Exploring the molecular mechanism of <i>Citri Grandis</i> Exocarpium in relieving cough and resolving phlegm based on network pharmacology and molecular docking

CHEN Fangjian; ZHANG Yumei; WU Wenyan; ZHOU Yuqin; ZHANG Yang

doi:10.12206/j.issn.2097-2024.202604056

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Article Navigation > Journal of Pharmaceutical Practice and Service > 2026 > Accepted Manuscript

CHEN Fangjian, ZHANG Yumei, WU Wenyan, ZHOU Yuqin, ZHANG Yang. Exploring the molecular mechanism of Citri Grandis Exocarpium in relieving cough and resolving phlegm based on network pharmacology and molecular docking[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202604056

Citation:

CHEN Fangjian, ZHANG Yumei, WU Wenyan, ZHOU Yuqin, ZHANG Yang. Exploring the molecular mechanism of Citri Grandis Exocarpium in relieving cough and resolving phlegm based on network pharmacology and molecular docking[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202604056

Exploring the molecular mechanism of Citri Grandis Exocarpium in relieving cough and resolving phlegm based on network pharmacology and molecular docking

doi: 10.12206/j.issn.2097-2024.202604056

1.
Shanghai Huangpu District Geriatric Care Hospital, Shanghai 200010, China
2.
Department of Pharmacy, The First Affiliated Hospital of Naval Medical University, Shanghai 200433, China
3.
Shanghai Huangpu District Yuyuan Community Health Service Center, Shanghai 200010, China

Received Date: 2026-04-22
Accepted Date: 2026-06-23
Rev Recd Date: 2026-05-19

Abstract

Objective To analyze the molecular mechanism of Citri Grandis Exocarpium （Huajuhong） in relieving cough and resolving phlegm based on network pharmacology and molecular docking technology. Methods The active ingredients of Citri Grandis Exocarpium were retrieved from the TCMSP. The targets of these active ingredients were predicted via the PubChem database and SwissTargetPrediction platform. Targets associated with cough and phlegm syndrome were obtained from the GeneCards and DisGeNET databases, and the intersection targets were acquired using a Venn diagram. The PPI network of the targets was constructed with the STRING 12.0 database. Core targets were screened out for GO functional enrichment analysis and KEGG signaling pathway enrichment analysis. The “active ingredient-core target-pathway” network was established by Cytoscape 3.10.4, and molecular docking validation was performed on the CB-DUCK2 online platform. Human bronchial epithelial BEAS-2B cells were cultured in vitro. Cell viability was detected by the CCK-8 assay, and the mRNA expression levels of AKT1, BCL2 and EGFR were determined by qPCR. Results 10 active ingredients of Citri Grandis Exocarpium were screened, with 390 corresponding action targets and 214 intersection targets related to cough and phlegm syndrome. 10 core targets were identified, including GAPDH, AKT1, EGFR, BCL2, CASP3, SRC, ESR1, PTGS2, MMP-9, and CCND1. GO analysis and KEGG showed that these targets were involved in biological processes such as negativing regulation of intrinsic apoptotic signaling pathway, responding to UV-A and exogenous stimuli; were localized in cellular components including protein-containing complexes, nuclear envelope and cytosol; and participated in molecular functions such as enzyme binding, protein kinase activity and identical protein binding. The targets mainly regulated signaling pathways including the Estrogen signaling pathway, HIF-1 signaling pathway, and TNF signaling pathway. Molecular docking results indicated that the active ingredients of Citri Grandis Exocarpium had favorable binding affinity with the core targets GAPDH, AKT1, EGFR and BCL2. In vitro cell experimental results showed that Citri Grandis Exocarpium extract significantly increased cell viability （P<0.05）, downregulated the expression level of AKT1, upregulated the expression level of BCL2 （P<0.05）, and decreased the expression level of EGFR. Conclusion Citri Grandis Exocarpium exerted cough-relieving and phlegm-resolving effects possibly through a multi-ingredient, multi-target and multi-pathway mode.
- Citri Grandis Exocarpium,
- antitussive and expectorant effects,
- network pharmacology,
- molecular docking,
- molecular mechanism

