复方参鹿颗粒通过WNT信号通路治疗再生障碍性贫血的分子机制研究  

Molecular mechanism of Compound Shenlu Granule in the treatment of aplastic anemia through Wnt signaling pathway

作　　者：封舟[1] 胡晓莹[1] 朱小勤[1] 瞿玮颖 陆颖佳[1] 赵琳[1] 黄中迪[1] 陈珮[1] FENG Zhou;HU Xiaoying;ZHU Xiaoqin;QU Weiying;LU Yingjia;ZHAO Lin;HUANG Zhongdi;CHEN Pei(Department of Hematology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200021,China)

机构地区：[1]上海中医药大学附属曙光医院血液科,上海200021

出　　处：《上海中医药杂志》2020年第S01期193-198,共6页Shanghai Journal of Traditional Chinese Medicine

基　　金：上海市卫健委中医专项培育项目(3049).

摘　　要：目的通过再生障碍性贫血(AA)大鼠模型,研究复方参鹿颗粒通过Wnt/β-catenin-TRF1影响端粒酶的活性和端粒长度的机制。方法(1)体外研究:将SD大鼠100只随机分为4组,分别灌胃0、0.5 g/kg×1、0.5 g/kg×2、0.5 g/kg×3不同给药量的复方参鹿颗粒,以制备不同浓度的复方参鹿颗粒药物血清。分离大鼠骨髓的造血干细胞(HSC),予药物血清干预后检测HSC细胞活力、HSC细胞增殖情况及对端粒酶活性的影响。(2)体内实验:100只乳鼠构建AA动物模型,随机分为4组,分别为空白组、复方参鹿颗粒灌服组、Wnt/β-catenin抑制剂处理组、Wnt/β-catenin抑制剂处理+复方参鹿颗粒灌服组,各组分别予相应的药物干预,每日1次。给药4周后颈动脉采血,用CCK8法检测HSC细胞的活力,明确复方参鹿颗粒对HSC细胞活力的影响;流式检测各组细胞的凋亡和周期的影响,分子生物学技术检测HSC细胞的周期,Wnt信号通路及端粒结合蛋白的相关基因的mRNA和蛋白水平的变化,Q-Fish检测细胞的端粒的长度,TRAR法检测端粒酶的活性。结果复方参鹿颗粒处理组可以抑制HSC的凋亡,增加再生障碍性贫血动物模型的HSC的细胞周期,促进TRF1、TERT、Wnt5a的表达。结论复方参鹿颗粒可激活Wnt/β-catenin通路促进TRF1的转录,促进端粒酶的活性和端粒的长度,促进HSC的增殖。Objective To study the mechanisms of Compound Shenlu Granule on telomerase activity and telomere length through Wnt/β-catenin-TRF1 based on aplastic anemia(AA)rat model.Methods①Study in vitro:One hundred SD rats were randomly divided into four groups,and were treated with Compound Shenlu Granule at different doses(0 g/kg,0.5 g/kg once a day,0.5 g/kg twice a day,0.5 g/kg for 3 times a day),so as to prepare the medicated serum of Compound Shenlu Granule at different concentrations.The hematopoietic stem cells(HSC)were isolated from rat bone marrow,and treated with medicated serum.The activity of HSC,the proliferation of HSC and the effect on telomerase activity were detected.②Study in vivo:One hundred suckling mice were used to establish AA animal model,and were randomly divided into the blank group,Compound Shenlu Granule group,Wnt/β-catenin inhibitor group,Wnt/β-catenin inhibitor+Compound Shenlu Granule group.Each group was treated with the corresponding drugs,once a day.After administration for 4 weeks,blood was collected from carotid artery.The activity of HSC was detected by CCK8 assay,and the effect of Compound Shenlu Granule on HSC activity was clarified.The effect on cell apoptosis and cell cycle in each group was detected by flow cytometry.The changes of HSC cell cycle,Wnt signaling pathway and mRNA and protein levels of telomere binding protein related genes were detected by molecular biology.The length of telomere was detected by Q-Fish and the activity of telomerase was detected by TRAR.Results Compound Shenlu Granule treatment group could inhibit the HSC apoptosis,increase the cell cycle of HSC in aplastic anemia model animal,and promote the expressions of TRF1,TERT and Wnt5 a.Conclusion Compound Shenlu Granule can activate Wnt/β-catenin pathway,improve TRF1 transcription,telomerase activity and telomere length,and promote HSC proliferation.

关 键 词：复方参鹿颗粒 再生障碍性贫血 WNT通路 端粒酶 造血干细胞 

分 类 号：R285.5[医药卫生—中药学]