左慈丸的化学成分及治疗围绝经期听力损失的作用机制研究：基于网络药理学与分子对接技术

doi:10.12114/j.issn.1007-9572.2023.0390

中国全科医学 ›› 2024, Vol. 27 ›› Issue (12): 1493-1503.DOI: 10.12114/j.issn.1007-9572.2023.0390

• 论著·中医·中西医结合研究 • 上一篇下一篇

左慈丸的化学成分及治疗围绝经期听力损失的作用机制研究：基于网络药理学与分子对接技术

刁翯¹^,^*(), 白文佩², 赵立东³

1．300199　天津市中心妇产科医院　南开大学附属妇产医院中医科　天津市人类发育与生殖调控重点实验室
2．100038　北京市，首都医科大学附属北京世纪坛医院妇产科
3．100853　北京市，中国人民解放军总医院耳鼻咽喉头颈外科医学部耳鼻喉科　国家耳鼻咽喉疾病临床医学研究中心聋病教育部重点实验室　聋病防治北京市重点实验室　中国人民解放军医学院

收稿日期:2023-06-13 修回日期:2023-09-11 出版日期:2024-04-20 发布日期:2024-01-23
通讯作者: 刁翯
作者贡献：刁翯负责设计文章思路与框架，绘制图表，动物实验，数据结果和可视化呈现，撰写论文，对文章整体负责；白文佩、赵立东负责论文终稿审校及质量控制。
基金资助:
国家自然科学基金青年基金资助项目(82004409); 天津市医学重点学科（专科）建设项目(TJYXZDXK-039A); 北京市自然科学基金资助项目(7202075)

Chemical Composition and Mechanism of Zuoci Pill in Treating Perimenopausal Hearing Loss Based on Network Pharmacology and Molecular Docking Technology

DIAO He¹^,^*(), BAI Wenpei², ZHAO Lidong³

1. Department of Traditional Chinese Medicine, Tianjin Central Hospital of Gynecology Obstetrics/Nankai University Maternity Hospital/Tianjin Key Laboratory of Human Development and Reproductive Regulation, Tianjin 300199, China
2. Department of Obstetrics and Gynecology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China
3. Academy of Otolaryngology-Head and Neck Surgery, Chinese PLA General Hospital/National Clinical Research Center for Otolaryngologic Diseases/State Key Lab of Hearing Science Affiliated to Chinese Ministry of Education/Beijing Key Lab of Hearing Impairment Prevention and Treatment, Chinese PLA Medical School, Beijing 100853, China

Received:2023-06-13 Revised:2023-09-11 Published:2024-04-20 Online:2024-01-23
Contact: DIAO He