References

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咳嗽是对呼吸道刺激的基本生理过程，有助于清除呼吸道的刺激物和多余的黏液，但频繁的咳嗽严重影响患者日常生活，对患者健康造成危害^[1]。目前临床常用的对症治疗药物主要有中枢镇咳药、外周镇咳药和祛痰药，虽然有较强镇咳作用，但具有成瘾性，只能起到短暂缓解症状的作用，大部分患者症状反复，严重影响生活质量，还可能进一步发展为慢性阻塞性肺疾病等^[2]。因此，探索安全有效的药物对咳嗽患者的治疗具有重要意义。中医学认为肺失宣降，肺气上逆而作咳嗽，中医中药对咳嗽辨证论治，通过多环节、多靶点发挥效应治疗咳嗽，临床常以疏风、宣肺、降浊化痰为主进行治疗，同时标本兼治，对肝脾肾进行调治^[2]。

化橘红（Citrus grandis ‘Tomentosa’）是芸香科植物化州柚的未成熟或近成熟的干燥外层果皮，为我国十大广药之一，化橘红味苦性温，归肺、脾经，具有理气宽中，燥湿化痰作用，常用于治疗气喘、咳嗽痰多、呕恶痞闷^[3-4]。已有研究显示，化橘红含有类黄酮、挥发油、香豆素等多种具有抗炎、抗氧化等多重作用的化学成分，具有止咳、祛痰等多种药理作用^[5-7]。然而化橘红止咳化痰具体分子机制尚未阐明。网络药理学通过构建“多成分-多靶点-多通路”网络，探索中药复方协同作用、完善中药单体多靶点协同机制，其整体性的分析特点与中药多成分、多靶点的作用机制相契合，是分析中药效果的重要方式^[8]。为进一步研究化橘红止咳化痰分子机制，本研究利用网络药理学和分子对接技术，预测化橘红止咳化痰的活性成分及潜在作用机制，并进行了细胞实验验证，为其临床应用与开发提供理论依据。

1. 材料

1.1. 细胞

人支气管上皮BEAS-2B细胞（批号26020C03），购自苏州海星生物科技有限公司。

1.2. 药品与试剂

化橘红饮片（批号251000201）、AMG9810（批号F28IB208092）、亚硫酸氢钠（批号K18WA7001）、无水亚硫酸钠（批号L822BA10005）、RNA Easy Fast动物组织/细胞总RNA提取试剂盒（批号B0109A）、UniPeak U+ One Step RT-qPCR SYBR Green Kit（批号7E0321D5）、CCK8试剂（批号2894240801）。

1.3. 仪器

CFX Opus 96型qPCR仪（美国BIO-RAD公司）、5425R型低温离心机（德国艾本德公司）、311型培养箱（美国赛默飞世尔科技公司）。

2. 方法

2.1. 网络药理学分析

2.1.1. 化橘红活性成分及靶点筛选

在中药系统药理学平台（TCMSP，https://tcmsp-e.com/index.php），以“化橘红”为关键词进行检索，以口服生物利用度（oral bioavailability，OB）≥30%且类药性（drug-likeness，DL）≥0.18为筛选标准，初步筛选出具有化橘红的活性成分和靶点。通过PubChem数据库（https://pubchem.ncbi.nlm.nih.gov）检索上述活性成分结构的Canonical SMILES序列及2D结构，并将Canonical SMILES序列输入SwissTargetPrediction平台（http://www.swisstargetprediction.ch），进行化橘红活性成分靶点预测，经合并冗余及去重处理后，得到化橘红的最终靶点蛋白集合。

2.1.2. 疾病靶点预测

以“咳嗽”（cough）、“痰证”（phlegm）为检索词，分别从GeneCards数据库（https://www.genecards.org）及DisGeNET数据库（https://www.disgenet.org）中挖掘与疾病相关的靶点基因。获取的靶点蛋白中，Score值越高则代表该靶点与疾病联系密切，设定Score值大于中位数的目标靶点为咳嗽、痰症相关的潜在靶点，对两数据库获取的靶点进行整合与去重后，得到咳嗽、痰症相关靶点。

