当归补血汤及其主要成分对骨髓抑制小鼠的抗造血细胞凋亡的研究

The Antiapoptotic Effect of Danggui Buxue Tang and Its Main Components on Hematopoietic Cells in Mice with Bone Marrow Suppression

目的:探讨当归补血汤(danggui buxue tang,DBT)及其主要成分当归多糖(angelica polysaccharide,APS)和黄芪多糖(astragalus polysaccharide,ASPS)对骨髓抑制小鼠的造血保护作用和对巨核细胞系Meg-01细胞抗凋亡的机制研究。方法:用4 Gy的^(137)Csγ射线辐照小鼠,建立小鼠骨髓抑制模型。DBT、APS或ASPS(10mg/kg)注射小鼠,在小鼠辐照前(d0)和辐照后d7、14和21对小鼠进行一般状况评估、外周血细胞计数,以及d 21收集小鼠贫血小板血浆检测血小板生成素(TPO)浓度;取d21处死后的小鼠股骨骨髓细胞进行集落细胞形成单位的培养。无血清培养Meg-01细胞24h诱导细胞凋亡,DBT、APS或ASPS处理Meg-01细胞72h后,采用流式细胞术检测细胞的早期凋亡(Annexin V)、线粒体膜电位(JC-1)和凋亡蛋白酶(Caspase-3)的活性。结果:DBT可刺激骨髓抑制小鼠白细胞、红细胞和血小板的恢复,对白细胞和血小板尤为明显(P<0.01,P<0.05)。DBT治疗组小鼠骨髓细胞BFU-E、CFU-MK和CFU-GM的形成数量较对照组明显增多(BFU-E&CFU-GM:P<0.05;CFU-MK:P<0.01)。DBT对小鼠血液中TPO浓度的影响不明显(P=0.89)。与对照组相比,APS组红细胞、白细胞和血小板数均有明显升高(P<0.05);ASPS组白细胞数较对照组明显升高(P<0.05)。CFU培养结果显示,APS可刺激CFU-F、CFU-MK和CFU-GM的形成(P<0.05);而ASPS组只有CFU-GM的数量较对照组增多(P<0.05)。凋亡实验结果显示,DBT可明显降低Meg-01细胞的凋亡(P<0.05)。APS(100μg/ml)处理组Meg-01细胞的早期凋亡率和总死亡率较对照组明显降低(P<0.01,P<0.05);ASPS(100μg/ml)组早期凋亡率较对照组明显降低(P<0.05)。JC-1和Caspase-3结果显示,APS(100μg/ml)处理后,细胞凋亡率明显降低(P<0.01,P<0.05)。结论:DBT对放射诱导骨髓抑制小鼠的造血系统具有保护作用,尤其是白细胞和血小板;对Meg-01细胞有抗凋亡作用。DBT上述作用主要是由APS引起的,其对抗细胞凋亡的机制主要是通过JC-1和Caspase-3途径进行的。

Objective:To explore the hematopoiesis protection effect of Danggui Buxue Tang(DBT)and its main components Angelica polysaccharide(APS)and Astragalus polysaccharide(ASPS)on myelosuppression mice,and the mechanism of anti-apoptosis of Meg-01 cells.Methods:Mice were radiated with 4 Gy of^(137)Csγray to establish the model of radiation-induced myelosuppression.DBT,APS or ASPS(10 mg/kg)were injected into irradiated mice.Peripheral blood cell counts were performed on mice before radiation(day 0)and day 7,14 and 21 after radiation.On the 21 st day,poor plasma platelets were collected from mice to detect TPO concentration and then the mice were sacrificed.The femoral bone marrowcells were cultured for colony cell forming units(CFU).Meg-01 cells were cultured without FBS for 24 h to induce apoptosis,and then treated with DBT/APS/ASPS for 72 h.Flowcytometry(FCM)was used to detect early apoptosis(Annexin V),mitochondrial membrane potential(JC-1)and the expression of Caspase-3 to analyze the effect of DBT/APS/ASPS on cell apoptosis.Results:DBT can stimulate the recovery of white blood cells(WBC),red blood cells(RBC)and platelets(PLT)of myelosuppression mice,especially for WBC and PLT(P<0.01,P<0.05).Compared with the control group,the number of BFU-E,CFU-M K and CFU-GM increased after adding DBT(BFU-E&CFU-GM:P<0.05;CFU-M K:P<0.01).The effect of DBT on blood TPO concentration in mice was not obvious(P=0.89).RBC,WBC and PLT were increased in APS group compared with control group(P<0.05).WBC increased after the treatment of ASPS(P<0.05).APS stimulated the formation of CFU-F,CFU-M K and CFU-GM(P<0.05).Only CFU-GM increased in ASPS group(P<0.05).Besides,DBT decreased the apoptosis of M eg-01 cells(P<0.05).The early apoptosis rate and total death rate in APS(100μg/ml)group were lower than that of control group(P<0.01,P<0.05).The early apoptosis rate of ASPS(100μg/ml)group was lower than that of control group(P<0.05).JC-1 and Caspase-3 showed that APS(100μg/ml)significantly reduced apoptosis rate(P<0.01,P<0.05).Conclusion:DBT has protective effect on hematopoietic system,especially WBC and PLT,and has anti-apoptotic effect on M eg-01.It was found that the above effects of DBT were mainly caused by APS,and its anti-apoptosis mechanism was carried out mainly through JC-1 and Caspase-3 pathways.