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摘要/Abstract

摘要： 背景近年来，围绝经期听力损失（PMS-HL）症状得到普遍关注，但无针对性治疗。左慈丸治疗耳聋已有百年历史，但尚无该药治疗PMS-HL的相关研究。目的在网络药理学和分子对接技术的基础上，进一步通过动物实验验证，初步探讨左慈丸对PMS-HL的作用机制及治疗靶点。方法检索数据库时限均为建库至2023年2月。根据左慈丸的组方，通过TCMSP和Uniprot数据库挖掘该药物活性成分和作用靶点，根据GeneCards、OMIM、TTD、DrugBank、PharmGKB数据库获取PMS-HL的蛋白靶点，取其交集，筛选左慈丸治疗PMS-HL的潜在治疗靶点，构建"药物-活性成分-靶点"相互作用网络图。利用String数据库的蛋白相互作用分析功能筛选核心靶点。采用Metascape数据库对蛋白的功能及其通路进行富集分析。使用Autodock和Pymol对核心蛋白进行分子对接，确定核心靶点与左慈丸的关键活性成分之间的结合能力。于2022年9月—2023年1月建立绝经大鼠模型：假手术组（SHAM）组、去卵巢组（OVX）组、左慈丸（ZCW）组，并检测血清中关键蛋白白介素1β（IL-1β）水平，使用SPSS 26.0进行统计分析。结果左慈丸组方中化合物的活性成分90个，潜在蛋白靶点226个，PMS-HL相关靶点2 481个，左慈丸与PMS-HL交集靶点150个。基因本体论（GO）功能中获得生物过程共183个条目，分子功能103个条目，细胞成分103个条目；京都基因和基因组百科全书（KEGG）前三位分别是癌症通路、脂质与动脉粥样硬化病变通路和化学致癌的受体激活通路。分子对接显示，左慈丸治疗PMS-HL的主要活性成分有槲皮素、山柰酚、豆甾醇、β-谷甾醇、异鼠李素、薯蓣皂苷、四氢鸭脚木碱和海风藤酮；左慈丸的活性成分与核心靶蛋白：丝氨酸/苏氨酸蛋白激酶1（AKT1）、细胞肿瘤抗原p53（TP53）、白介素6（IL-6）、血管内皮生长因子（VEGFA）、胱天蛋白酶3（CASP3）、IL-1β、表皮生长因子受体（EGFR）、雌激素受体1（ESR1）结合能稳定。3组大鼠血清IL-1β水平比较，差异有统计学意义（F=11.73，P<0.001）。结论左慈丸中的槲皮素等90个活性成分通过作用于AKT1等226个潜在蛋白，实现调控组织细胞的抗氧化应激、调节血脂血糖代谢、抗肿瘤等途径，直接或间接地起到保护围绝经期听力功能的作用，IL-1β可能是其中发挥作用的关键靶蛋白。

关键词: 围绝经期, 听力损失, 左慈丸, 中药化学成分, 网络药理学, 分子对接

Abstract:

Background

In recent years, the symptoms of perimenopausal hearing loss (PMS-HL) have received widespread attention, but there is no targeted treatment. Zuoci pill has been used clinically for a hundred years, but there is no relevant research on the treatment of PMS-HL with this drug.

Objective

To explore the mechanism and therapeutic targets of Zuoci pill on PMS-HL through further validation of animal experiments based on the network pharmacology and molecular docking technology.

Methods

The active components and action targets of Zuoci pill were obtained through TCMSP and Uniprot databases, and the protein targets of PMS-HL were obtained through GeneCards, OMIM, TTD, DrugBank, and PharmGKB databases according to the composition of Zuoci pill from inception to February 2023, the intersection was taken to screen the potential therapeutic targets of Zuoci pill for the treatment of PMS-HL, and the "drug-active ingredient-target" interaction network diagram was constructed. The protein interaction analysis function of the String database was utilized to screen core targets. The Metascape database was used for enrichment analysis of the function of the proteins and pathways. Molecular docking of the core proteins was performed using Autodock and Pymol software to determine the binding capacity between key active ingredients and core targets. The menopausal animal model was established from September 2022 to January 2023, including Sham operation group (SHAM) group, ovariectomized (OVX) group, and Zuoci pill (ZCW) group, and serum level of the key protein interleukin 1β (IL-1β) was detected and statistically analyzed using SPSS 26.0.

Results

There were 90 active components, 226 potential protein targets, 2 481 PMS-HL related targets, and 150 Zuoci pill and PMS-HL intersection targets for the compounds in the Zuoci pill formula. A total of 183 items for biological process, 103 items for molecular function, and 103 items for cellular components were obtained in the Gene Ontology (GO) function. The top three Kyoto Encyclopedia of Genes and Genomes (KEGG) were cancer pathways, lipid and atherosclerotic lesion pathway, and receptor activation pathway for chemical carcinogenesis. Molecular docking showed that the main active ingredients of Zuoci pill in treating PMS-HL were quercetin, kaempferol, stigmasterol, β- Sitosterol, isorhamnetin, diosgenin, tetrahydrostilbene and kaempferone; the active components of Zuoci pill can bind to the core target proteins of serine/threonine kinase 1 (AKT1), cellular tumor antigen p53 (TP53), interleukin 6 (IL-6), vascular endothelial growth factor (VEGFA), cystatinase 3 (CASP3), IL-1β, epidermal growth factor receptor (EGFR), and estrogen receptor 1 (ESR1) stably. Comparison of serum IL-1β level among the three groups of rats showed statistically significant difference (F=11.73, P<0.001) .