2.1.3. 化橘红与咳嗽、痰证交集靶点

为探究化橘红相关靶点与咳嗽、痰症靶点间的相互作用，利用微生信在线平台（https://www.bioinformatics.com.cn）韦恩图^[9]可视化分析化橘红活性成分靶点与咳嗽、痰证相关靶点的重叠关系，二者的交集靶点即为化橘红干预疾病的潜在靶点，并绘制韦恩图。

2.1.4. 交集靶点蛋白质-蛋白质相互作用（PPI）网络构建及核心靶点筛选

将交集靶点导入STRING 12.0数据库（https://string-db.org/），设置物种为“Homo sapiens”并采用默认参数（中等置信度阈值0.4），构建共有靶点蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络图，保存为“tsv”文件。随后将网络数据导入Cytoscape 3.10.4软件，利用其内置CytoHubba插件进行拓扑学分析，基于节点度值（Degree）筛选排名前10的核心靶点基因。网络图中节点颜色深浅反映靶点重要性程度，颜色越深表明该靶点在网络中具有更高的连接丰富性。

2.1.5. 核心靶点GO功能和KEGG通路富集分析

将筛选得到的核心靶点上传至DAVID数据库（https:/http://david.ncifcrf.gov），并设定为“Homo sapines”属性，开展基因本体论（gene ontology，GO）功能富集分析和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes, KEGG）信号通路富集分析（P<0.05），解析核心靶点涉及的生物功能及信号通路。

2.1.6. 构建化橘红对咳嗽、痰证“活性成分-核心靶点-通路”网络

运用Cytoscape 3.10.4软件整合活性成分与10个核心靶点关联关系及核心靶点-信号通路映射关系，构建多层级互作网络模型，直观展示化橘红止咳化痰的物质基础与作用途径。

2.1.7. 分子对接验证

从RCSB蛋白数据库（https://www.rcsb.org）获取核心靶点蛋白排序居前4位的X射线晶体结构。利用Yang Cao Lab（http://cao.labshare.cn/clab/index.html）CB-Dock2在线平台进行分子对接，按照平台的操作流程对活性成分与前4位的核心靶点蛋白进行分子对接模拟^[10]。

2.2. 细胞实验

2.2.1. 化橘红提取物的制备

取适量化橘红中药饮片加水浸泡1 h，然后使用文火煎煮2次，每次1 h；合并两次的水煎液，用双层纱布过滤；浓缩滤液至终浓度为1 g/ml。为保证药液无菌，将浓缩后的滤液密封后置于烧杯中，烧杯加水，置于微波炉中煮沸15 min灭菌。药液4℃保存待用。

2.2.2. BEAS-2B细胞模型的建立

BEAS-2B细胞以每孔3×10⁴个的密度接种于96孔板，每孔注入100 μl培养液，于37℃、5 %细胞培养箱孵育过夜。分别加入100 μl不同浓度（300、100、33.3、11.1、3.7、1.2、0.4、0.14 μmol/L）SO₂衍生物。培养24 h后取出细胞培养板，每孔加入10 μl的CCK-8溶液，在细胞培养箱中放置1 h，用酶标仪检测450 nm波长下吸光度（A）。

2.2.3. AMG9810、化橘红提取物干预浓度筛选

BEAS-2B细胞以每孔3×10⁴个的密度接种于96孔板，孵育过夜。分别加入不同浓度化橘红提取物（10、1 mg/ml和100、50、20、10、5、2.5、1、0.5 μg/ml）、AMG9810（100、80、60、40、20、15、10、7.5 μmol/L）。培养24 h后取出细胞培养板，CCK8法测定细胞存活率。

2.2.4. CCK8法检测BEAS-2B细胞存活率

BEAS-2B细胞以每孔1.5×10⁴个的密度接种于96孔板，每孔注入100 μl培养液，于37℃、5 %细胞培养箱孵育过夜。分别加入4 μmol/L SO₂衍生物、不同浓度化橘红提取物（10 mg/ml和100、1 μg/ml）、AMG9810（100、50、5 μmol/L），培养24 h后取出细胞培养板，CCK8法测定细胞存活率。

