补肾活血方对再生障碍性贫血合并铁负荷过载小鼠铁代谢的调节作用  

Iron metabolism regulation effect of Bushen Huoxue Formula in aplastic anemia mouse complicated with iron overload

作　　者：吴迪炯[1] 温晓文 刘文宾[1] 胡慧瑾[1] 沈一平[1] 李秋爽[1] 周郁鸿[1] 叶宝东[1] WU Di-jiong;WEN Xiao-wen;LIU Wen-bin;HU Hui-jin;SHEN Yi-ping;LI Qiu-shuang;ZHOU Yu-hong;YE Bao-dong(First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310006,China;Central Hospital of Jinhua Affiliated to Zhejiang University,Jinhua 321000,China)

机构地区：[1]浙江中医药大学附属第一医院,杭州310006 [2]浙江大学附属金华中心医院,金华321000

出　　处：《中华中医药杂志》2021年第7期3856-3862,共7页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.81774092);浙江省中医药科学研究基金项目(No.2016ZA069,No.2020ZB085,No.2020ZB095);国家中医临床研究基地科研专项(No.JDZX2015109)。

摘　　要：目的:观察补肾活血方对再生障碍性贫血(简称"再障")合并铁负荷过载(简称"铁过载")小鼠铁代谢的调节作用。方法:建立再障合并铁过载的小鼠模型,设置正常组、去铁胺(DFO)组、中药治疗组以及模型对照组,每组8只,完成DBA/2小鼠胸腺细胞输注后,进行相应药物治疗。检测治疗后第7、14、35天血常规水平变化;第35天,处死各组小鼠,收集血清、股骨及肝脏组织。采用酶标法检测各组血清铁水平、ELISA法检测铁蛋白表达;TUNEL染色检测骨髓细胞凋亡,免疫组化检测相关凋亡蛋白表达水平;Western Blot及RT-PCR法检测铁负荷(血清铁、血清铁蛋白)及铁代谢调控(Hepcidin、BMP-6、SMAD4、TfR2)的相关蛋白、mRNA表达差异。结果:中药及去铁胺治疗后,各组外周血常规(白细胞、血红蛋白、血小板)均有不同程度上升,尤以第35天最为显著。中药治疗能显著下降细胞凋亡水平(TUNEL),与其调控凋亡相关蛋白Bax、caspase-9表达相关,同时DFO能显著提高模型小鼠骨髓有核细胞计数。DFO及中药治疗35天后,两组骨髓铁沉积减少,以DFO组尤为明显。中药治疗能显著下调模型小鼠血清铁和铁蛋白水平,在第14、35天与正常组无显著性差异,并能上调肝脏Hepcidin及SMAD4表达,下调TfR2水平。结论:中药补肾活血方能有效调控再障的铁代谢过程,减少铁过载对骨髓细胞促凋亡作用,继而改善外周血象。Objective: To assess the effects of Bushen Huoxue Formula on iron metabolism in aplastic anemia(AA) complicated with iron overload. Methods: A mouse model of AA with iron overload complication was firstly established. Normal control group, deferoxamine-treated(DFO) group, Bushen Huoxue Formula-treated group(TCM-treated group) as well as model control group were set up, 8 in each group, after completing the infusion of thymocytes in DBA/2 mice, the corresponding drugs were given. The changes of blood routine level on the 7 th, 14 th and 35 th day after treatment were detected. On the 35 th day, mice in each group were killed, and serum, femur and liver tissues were collected. The serum iron level of each group was detected by enzyme labeling method and ELISA method. Detection of ferritin expression;Apoptosis of bone marrow cells was detected by TUNEL staining and expression level of apoptosis-related proteins was detected by immunohistochemistry. Western Blot and RT-PCR were used to detect the differences of iron load(serum iron, serum ferritin) and related proteins and mRNA expression of iron metabolism regulation(Hepcidin, BMP-6, SMAD4, TfR2). Results: Both TCM and DFO treatment can improve the peripheral hemogram(white cells, homoglobin, platelet), especially on day 35. TCM-treatment can decrease the apoptosis(TUNEL) in bone marrow, which might related to the Bax and caspase-9 expression, and DFO could increase the amounts of nucleated cells significantly. After 35-day treatment, the iron depositing in bone marrow were significantly alleviated in both treated group, especially the DFO group. TCM treatment can decrease the serum iron and ferritin, which were not comparable to the normal level at day 14 and 35, and can upregulated hepcidin and its regulators SMAD, and down-regulate TfR2 expression. Conclusion: Bushen Huoxue Formula protects hematopoiesis from immune and iron overload-induced apoptosis, exerting iron chelation effects in vivo.

关 键 词：再生障碍性贫血 铁负荷过载 补肾活血 中医 

分 类 号：R285.5[医药卫生—中药学]