Conclusion

The 90 active ingredients in Zuoci pill, such as quercetin, act on 226 potential proteins such as AKT1 to regulate the antioxidant stress of tissues and cells, metabolism of blood lipids and glucose, and anti-tumor pathway, directly or indirectly protect the perimenopausal hearing function, IL-1β may be one of the key target proteins.

Key words: Perimenopause, Hearing loss, Zuoci pill, Chemical compositions, Network pharmacology, Molecular docking

刁翯,白文佩,赵立东. 左慈丸的化学成分及治疗围绝经期听力损失的作用机制研究：基于网络药理学与分子对接技术[J]. 中国全科医学, 2024, 27(12): 1493-1503. DOI: 10.12114/j.issn.1007-9572.2023.0390.
DIAO He,BAI Wenpei,ZHAO Lidong. Chemical Composition and Mechanism of Zuoci Pill in Treating Perimenopausal Hearing Loss Based on Network Pharmacology and Molecular Docking Technology[J]. Chinese General Practice, 2024, 27(12): 1493-1503. DOI: 10.12114/j.issn.1007-9572.2023.0390.

图/表 13

表1 左慈丸中药共有的活性成分信息

Table 1 Active ingredients of Zuoci pill traditional Chinese medicine

Mol-ID	化合物	靶蛋白个数（个）	OB（%）	DL	药物来源	编号
MOL000098	槲皮素（quercetin）	149	46.43	0.28	丹皮、柴胡	A1
MOL000422	山柰酚（kaempferol）	61	41.88	0.24	丹皮、柴胡	A2
MOL000359	β-谷甾醇（β-sitosterol）	38	36.91	0.75	柴胡、丹皮、山茱萸、泽泻	B1
MOL000449	豆甾醇（Stigmasterol）	31	43.83	0.76	柴胡、熟地、山药、山茱萸	C1

图1 药物-活性成分-靶点的韦恩图注：PMS=围绝经期综合征，HL=听力损失，ZCW=左慈丸。

Figure 1 Wayne diagram of drug-active ingredient-target

表2 网络节点连接度值排名前8位的活性成分

Table 2 Top 8 active ingredients in terms of network node connectivity value

序号	编号	Mol-ID	化合物	所属中药	中心度值	紧密中心度值	节点连接度值
1	A1	MOL000098	槲皮素（quercetin）	柴胡、丹皮	0.644 182 212	0.549 597 855	236
2	A2	MOL000422	山柰酚（kaempferol）	柴胡、丹皮	0.122 348 153	0.405 940 594	86
3	C1	MOL000449	豆甾醇（stigmasterol）	柴胡、山药、山茱萸、熟地	0.081 989 992	0.378 927 911	60
4	B1	MOL000359	β-谷甾醇（beta-sitosterol）	泽泻、丹皮、山茱萸、熟地	0.057 065 242	0.377 532 228	21
5	CH4	MOL000354	异鼠李素（isorhamnetin）	柴胡	0.045 482 407	0.352 839 931	20
6	SY10	MOL000546	薯蓣皂苷（diosgenin）	山药	0.080 706 371	0.370 705 244	17
7	SZY10	MOL008457	四氢鸭脚木碱（tetrahydroalstonine）	山茱萸	0.034 819 519	0.359 019 264	16
8	SY4	MOL000322	海风藤酮（kadsurenone）	山药	0.028 794 402	0.350 427 350	13

