当归多糖、黄芪多糖及其配伍对化疗性骨髓抑制小鼠骨髓干细胞增殖和PI3K/AKT信号转导通路的影响

Effects of Angelica Polysaccharides, Astragalus Polysaccharides and Their Compatibility on Proliferation of Bone Marrow Stem Cells and PI3K/AKT Signal Transduction Pathway in Mice with Chemotherapy-Induced Bone Marrow Suppression

目的:从PI3K/AKT信号转导通路探讨当归多糖、黄芪多糖及其配伍对化疗性骨髓抑制小鼠骨髓干细胞的调控机制。方法:C57BL/6小鼠20只,雌雄各半,28~32 g。给模型组小鼠每日腹腔注射1次环磷酰胺380 mg·kg-1,正常小鼠腹腔注射等量生理盐水,连续3 d。造模后,以脱颈椎法处死各组小鼠,取出股骨,剔除肌肉和结缔组织,剪开股骨两端,用6号针头以生理盐水反复冲洗骨髓腔,并将冲出的骨髓打碎,再用4号针头过滤制成单个细胞悬液,在离心机上2000 r/min,离心10 min,去上清液,加入4 mL冷70%乙醇固定,上振荡器摇匀制成骨髓干细胞样本。细胞实验分为6组,包括正常组、模型组、EPO组、EPO+(G-CSF)组、当归多糖组、黄芪多糖组、两药配伍组。直接给药当归多糖0.2 mg/mL,黄芪多糖0.2 mg/mL,EPO 50 U/mL,(G-CSF)50 U/mL于骨髓干细胞体外培养体系中,培养3 d,室内18~20℃,20%相对湿度,5%CO2,37℃孵育箱。采用MTT法检测骨髓干细胞生长和增殖的情况;采用ELISA检测骨髓干细胞中pPI3K和p-AKT的蛋白含量;采用RT-qPCR法检测骨髓干细胞中PI3K、AKT、PTEN的mRNA表达。结果:(1)对小鼠骨髓干细胞生长和增殖的影响。①造模后24 h,各给药组与模型组比较均有明显差异。当归多糖与黄芪多糖有交互作用,合用不如单用当归多糖。②造模后48 h,各给药组与模型组比较均有明显差异,药物组之间差异均没有统计学意义。当归多糖与黄芪多糖有交互作用,合用比单一用药好。③造模后72 h,各给药组与模型组比较均有明显差异。当归多糖与黄芪多糖有交互作用,合用比单一用药好。(2)对PI3K、AKT的mRNA表达的影响。各给药组与模型组PI3K、Akt的mRNA比较均有明显差异,当归多糖与黄芪多糖均有提升PI3K、Akt的mRNA作用,两药交互作用无统计学意义。(3)对pPI3K、p-AKT蛋白含量的影响。各给药组与模型组pPI3K、p-ATK的蛋白含量比较均有明显差异,当归多糖与黄芪多糖有交互作用,合用比单一用药好。(4)对PTEN mRNA表达的影响。各给药组与模型组PTEN mRNA比较均有明显差异,当归多糖与黄芪多糖交互作用明显,合用比单一用药好。结论:(1)当归多糖、黄芪多糖及其配伍能够促进化疗性骨髓抑制小鼠骨髓干细胞增殖,两药配伍的作用优于单一用药。(2)当归多糖、黄芪多糖及两药配伍能够上调PI3K、AKT mRNA的表达,增加pPI3K和p-AKT的蛋白含量,下调PTEN mRNA的表达,两药配伍的作用优于单一用药。(3)当归多糖、黄芪多糖及两药配伍激活了PI3K/AKT信号转导通路,两药治疗化疗性骨髓抑制的作用可能与PI3K/AKT通路有关。

