δ-生育三烯酚促进小鼠粒细胞集落刺激因子生成作用及其促造血活性

δ-Tocotrienol promotes granulocyte colony-stimulating factor production in mice and its hematopoietic activity

目的探讨δ-生育三烯酚(δ-T3H)诱导小鼠体内粒细胞集落刺激因子(G-CSF)生成的作用,评价δ-T3H促健康小鼠造血活性作用。方法雄性C57BL/6J小鼠分别单次sc给予δ-T3H 12.5,25,50,100和200 mg·kg^-1,于药后24 h采血,酶联免疫吸附法(ELISA)检测血清中G-CSF水平,分析δ-T3H诱导小鼠体内生成G-CSF的剂量效应。小鼠单次sc给予δ-T3H 100 mg·kg^-1,于给药后3,6,12,24,36,48,72和96 h采血,ELISA检测血清中G-CSF水平和细胞趋化因子1(CXCL1)、粒巨噬细胞集落刺激因子(GM-CSF),白细胞介素6(IL-6),IL-10,IL-12和肿瘤坏死因子α(TNF-α)水平。雄性小鼠单次sc给予δ-T3H 100 mg·kg^-1,于给药后24 h取材分析组织匀浆中G-CSF水平。雄性小鼠连续sc给予δ-T3H 100 mg·kg^-15 d,于给药后利用ELISA检测血清中G-CSF、红细胞生成素(EPO)、血小板生成素(TPO)和基质细胞衍生因子1β(SDF-1β)的含量,利用血细胞分析仪检测外周血常规参数。δ-T3H按照100 mg·kg^-1分为单次sc给药组和连续2 d sc给药组,于给药24 h后利用流式细胞术分析外周血造血干细胞数量。结果δ-T3H单次给药升高G-CSF的作用呈给药剂量效应,最低有效剂量≤12.5 mg·kg^-1,100 mg·kg^-1给药后升高G-CSF的作用达到饱和;δ-T3H单次给药后48 h内以及连续给药期间小鼠血清G-CSF浓度均可维持在较高水平(36±12)μg·L^-1;细胞因子检测发现,δ-T3H对CXCL1生成有显著促进作用(P<0.05),不影响血清中其他炎症细胞因子(GM-CSF,IL-6,IL-10,IL-12和TNF-α)的生成;δ-T3H显著升高小鼠淋巴-造血组织如胸腺、脾、淋巴结和骨髓组织液中G-CSF水平(P<0.05),对其他组织器官匀浆中G-CSF含量无显著影响;δ-T3H诱导小鼠血清中TPO和EPO略有升高;血常规检测发现,δ-T3H连续给药可显著增加小鼠外周血白细胞数量(主要为中性粒细胞和单核细胞的升高),血小板小幅升高,而淋巴细胞和红细胞则有一过性降低(P<0.05,P<0.01);流式细胞术分析发现,δ-T3H连续2 d sc给药组小鼠外周血造血干细胞数量显著升高(P<0.01)。结论δ-T3H促进小鼠体内G-CSF生成具有专一性以及组织特异性,同时δ-T3H通过刺激体内G-CSF的生成升高小鼠外周血粒细胞数,并促进外周血造血干细胞数量的增加,具有一定的促造血活性。

OBJECTIVE To explore the dose and time-dependent effect,tissue specificity and specificity ofδ-tocotrienol(δ-T3H)-induced granulocyte colony stimulating factor(G-CSF)in mice,and to evaluate the effect of δ-T3H on the increase in peripheral blood granulocytes and mobilization of hematopoietic stem cells(HSCs)into peripheral blood in normal healthy mice.METHODS Mice were givenδ-T3H 12.5,25,50,100 and 200 mg·kg^-1 in a single subcutaneous injection,and blood was collected 24 h after drug administration.The levels of G-CSF in the serum were detected by enzyme-linked immunosorbent assay(ELISA)and theδ-T3H-induced dose effect of G-CSF in mice was analyzed.δ-T3H 100 mg·kg^-1 was subcutaneously injected at a single dose and blood was collected at 3,6,12,24,36,48,72 and 96 h after administration.ELISA was used to detect the levels of G-CSF,C-X-C motif chemokine ligand 1(CXCL1),granulocyte macrophage colony stimulating factor(GM-CSF),interleukin 6(IL-6),IL-10,IL-12 and tumor necrosis factor-α(TNF-α)in serum.The effect ofδ-T3H on the production of inflammation-related cytokines was analyzed.The mice were taken to analyze the level of G-CSF in the homogenate 24 h after administration by ELISA.δ-T3H 100 mg·kg^-1 was sc injected for 5 consecutive days before the levels of G-CSF,erythropoietin(EPO),thrombopoietin(TPO),and stromal cell-derived factor-1β(SDF-1β)in the serum were detected by ELISA.The effect of δ-T3H on the production of hematopoiesis related cytokines was analyzed.The blood cell analyzer was used to analyze the effect ofδ-T3H on peripheral blood leukocytes.δ-T3H 100 mg·kg-1 was administered subcutaneously at a single dose or once daily for 2 d.24 h after administration,flow cytometry was used to count the number of hematopoietic stem cells in peripheral blood.RESULTS A single-dose of δ-T3H increased G-CSF in a dose-dependent manner.The lowest effective dose was not higher than 12.5 mg·kg^-1.After 100 mg·kg^-1 administration,the effect of increasing G-CSF was saturated.The serum G-CSF concentration of mice within 48 hours after a single administration ofδ-T3H and during continuous administration could be maintained at a high level(36±12)μg·L^-1.Cytokine detection found thatδ-T3H could promote the production of CXCL1(P<0.05),but on other inflammatory cytokines(GM-CSF,IL-6,IL-10,IL-12 and TNF-α)production had no significant effect.δ-T3H significantly increased the level of G-CSF in lymphatic-hematopoietic tissues of mice such as the thymus,spleen,lymph nodes and bone marrow tissue fluid(P<0.05),but had no significant effect on other tissues and organs.In addi⁃tion,TPO and EPO in mouse serum increased slightly after δ-T3H administration.Routine blood tests found that continuous administration ofδ-T3H significantly increased the number of white blood cells in peripheral blood,especially neutrophils and monocytes,but platelets increased slightly,while lympho⁃cytes and red blood cells temporarily decreased(P<0.05,P<0.01).Flow cytometry analysis found that δ-T3H increased the number of hematopoietic stem cells in the peripheral blood of mice after two-time subcutaneous administration(P<0.01).CONCLUSIONδ-T3H can promote G-CSF production in mice with specificity and tissue selectivity.In addition,δ-T3H has some hematopoietic activity,able to increase peripheral blood leukocytes and mobilize bone marrow HSCs into peripheral blood by stimulating G-CSF production in mice.