2.2.5. 实验分组

BEAS-2B细胞培养24 h后进行分组。实验分为：对照组、模型组（含4 μmol/L SO₂衍生物）、阳性组（AMG9810 100 μmol/L和4 μmol/L SO₂衍生物）、化橘红提取物组（10 mg/ml化橘红提取物和4 μmol/LSO₂衍生物）。

2.2.6. qPCR法检测BEAS-2B细胞BCL2、AKT1、EGFR基因表达

BEAS-2B细胞以每孔1×10⁶个的密度接种于6孔板，按照“2.2.5”项下方法分组、给药，培养24 h后按照总RNA提取试剂说明书要求提取总RNA，测定浓度，去除DNA。对各样本进行qPCR扩增反应。扩增条件为：50℃，5 min，95℃×30 s，95℃×10 s，60℃×30 s，共40个循环。使用2^−ΔΔCt方法计算各基因相对表达量。引物序列见表1。

基因名称	上游引物	下游引物
BCL2	TTTCGGTGACTTCC GCATCA	CGGTCTCCTAAAAGCAGGCA
AKT1	AGAGCAAACGGGG CCATC	CCAGGTCTTGATGTACTCCCC
EGFR	GCCCAGACCGGAC GACAG	CCAACTGCGTGAGCTTGTTAC
β-actin	ACCTAACTTGCGC AGAAAACA	TTGTGAACTTTGGGGGATGCT

2.2.7. 统计学分析

通过Graphpad Prism 10.2.0软件进行处理，数据以（x̅±s）表示，组间比较采用单因素方差分析。P<0.05表示差异具有统计学意义。

4. 讨论

咳嗽作为机体重要的防御反射，其发生机制复杂且涉及多系统协同作用。中医理论认为，咳嗽的病因源于外邪侵袭肺系，导致肺气宣降失常，进而引发肺气上逆；虽病位主要责之于肺，但其病理过程亦与肝、脾、肾等脏腑功能失调密切相关^[11]。化橘红作为我国四大南药之一，兼具抗炎、抗氧化及抑制细胞凋亡等多重药理活性，在肺病领域展现出显著疗效。临床实践证实，以化橘红为核心成分的橘红痰咳液对轻、中、重度咳嗽伴咯痰证状均具有良好治疗效果，且安全性良好，未见明显不良反应报道^[12]。

本研究采用网络药理学方法系统解析化橘红止咳化痰的作用机制。通过构建“活性成分-靶点-通路”网络模型，共筛选出10种具有潜在药效的活性成分。具体而言，柚皮素（naringenin）可通过抑制PI3K/Akt/PTEN信号通路及NF-κB介导的炎症反应，发挥抗炎与抗氧化双重作用^[13-14]。枸橘苷（poncirin）通过调节Th17/Treg细胞平衡，抑制NF-κB通路激活，有效减轻小鼠结肠炎症反应^[15]。异橙黄酮（isosinensetin）与5,7,4'-三甲氧基黄酮（5,7,4'-Trimethylapigenin）同属黄酮类化合物，二者均可通过激活抗氧化酶系、抑制促炎细胞因子释放，展现抗菌、抗病毒及气道保护作用^[16]。川陈皮素（nobiletin）具有抗炎、抗氧化作用，可以通过抑制NF-κB信号通路发挥抗炎活性，其抗氧化活性可抑制氧化应激反应，对氟尿嘧啶诱导的肺损伤具有保护作用^[17]。β-谷甾醇（β-sitosterol）通过激活LXRs/ABCA1通路发挥抗炎效果，缓解LPS诱导的急性肺损伤^[18]。Kan等研究显示，β-谷甾醇通过抑制FGFR1和EGFR表达，下调PI3K/AKT/mTOR/CD1信号通路，从而抑制肿瘤细胞增殖、诱导凋亡和减少迁移，表现出抗癌潜力^[19]。此外，化橘红活性成分柚皮素、β-谷甾醇可以通过激活糖皮质激素受体，进一步发挥抗炎作用^[20-21]。

