基于网络药理学探析五味子防治稽留流产的机制及验证

doi:10.12280/gjfckx.20230445

摘要/Abstract

摘要：

目的： 基于网络药理学方法分析五味子防治稽留流产的潜在作用机制，并通过实验进行验证。方法： 运用系统药理学方法查找并从中药系统药理学数据库和分析平台（Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform，TCMSP， https://old.tcmsp-e.com/tcmsp.php）筛选五味子的活性成分及靶点，查找稽留流产相关基因，确定五味子防治稽留流产的靶点。利用Cytoscape构建“药物成分-靶点”网络，筛选关键化合物。利用String建立蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络，并通过Cytoscape-CytoNCA拓扑分析筛选核心靶点。通过对靶基因进行基因本体（gene ontology，GO）和京都基因与基因组百科全书（kyoto encyclopedia of genes and genomes，KEGG）分析，以预测其可能的信号通路及机制。最后借助人绒毛膜外滋养层细胞系（HTR-8/SVneo）利用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，qRT-PCR）检测和蛋白质印迹（Western blotting）检测进行机制验证。结果： 从TCMSP数据库中鉴定了7种五味子活性成分。PPI网络表明雄激素受体（androgen receptor，AR）、雌激素受体（estrogen receptor，ER）、环氧合酶-2（cyclooxygenase-2，COX-2）、核受体辅激活因子2（nuclear receptor coactivator 2，NCOA2）和乙酰胆碱酯酶（acetylcholine esterase，ACHE）可能是五味子防治稽留流产的核心作用靶点。GO富集分析获得44个细胞生物学过程，KEGG途径富集分析获得2个相关信号通路，主要包括甲状腺激素信号通路和雌激素信号通路；在利用细胞模型验证机制过程中发现，二羟环氧苯并芘[benzo(a)pyren-7, 8-dihydrodiol-9, 10-epoxide，BPDE]染毒后的细胞中AR、ER和COX-2的蛋白质和（或）mRNA水平高于正常细胞，而先使用五味子乙素（Schisandrin B，Sch B）预处理后再染毒BPDE的细胞中AR、ER和COX-2的蛋白质和（或）mRNA水平均较未用Sch B预处理的细胞降低，而NCOA2和ACHE的蛋白质和（或）mRNA水平变化不明显。结论： 五味子可通过抗炎和调节AR、ER发挥防治稽留流产的作用，这一保护作用可能是通过雌激素信号通路来实现的。

关键词: 五味子, 流产, 稽留, 机制, 网络药理学

Abstract:

Objective: To explore the potential mechanism of Schisandrae chinensis fructus on the treatment of missed abortion based on a network pharmacology approach and verified experimentally. Methods: The system pharmacology approach was used to find and screen the active components and targets of Schisandrae chinensis from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://old.tcmsp-e.com/tcmsp.php), find the genes related to missed abortion, and identify the targets of Schisandra chinensis for prevention and treatment of missed abortion. The "drug component-target" network was constructed by using Cytoscape to screen key compounds. The protein-protein interaction (PPI) network was established by String, and the core targets were screened by the Cytoscape-CytoNCA topology analysis. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis were performed on the target genes to predict their possible signal pathways and explore their molecular mechanisms. Finally, the mechanism was validated with the help of human chorionic villous trophoblast cell line (HTR-8/SVneo) using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) detection and Western blotting assays. Results: Seven active compounds of Schisandra chinensis were identified from TCMSP database. Schisandrae chinensis has five potential targets for the prevention and treatment of missed abortion. The PPI network shows that androgen receptor (AR), estrogen receptor (ER), cyclooxygenase-2 (COX-2), nuclear receptor coactivator 2 (NCOA2) and acetylcholine esterase (ACHE) may be the core targets of Schisandrae chinensis for the prevention and treatment of missed abortion. Forty-four cell biological processes were obtained by GO enrichment analysis, and two related signal pathways were obtained by KEGG pathway enrichment analysis, mainly including thyroid hormone signal pathway and estrogen signal pathway. During the process of validating the mechanism using a cell model, it was found that the protein and/or mRNA levels of AR, ER, and COX-2 in cells treated with benzo(a)pyren-7, 8-dihydrodiol-9, 10-epoxide (BPDE) were higher than those of normal cells, while the protein and/or mRNA levels of AR, ER, and COX-2 were reduced in cells pretreated with Schisandrin B (Sch B) first and then tainted with BPDE compared with those not pretreated with Sch B. The NCOA2 and ACHE protein and/or mRNA levels did not change significantly. Conclusions: Schisandrae chinensis may exert its preventive and therapeutic effects on missed abortion through anti-inflammatory and hormonal receptor regulation, and this protective effect may be achieved through the estrogen signaling pathway.