Objective:To investigate the regulatory mechanism of Angelica polysaccharides,Astragalus polysaccharides and their compatibility on bone marrow stem cells of mice with chemotherapy-induced bone marrow suppression through PI3 K/AKT signal transduction pathway.Methods:Twenty C57 BL/6 mice,male and female in half,were weighed 28 to 32 g.The mice in the model group were given intraperitoneal injection of cyclophosphamide 380 mg/kg once a day,and the normal mice were given intraperitoneal injection of normal saline for 3 consecutive days.After modeling,mice in each group were killed by cervical spondylolisthesis.The femur was removed and the muscle and connective tissue were removed.Both ends of the femur were cut off.The bone marrow cavity was washed repeatedly by No.6 needle with normal saline,and the washed bone marrow was broken.Then the washed bone marrow was filtered with No.4 needle to form a single cell suspension.The cell suspension was centrifuged for 2000 r/min,centrifuged for 10 min.The supernatant was removed and added with 4 mL cold 70%ethanol.Ethanol was immobilized and the upper oscillator was shaken to make bone marrow stem cell samples.Cell experiments were divided into six groups,including normal group,model group,EPO group,EPO+(G-CSF)group,angelica polysaccharide group,Astragalus polysaccharide group,two drugs compatibility group.We directly administered Angelica polysaccharide 0.2 mg/mL,Astragalus polysaccharide 0.2 mg/mL,EPO 50 U/mLand G-CSF 50 U/mL in vitro culture system of bone marrow stem cells for 3 days,incubation chamber at 18-20℃,20%relative humidity,5%CO2,37℃.MTT assay was used to detect the growth and proliferation of bone marrow stem cells.ELISA was used to detect the protein contents of pPI3 K and pAKT in bone marrow stem cells.RT-q PCR was used to detect the expressions of PI3 K,AKT and PIEN in bone marrow stem cells.Results:(1)Effects on the growth and proliferation of bone marrow stem cells in mice:①After modeling,there was significant difference between the 24 h groups and the model group.The polysaccharides of Angelica sinensis and Astragalus polysaccharides interacted with each other.②48 hours after the establishment of the model,there were significant differences between the drug group and the model group,and there was no significant difference among the drug groups.The polysaccharides of Angelica sinensis and Astragalus polysaccharides interacted with each other.③After modeling,there was significant difference between the 72 h groups and the model group.The polysaccharides of Angelica sinensis and Astragalus polysaccharides interacted with each other.(2)The effects of PI3 K on mRNA expressions of AKT and PIEN:Compared with the model group,the PI3 K and Akt mRNA of each drug group were significantly different.Angelica polysaccharide and Astragalus polysaccharide could enhance PI3 K and Akt mRNA,but there was no significant difference in the interaction between the two drugs.(3)Effects on pPI3 K and p-AKT protein content:The protein contents of pPI3 K and pATK in each drug group was significantly different from those in the model group.Angelica polysaccharide and Astragalus polysaccharide had interaction,and the combination was better than single drug.4.The effect of PIEN mRNA expression:Compared with the model group,PIEN mRNA in each group showed significant difference.The interaction between Angelica polysaccharides and Astragalus polysaccharides was obvious,and the combination of Angelica polysaccharides and Astragalus polysaccharides was better than single drug.Conclusion:(1)Angelica polysaccharides,Astragalus polysaccharides and their compatibility can promote the proliferation of bone marrow stem cells in mice with chemotherapy-induced bone marrow suppression,and the effect of the compatibility of the two drugs is better than that of single drug.(2)Angelica polysaccharides,Astragalus Polysaccharides and their compatibility can up-regulate the expressions of PI3 K and AKT,increase the protein contents of pPI3 K and pAKT,and down-regulate the expression of PIEN.The effect of the compatibility of Angelica polysaccharides and Astragalus polysaccharides is better than that of single drug.(3)Angelica sinensis polysaccharide,astragalus polysaccharide and their combination activate PI3 K/AKT signal transduction pathway.The effect of the two drugs on chemotherapy-induced bone marrow suppression may be related to PI3 K/AKT pathway.