Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (8): 1256-1263.doi: 10.3969/j.issn.2095-4344.3057
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Li Zhongfeng1, Chen Minghai1, Fan Yinuo1, Wei Qiushi2, He Wei2, Chen Zhenqiu2
Received:
2020-03-27
Revised:
2020-04-01
Accepted:
2020-05-09
Online:
2021-03-18
Published:
2020-12-14
Contact:
Chen Zhenqiu, MD, Professor, Master’s supervisor, The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, Guangdong Province, China
About author:
Li Zhongfeng, Master candidate, The First Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, Guangdong Province, China
Supported by:
CLC Number:
Li Zhongfeng, Chen Minghai, Fan Yinuo, Wei Qiushi, He Wei, Chen Zhenqiu. Mechanism of Yougui Yin for steroid-induced femoral head necrosis based on network pharmacology[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(8): 1256-1263.
2.2 有效成分靶基因和激素性股骨头坏死靶基因的交集 借助Genecard和OMIM两个数据库,输入关键词“femur head steroid necrosis”,搜索疾病激素性股骨头坏死相关的靶基因,分别得到1 187个和209个结果(存在重复的靶基因)。借助R语言将激素性股骨头坏死靶基因及所有中药有效成分靶基因筛选去重,再取所有有效成分靶基因和激素性股骨头坏死靶基因的交集,同时绘制韦恩图(图2),得到激素性股骨头坏死靶基因共1 376个,所有中药有效成分靶基因共185个,激素性股骨头坏死和中药有效成分的交集靶基因共102个(其中关于制附子的有效成分靶基因7个,熟地黄的有效成分靶基因10个,山茱萸的有效成分靶基因23个,炒山药的有效成分靶基因27个,枸杞子的有效成分靶基因100个),如NCOA2、PTGS2、PTGS1、NCOA1、RXRA、ESR1、JUN、AKT1、MAPK1、RELA、IL6、EGFR等。推测右归饮的有效成分主要通过这些交集靶基因来治疗激素性股骨头坏死。"
2.3 中药调控网络图的构建 通过得出的有效成分和交集靶基因,借助perl语言和Cytoscape软件绘制中药调控网络图(图3)。右归饮治疗激素性股骨头坏死相关的中药有效成分共43个(其中关于制附子的有效成分6个,熟地黄的有效成分2个,山茱萸的有效成分13个,炒山药的有效成分10个,枸杞子的有效成分21个)。对应交集靶基因数目排名前十的有效成分及其来源:quercetin(枸杞子)、glycitein(枸杞子)、beta-sitosterol (枸杞子、山茱萸)、Stigmasterol(枸杞子、炒山药、山茱萸、熟地黄)、Tetrahydroalstonine(山茱萸)、Diosgenin(炒山药)、Kadsurenone(炒山药)、AIDS180907(炒山药)、hancinone C(炒山药)、Atropine(枸杞子);其中最多者quercetin对应84个靶基因,最少者Atropine对应6个靶基因。而右归饮中补火助阳的君药制附子有效成分中最多对应4个靶基因,排在第11位。交集靶基因中连接中药有效成分的数量排名前10的依次为NCOA2、PTGS2、PTGS1、ADRB2、CHRM3、NCOA1、NOS2、RXRA、OPRM1、AR、ESR1、SLC6A4(最高者对应29个有效成分,最少者为最后4个均对应4个有效成分),其中关于制附子的有效成分靶基因6个,枸杞子的有效成分靶基因12个,炒山药的有效成分靶基因12个,山茱萸的有效成分靶基因11个,熟地黄的有效成分靶基因7个。推测右归饮主要通过以上的交集靶基因和有效成分来发挥激素性股骨头坏死的治疗作用,应予以重视和进一步研究。"
2.4 蛋白互作网络(PPI)的构建 借助STRING在线软件,输入右归饮-激素性股骨头坏死交集靶基因,生物种类选择Homo sapiens,即可绘制出PPI,但发现网络较复杂,且存在少量游离无连接节点,遂将默认设置的蛋白相互作用综合分数≥0.4过滤数值提升至≥0.9,同时去除游离无连接的节点,得到较为满意的蛋白互作网络图(图4),其中共有95个节点,374个边(节点代表靶基因及蛋白,边代表蛋白互作关系)。边分别代表着不同的证据支持,通过数据库、实验测定已证明的蛋白互作关系(基因相邻证据、基因融合证据、基因共现证据、文本挖掘证据、共表达证据、蛋白同源性证据)。再借助R语言统计各节点的边的数量,并将排名前30的靶基因绘制成柱形图(图5),这些与其他节点连接较多的靶基因将会是重点研究对象。"
可见右归饮治疗激素性股骨头坏死是通过影响多种生物学功能和参与多种通路来协调发挥作用的。通过比较文章中产生的4种靶基因排名情况(交集靶基因中连接中药有效成分的数量排名前10者、PPI蛋白互作关系排名前30者、GO富集分析和KEGG通路分析排名前20者对应的靶基因),综合考虑ESR1、AKT1、NCOA2、NCOA1、PTGS2、PTGS1、RXRA、JUN、MAPK1、RELA、IL6、EGFR等具有较为重要的作用,例如ESR1对应的生物学功能是类固醇激素受体、核激素受体结合、激素受体结合等,对应的通路是内分泌抵抗(图10)等,图中深色位点代表右归饮有效成分的重要靶基因在内分泌抵抗通路中的作用位点,可见作用位点较多,这些靶基因在此通路中起着重要作用,应予以重视和进一步研究。当然,其他靶基因、生物学功能和通路也发挥着不可忽视的作用。"
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