Key words: Schisandrae chinensis fructus, Abortion, missed, Mechanism, Network pharmacology

梁婧, 乔林静, 侯海燕. 基于网络药理学探析五味子防治稽留流产的机制及验证[J]. 国际妇产科学杂志, 2024, 51(2): 167-175.

LIANG Jing, QIAO Lin-jing, HOU Hai-yan. Mechanism of Schisandrae Chinensis Fructus on Treating Missed Abortion Based on Network Pharmacology and Preliminary Verification[J]. Journal of International Obstetrics and Gynecology, 2024, 51(2): 167-175.

图/表 8

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基因名称	上游引物（5′~3′）	下游引物（5′~3′）	片段长度（bp）
COX-2	AGAGCAGAGAGATGAAATACC	AGGAGAACAGATGGGATTAC	266
AR	CTACATCAAGGAACTCGATCGT	CATGTGTGACTTGATTAGCAGG	159
ER	TACTGCATCAGATCCAAGGGAA	CCTCGGGGTAGTTGTACAC	128
NCOA2	GCTGCACAAACGAAGAGCAA	CATGTCCTGACCTGGGTTCC	210
ACHE	AGCGACTGATGCGATACTGG	CCATGGGTGAAGCCTGGG	250
GAPDH	CAGGAGGCATTGCTGATGAT	GAAGGCTGGGGCTCATTT	137

基因名称	上游引物（5′~3′）	下游引物（5′~3′）	片段长度（bp）
COX-2	AGAGCAGAGAGATGAAATACC	AGGAGAACAGATGGGATTAC	266
AR	CTACATCAAGGAACTCGATCGT	CATGTGTGACTTGATTAGCAGG	159
ER	TACTGCATCAGATCCAAGGGAA	CCTCGGGGTAGTTGTACAC	128
NCOA2	GCTGCACAAACGAAGAGCAA	CATGTCCTGACCTGGGTTCC	210
ACHE	AGCGACTGATGCGATACTGG	CCATGGGTGAAGCCTGGG	250
GAPDH	CAGGAGGCATTGCTGATGAT	GAAGGCTGGGGCTCATTT	137

成分代码	化合物名称	口服生物利用度（%）	药物相似性	靶点	靶点通用名
MOL004624	Longikaurin A	47.72	0.53	Muscarinic acetylcholine receptor M1	CHRM1
				Muscarinic acetylcholine receptor M2	CHRM2
				Trypsin-1	PRSS1
MOL005317	Deoxyharringtonine	39.27	0.81	Androgen receptor	AR
				Mineralocorticoid receptor	NR3C2
MOL008956	Angeloylgomisin O	31.97	0.85	Androgen receptor	AR
MOL008957	Schizandrer B	30.71	0.83	Prostaglandin G/H synthase 2	PTGS2*
MOL008968	Gomisin A	30.69	0.78	Acetylcholinesterase	ACHE
MOL008978	Gomisin R	34.84	0.86	Estrogen receptor 1	ESR1**
				Androgen receptor	AR
				Prostaglandin G/H synthase 2	PTGS2*
				Trypsin-1	PRSS1
				Nuclear receptor coactivator 2	NCOA2
MOL008992	Wuweizisu C	46.27	0.84	Acetylcholinesterase	ACHE
				Gamma-aminobutyric acid receptor subunit alpha-1	GABRA1

成分代码	化合物名称	口服生物利用度（%）	药物相似性	靶点	靶点通用名
MOL004624	Longikaurin A	47.72	0.53	Muscarinic acetylcholine receptor M1	CHRM1
				Muscarinic acetylcholine receptor M2	CHRM2
				Trypsin-1	PRSS1
MOL005317	Deoxyharringtonine	39.27	0.81	Androgen receptor	AR
				Mineralocorticoid receptor	NR3C2
MOL008956	Angeloylgomisin O	31.97	0.85	Androgen receptor	AR
MOL008957	Schizandrer B	30.71	0.83	Prostaglandin G/H synthase 2	PTGS2*
MOL008968	Gomisin A	30.69	0.78	Acetylcholinesterase	ACHE
MOL008978	Gomisin R	34.84	0.86	Estrogen receptor 1	ESR1**
				Androgen receptor	AR
				Prostaglandin G/H synthase 2	PTGS2*
				Trypsin-1	PRSS1
				Nuclear receptor coactivator 2	NCOA2
MOL008992	Wuweizisu C	46.27	0.84	Acetylcholinesterase	ACHE
				Gamma-aminobutyric acid receptor subunit alpha-1	GABRA1