核因子NF-E2相关因子（Nrf2）、血红素加氧酶（HO）-1表达。结果（1）I3C浓度>10-4 mol/L时出现明显的细胞毒性作用（P>0.05）；相同剂量射线照射下，10-7~10-6 mol/L I3C可减轻射线对细胞的损伤；因此选取10-6 mol/L为本研究I3C的实验浓度。（2）相同剂量射线照射下，10-6 mol/L I3C组CFU-GM形成数量较空白对照组明显升高（P<0.05）。（3）流式细胞结果显示，4 Gy照射组细胞移植后，受体小鼠外周血中供体细胞比例较对照组明显降低（P<0.05），而10-6 mol/L I3C预处理的供体小鼠细胞移植后，受体小鼠中供体细胞比例较4 Gy照射组升高（P<0.05）。（4）Western blot结果显示，1 Gy照射+10-6 mol/L I3C组Nrf2和HO-1蛋白表达水平明显高于其他3组（P<0.05）。结论 I3C可以减轻辐射引起的小鼠造血细胞损伤和功能下降，其机制可能与激活Nrf2/HO-1通路有关。%Objective To investigate the protective effect of indole-3-carbinol (I3C) on radiation-induced mouse bone marrow hematopoietic cell injury and the involved mechanisms. Methods (1) The bone marrow nuclear cells (BMNCs) from CD45.1 subtype of C57BL/6J mice were collected by a density gradient centrifugation method. The BMNCs were pretreated with a series doses of I3C (0 mol/L, 10-8 mol/L-10-3 mol/L) and then exposed with radiation of 137Csγ-ray (doses of irradiation were 0 Gy, 1 Gy and 4 Gy). After 18-hour culturing, the bioluminescence method was used to detect the cell viability. (2) These cells were divided into control group and 10-6 mol/L I3C group. Both groups were received the irradiation (0 Gy, 1 Gy and 4 Gy) and inoculated into the methylcellulose semi-solid culture medium to incubate 7 days, the colony forming unit-granulocyte monocytes (CFU-GM) were observed. (3) Twenty-four CD45.2 subtype mice used as the receptor were exposed with 8 Gy radiation. The CD45.1 BMNCs were divided into control group, 4 Gy irradiation group, 4 Gy irradiation and 10-6 mol/L I3C group. Donor cells were harvested from C57BL/6J (CD45.1) mice after they received various treatments, and were then mixed with competitive BMNCs from C57BL/6J (CD45.2) mice. The mixed cells were transplanted into recipient mice (8 mice/group). Flow cytometry was used to analyze the proportion of donor cells in peripheral blood of receptor. (4) The cells were divided into control group, 10-6 mol/L I3C group, 1 Gy irradiation group, 1 Gy irradiation with 10-6 mol/L I3C group. After 24-hour culturing, Western blot assay was used to detect the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Results (1) I3C showed a significant cytotoxic effect on the BMNCs when its concentration was above 10-4 mol/L. 10-7-10-6 mol/L I3C could reduce the radiation injury of BMNCs under the same dose of irradiation. Therefore, 10-6 mol/L I3C was chosen for subsequent experiments. (2) The CFU-GM was significantly higher in 10-6 mol/L I3C group than that of control group (P<0.05). (3) Results of flow cytometry showed that the proportion of donor cells in receptor was significantly higher in 4 Gy irradiation group than that of control group, which decreased the engraftment capability of irradiated HSCs (P<0.05), although the engraftment capability of irradiated HSCs improved after 10-6 mol/L I3C treatment. (4) I3C significantly enhanced the increased protein expression of Nrf2 and HO-1 caused by radiation (P<0.05). Conclusion I3C has a protective effect on hematopoietic cells following radiation-induced injury, which may be related with activating the Nrf2/HO-1 signal pathway.
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