基于Cytoscape软件的拓扑学分析进一步揭示，GAPDH、AKT1、EGFR和BCL2为化橘红止咳化痰的核心作用靶点。其中，GADPH是糖酵解的关键酶，在细胞中参与氧化应激等多种作用，最新研究显示，GADPH过表达促进肿瘤细胞生长和转移^[22]。AKT1广泛分布于肺组织，参与代谢、转录及翻译等关键细胞功能，并在炎症性疾病中发挥重要调控作用。研究显示，抑制AKT1表达可显著减少博来霉素诱导的小鼠肺泡巨噬细胞促炎细胞因子生成，进而阻断纤连蛋白沉积，对延缓肺纤维化进程具有积极意义^[23]。EGFR属于酪氨酸激酶受体家族成员，可以调节细胞增殖等功能，参与MAPK/ERK、PI3K/Akt等信号通路，其异常高表达与特发性肺纤维化关系密切^[24]。BCL2蛋白家族是调节线粒体凋亡途径的关键蛋白，可促进细胞存活，在间质性肺病炎症、凋亡和纤维化的发病机制中起着关键作用，BCL2在不同细胞类型中表达不同，在成纤维细胞中表达上升，抑制其表达有助于改善肺纤维化^[25]。经LPS诱导后，在小鼠单核巨噬细胞炎症中观察到BCL2表达水平降低^[26]。分子对接结果显示，化橘红活性成分与GAPDH、AKT1、EGFR和BCL2结合活性较佳，表示化橘红活性成分与核心蛋白结合较稳定，可以发挥止咳化痰作用。细胞实验表明，化橘红提取物可以提高BEAS-2B细胞存活率，降低AKT1表达、升高BCL2表达，表明化橘红可以缓解细胞炎症反应，促进细胞存活，对改善炎症反应有一定作用。

GO功能富集与KEGG通路分析表明，化橘红通过调控雌激素信号通路、HIF-1信号通路和TNF信号通路等多条信号通路发挥止咳化痰作用。雌激素信号通路参与调节肺部发育和功能，雌激素与其相应受体结合，通过ERβ/circ-TMX4/miR-622/CXCR4信号促进非小细胞肺癌细胞入侵^[27]，此外，研究显示雌激素还参与调节支气管平滑肌，雌激素受体高表达导致支气管收缩性增加，加重呼吸道疾病^[28]。HIF信号通路是细胞对缺氧反应的重要调节通路，McClendon等发现HIF-1α在急性肺损伤后肺泡上皮细胞中被激活，并促进其增殖和扩散，加快肺修复^[29]。TNF信号通路是细胞信号传导的关键途径之一，Lu等发现TNF-α在LPS诱导的急性肺损伤中高表达^[30]。结果显示，化橘红可能通过干预TNF信号通路减轻炎症反应，通过HIF信号通路改善缺氧环境，通过雌激素信号通路调节支气管平滑肌扩张等多靶点、多通路的协同作用，实现对咳嗽病理过程的综合干预。

综上，本研究表明化橘红可能通过靶向GAPDH、AKT1、EGFR和BCL2等关键节点，调控雌激素信号通路、HIF-1信号通路和TNF信号通路等信号通路，发挥止咳化痰作用。细胞实验结果表明，化橘红提取物通过下调AKT1表达，上调BCL2表达，发挥抗炎、提升细胞存活率作用。

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序号	化合物编号	化合物名称	口服生物利用度	类药性	化学式
1	MOL013276	Poncirin	36.55	0.74	C₂₈H₃₄O₁₄
2	MOL001798	Neohesperidin_qt	71.17	0.27	C₁₆H₁₄O₆
3	MOL004328	Naringenin	59.29	0.21	C₁₅H₁₂O₅
4	MOL005849	Didymin	38.55	0.24	C₁₆H₁₄O₅
5	MOL013279	5,7,4'-Trimethylapigenin	39.83	0.3	C₁₈H₁₆O₅
6	MOL005828	Nobiletin	61.67	0.52	C₂₁H₂₂O₈
7	MOL001803	Sinensetin	50.56	0.45	C₂₀H₂₀O₇
8	MOL013277	Isosinensetin	51.15	0.44	C₂₀H₂₀O₇
9	MOL000358	β-sitosterol	36.91	0.75	C₂₉H₅₀O
10	MOL010267	LYC	32.57	0.51	C₄₀H₅₆