图2 "左慈丸-活性成分-药物靶点"网络图注：三角形为药材，圆形为其所含活性成分，六边形为不同中药共有的活性成分（A1为槲皮素，A2为山柰酚，C1为豆甾醇，B1为β-谷甾醇），菱形为靶基因，其连接度值大小以面积大小表示，面积越大，连接度值越高；DP=丹皮，CH=柴胡，SZY=山茱萸，FL=茯苓，ZX=泽泻，SY=山药，SD=熟地。

Figure 2 Network of "Zuoci pill-active ingredient-drug target"

表3 PPI网络图节点连接度值排名前10位

Table 3 Top10 targets ranked by PPI network connectivity value

序号	靶基因	中心度值	紧密中心度值	节点连接度值
1	AKT1	0.059 715 47	0.780 104 71	107
2	TP53	0.030 890 95	0.756 345 18	103
3	IL-6	0.042 907 07	0.745 657 64	99
4	VEGFA	0.024 962 20	0.726 829 27	96
5	CASP3	0.025 602 74	0.726 829 27	94
6	IL-1β	0.034 180 63	0.719 806 76	92
7	EGFR	0.034 828 92	0.716 346 15	90
8	ESR1	0.040 838 92	0.712 918 66	90
9	MYC	0.033 866 46	0.702 830 19	89
10	HIF1A	0.009 612 23	0.680 365 30	84

图3 左慈丸治疗PMS-HL潜在作用靶点PPI网络图注：A为左慈丸治疗PMS-HL潜在作用靶点蛋白质-蛋白质相互作用（PPI）网络图，B为核心PPI网络图；图中的连线颜色深浅与相互关系得分大小相关，颜色越深表示两种基因之间互作关系越强；A图中节点面积大小与Mcode分数相关，面积越大，连通性越高。

Figure 3 PPI network of potential targets of Zuoci pill for PMS-HL

图4 左慈丸治疗PMS-HL潜在靶点GO功能富集分析

Figure 4 GO enrichment analysis of potential targets of Zuoci pill for PMS-HL

图5 左慈丸治疗PMS-HL潜在靶点KEGG功能富集分析

Figure 5 KEGG enrichment analysis of potential targets of Zuoci pill for PMS-HL

图6 "药物-活性成分-靶点-疾病-信号通路"图注：图中三角形代表中药，圆形代表中药活性成分，菱形为靶基因，正方形为KEGG信号通路；线的深浅表示两者间的关系程度，颜色越深，关系程度越高；图形的大小代表网络中的连通能力，面积越大，在网络中的连通性越高。

Figure 6 Map of "drug-active ingredients-targets-diseases-signaling pathways"

图7 左慈丸治疗PMS-HL的核心活性成分与靶蛋白分子结合能热图注：AKT1=丝氨酸/苏氨酸蛋白激酶1，TP53=细胞肿瘤抗原p53，IL-6=白介素6，VEGFA=血管内皮生长因子，CASP3=胱天蛋白酶3，IL-1β=白介素1β，EGFR=表皮生长因子受体，ESR1=雌激素受体1。

Figure 7 Heat map of binding energy of core active components and target protein of Zuoci pill in treating PMS-HL

图8 部分化合物与关键蛋白的分子对接图

Figure 8 Molecular docking diagram of key targets and active ingredients

图9 大鼠阴道脱落细胞巴氏染色（×40）注：SHAM组=假手术组，OVX组=去卵巢组，ZCW组=左慈丸组。

Figure 9 Vaginal smears with papanicolaou stain of rats

表4 3组大鼠血清IL-1β组间比较（±s，pg/mL）

Table 4 Serum IL-1β level comparison among three groups

组别	只数	IL-1β
SHAM组	20	29.58±5.66
OVX组	20	39.65±8.35^a
ZCW组	20	34.86±5.30^ab
F值			11.73
P值			<0.001

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左慈丸的化学成分及治疗围绝经期听力损失的作用机制研究：基于网络药理学与分子对接技术

Chemical Composition and Mechanism of Zuoci Pill in Treating Perimenopausal Hearing Loss Based on Network Pharmacology and Molecular Docking Technology

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