扶正化瘀方调节差异微小RNAs表达的抗肝纤维化的作用机制

王清兰; 陶艳艳; 吕靖; 刘平; 刘成海

doi:10.3969/j.issn.1001-5256.2016.03.022

扶正化瘀方调节差异微小RNAs表达的抗肝纤维化的作用机制

DOI: 10.3969/j.issn.1001-5256.2016.03.022

1. 上海中医药大学附属曙光医院肝病研究所
2. 上海中医药大学上海高校中医内科学E研究院

基金项目:

国家自然科学基金资助项目(30901943;81270053;81573810)；

详细信息

中图分类号: R285.5
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出版历程
- 出版日期: 2016-03-20

Antifibrotic mechanism of Fuzheng Huayu prescription by regulation of the differential expression of microRNAs in mouse liver

Institute of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine

Research funding:

摘要

摘要: 目的筛选出扶正化瘀方（FZHY）调控的差异微小RNAs（miRNAs）,并对这些差异miRNAs进行生物功能分析,部分揭示FZHY抗肝纤维化的作用机制。方法 CCl4皮下注射制备小鼠肝纤维化模型,分为正常组、模型组及FZHY组;自造模前3 d开始给药,1次/d,共8周。天狼星红染色及羟脯氨酸（Hyp）含量测定观察肝组织胶原沉积。应用miRNAs芯片检测各组小鼠肝脏miRNAs表达谱。基于miRNAs表达谱筛选出正常组、FZHY组与模型组具有相同变化趋势的差异miRNAs,即FZHY调控的miRNAs,并以qRT-PCR进行验证。采用Target Scan及PITA数据库对miRNAs靶基因进行预测,并采用DAVID数据库对这些预测的靶基因进行功能分析及信号通路分析,发现其调控的显著性功能及信号通路。多组间比较采用方差分析,miRNA差异基因筛选采用随机方差模型的t检验。结果 FZHY可明显降低纤维化肝组织Hyp含量,减轻胶原沉积。miRNAs芯片分析共发现3个FZHY调控的miRNAs,分别为:mmu-miR-322、mmu-miR-342-3p和mmu-miR-296-5p。信号通路...
- 肝硬化 /
- 扶正化瘀方 /
- 微RNAs /
- 寡核苷酸序列分析
Abstract: Objective To identify microRNAs( miRNAs) regulated by Fuzheng Huayu prescription( FZHY) and analyze their biological functions,and to partially reveal the antifibrotic mechanism of FZHY in liver fibrosis. Methods The mouse model of liver fibrosis was developed by subcutaneous injection of CCl4. The mice were divided into normal group,model group,and FZHY group. The mice received FZHY once a day at 3 days before model establishment,and the treatment lasted for 8 weeks. Collagen deposition in liver tissue was evaluated by Sirius Red staining and determination of hydroxyproline( Hyp) content. The expression profile of miRNAs in mouse liver was determined by miRNA microarray. According to the expression profile,miRNAs regulated by FZHY were identified by looking for miRNAs showing the same trends in the normal group and the FZHY group compared with the model group. The results were confirmed by quantitative real- time PCR. The miRNA targets were predicted using Target Scan program and PITA database. The DAVID database was used to analyze and identify the substantial functions and signaling pathways of those miRNA targets. Comparison between multiple groups was made by analysis of variance. Results FZHY substantially reduced Hyp content and inhibited collagen deposition in the fibrotic liver tissue. The miRNA microarray identified mmu- miR- 322,mmu- miR- 342- 3p,and mmu- miR- 296- 5p as miRNAs regulated by FZHY. According to the analysis of signaling pathway,the three miRNAs might regulate 32 signaling pathways,including chemokine signaling pathway,focal adhesion,MAPK signaling pathway,regulation of actin cytoskeleton,gap junction,ECM- receptor interaction,Wnt signaling pathway,and Jak- STAT signaling pathway,which were closely related to liver fibrosis; the functional enrichment analysis predicted 32 substantial functions of the three miRNAs,including GTPase regulator,cell junction,regulation of apoptosis,regulation of Ras signal transduction,and small GTPase regulator,which were closely related to liver fibrosis. Conclusion The antifibrotic effect of FZHY in the liver might be achieved by down- regulation of the expression of miR- 322,miR- 342- 3p,and miR- 296- 5p,which regulates their specific functions,such as MAPK signaling pathway,Wnt signaling pathway,regulation of apoptosis,and regulation of Ras signal transduction. The conclusion deserves further investigation.
- liver cirrhosis /
- Fuzheng Huayu Recipe /
- MicroRNAs /
- oligonucleotide array sequence analysis

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