序号	基因名称	靶点蛋白质名称	度值	介数中心性
1	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	146.0	4 497.1
2	AKT1	RAC-alpha serine/threonine-protein kinase	137.0	3 157.3
3	EGFR	Epidermal growth factor receptor	123.0	2 327.8
4	BCL2	Apoptosis regulator Bcl-2	116.0	1 354.7
5	CASP3	Caspase-3	110.0	951.0
6	SRC	Proto-oncogene tyrosine-protein kinase Src	109.0	3 486.2
7	ESR1	Estrogen receptor	104.0	1 469.5
8	PTGS2	Prostaglandin G/H synthase 2	98.0	2 226.2
9	MMP9	Matrix metalloproteinase-9	94.0	860.8
10	CCND1	G1/S-specific cyclin-D1	94.0	883.5

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Exploring the molecular mechanism of Citri Grandis Exocarpium in relieving cough and resolving phlegm based on network pharmacology and molecular docking

doi: 10.12206/j.issn.2097-2024.202604056

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通讯作者: 陈斌, bchen63@163.com

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Exploring the molecular mechanism of Citri Grandis Exocarpium in relieving cough and resolving phlegm based on network pharmacology and molecular docking

doi: 10.12206/j.issn.2097-2024.202604056

1. Shanghai Huangpu District Geriatric Care Hospital, Shanghai 200010, China 2. Department of Pharmacy, The First Affiliated Hospital of Naval Medical University, Shanghai 200433, China 3. Shanghai Huangpu District Yuyuan Community Health Service Center, Shanghai 200010, China

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1.1. 细胞

1.2. 药品与试剂

1.3. 仪器

2.1. 网络药理学分析

2.1.1. 化橘红活性成分及靶点筛选

2.1.2. 疾病靶点预测

2.1.3. 化橘红与咳嗽、痰证交集靶点

2.1.4. 交集靶点蛋白质-蛋白质相互作用（PPI）网络构建及核心靶点筛选

2.1.5. 核心靶点GO功能和KEGG通路富集分析

2.1.6. 构建化橘红对咳嗽、痰证“活性成分-核心靶点-通路”网络

2.1.7. 分子对接验证

2.2. 细胞实验

2.2.1. 化橘红提取物的制备

2.2.2. BEAS-2B细胞模型的建立

2.2.3. AMG9810、化橘红提取物干预浓度筛选

2.2.4. CCK8法检测BEAS-2B细胞存活率

2.2.5. 实验分组

2.2.6. qPCR法检测BEAS-2B细胞BCL2、AKT1、EGFR基因表达

2.2.7. 统计学分析

3.1. 化橘红活性成分筛选结果

3.2. 化橘红活性成分靶点获取结果

3.3. 化橘红对咳嗽、痰证交集靶点获取结果

3.4. 化橘红与咳嗽、痰证交集靶点PPI网络

3.5. 化橘红与咳嗽、痰证核心靶点获取结果

3.6. GO功能富集分析

3.7. KEGG信号通路富集分析

3.8. 化橘红对咳嗽、痰证活性成分-核心靶点-通路网络构建

3.9. 分子对接

3.10. 细胞实验结果

3.10.1. BEAS-2B细胞模型的建立

3.10.2. AMG9810和化橘红提取物干预浓度的确定

3.10.3. 化橘红提取物对BEAS-2B细胞存活率的影响

3.10.4. 化橘红提取物对BEAS-2B细胞BCL2、AKT1、EGFR表达的影响

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1. Shanghai Huangpu District Geriatric Care Hospital, Shanghai 200010, China

2. Department of Pharmacy, The First Affiliated Hospital of Naval Medical University, Shanghai 200433, China

3. Shanghai Huangpu District Yuyuan Community Health Service Center, Shanghai 200010, China