四仙颗粒及其拆方改善化疗后血小板减少的作用机制研究

Mechanism of Sixian(四仙)Granules and Modified Prescriptions in Improving Thrombocytopenia after Chemotherapy

目的:探讨四仙颗粒及其拆方对化疗致小鼠血小板减少的改善作用及其作用机制。方法:尾静脉注射卡铂建立化疗致血小板减少小鼠模型,以四仙颗粒及其拆方(温肾健脾方、活血生血方)干预14 d。采集小鼠眼眶静脉血,动物血细胞分析仪测定血小板数量;流式细胞法检测小鼠股骨骨髓中CD34^(+)和CD41a^(+)CD61^(+)细胞百分率;HE染色观察小鼠股骨骨髓中巨核细胞数量;RT-qPCR法检测小鼠骨髓分化相关基因的表达。结果:与正常对照组比较,模型对照组小鼠血小板数量显著降低(P<0.01),小鼠骨髓细胞中CD34^(+)细胞比例显著减少(P<0.01),骨髓中巨核细胞数量明显减少(P<0.05),骨髓细胞中CD41a^(+)CD61^(+)细胞比例显著减少,骨髓细胞中血小板生成素受体(Mpl)、血小板生成素(Tpo) mRNA的表达明显下调(P<0.05或P<0.01),表明造模成功;与模型对照组比较,各给药组血小板数量明显升高(P<0.05或P<0.01),四仙方组CD34^(+)细胞百分率明显升高(P<0.05),各给药组CD41a^(+)CD61^(+)细胞百分率均呈现升高趋势,各给药组骨髓中巨核细胞数量均呈现升高趋势,全方作用优于拆方,各给药组Tpo、Mpl mRNA的表达明显上调(P<0.05或P<0.01),温肾健脾组作用优于其他组,各给药组Gata1 mRNA的表达明显下调(P<0.05或P<0.01),四仙方组和重组人白介素-11(rhIL-11)组均能下调Gata2 mRNA的表达(P<0.01)。结论:四仙颗粒及其拆方均能促进造血干/祖细胞和巨核细胞的增殖、成熟及血小板的生成,2个拆方组作用效果各有侧重,其作用机制可能与调控TPO/MPL信号通路有关。

Objective:To investigate the therapeutic effects and mechanism of Sixian(四仙)Granules and the modified prescriptions on chemotherapy-induced thrombocytopenia in mice.Methods:A chemotherapy-induced thrombocytopenia mouse model was established by tail vein injection of cisplatin,followed by a 14-day intervention with Sixian Granules and the modified prescriptions[Wenshen Jianpi(温肾健脾)Prescription and Huoxue Shengxue(活血升血)Prescription].Mouse orbital venous blood was collected for platelet count using an animal blood cell analyzer.Flow cytometry was used to determine the expression of CD34^(+)and CD41a^(+)CD61^(+)cells in the femoral bone marrow.Hematoxylin-eosin(HE)staining was performed to observe the number of megakaryocytes in the femoral bone marrow.RT-qPCR was conducted to measure the expression of differentiation-related genes in mouse bone marrow.Results:Compared with the normal control group,the model control group showed a significant decrease in platelet count in mice(P<0.01),a significant reduction in the proportion of CD34^(+)cells in bone marrow cells(P<0.01),a significant decrease in the number of megakaryocytes in bone marrow(P<0.05),a significant reduction in the proportion of CD41a^(+)CD61^(+)cells in bone marrow cells,and down-regulated Mpl and Tpo mRNA expression in bone marrow cells(P<0.05 or P<0.01),indicating the successful establishment of the model.Compared with the model control group,all treatment groups showed a significant increase in platelet count(P<0.05 or P<0.01).The sixian Prescription group exhibited an increase in CD34^(+)cell expression(P<0.05).All treatment groups showed an increasing trend in the expression of CD41a^(+)CD61^(+)cells in bone marrow,an increasing trend in the number of megakaryocytes in bone marrow,and up-regulated Tpo and Mpl mRNA expression(P<0.05 or P<0.01),and the Wenshen Jianpi Prescription group was superior to other groups.All treatment groups showed significant downregulation of Gata1 mRNA expression(P<0.05 or P<.01).Both the Sixian Prescription group and the recombinant human interleukin-11(rhIL-11)group downregulated the expression of Gata2 mRNA(P<0.01).Conclusion:Sixian Granules and the modified prescriptions can promote the proliferation,maturation,and generation of hematopoietic stem/progenitor cells and megakaryocytes,with different emphases in the two modified prescriptions.The mechanism of action may be related to the regulation of the TPO/MPL signaling pathway.