基于斑马鱼模型及网络药理学研究植物乳植杆菌HCS03-001抗细菌炎症的作用

doi:10.19586/j.2095-2341.2023.0155

生物技术进展 ›› 2024, Vol. 14 ›› Issue (2): 295-303.DOI: 10.19586/j.2095-2341.2023.0155

• 研究论文 • 上一篇

基于斑马鱼模型及网络药理学研究植物乳植杆菌HCS03-001抗细菌炎症的作用

陈雪娇¹^,²(), 余萍¹^,²(), 赵迪¹^,², 宋佳¹^,², 闵祥博¹^,²

^1.江西仁仁健康微生态科技有限公司，江西樟树 331200
^2.仁仁微生物科技研究（沈阳）有限公司，沈阳 110170

收稿日期:2023-12-04 接受日期:2024-02-07 出版日期:2024-03-25 发布日期:2024-04-17
通讯作者: 余萍
作者简介:陈雪娇 E-mail: chenxuejiao0219@126.com；

Study on the Anti-inflammatory Effect of Lactiplantibacillus plantarum HCS03-001 Based on Zebrafish Model and Network Pharmacology

Xuejiao CHEN¹^,²(), Ping YU¹^,²(), Di ZHAO¹^,², Jia SONG¹^,², Xiangbo MIN¹^,²

^1.Jiangxi Renren Health Microecological Technology Co. ，Ltd. ，Jiangxi Zhangshu 331200，China
^2.Renren Microbial Technology Research （Shenyang） Co. ，Ltd. ，Shenyang 110170，China

Received:2023-12-04 Accepted:2024-02-07 Online:2024-03-25 Published:2024-04-17
Contact: Ping YU

摘要/Abstract

摘要：

过度炎症严重危害人体健康，为了验证植物乳植杆菌HCS03-001的抗炎功效并探讨其抗炎机理，以转基因中性粒细胞绿色荧光斑马鱼为模型，使用细菌脂多糖（lipopolysaccharide，LPS）诱导炎症发生，以吲哚美辛为阳性对照，探究10、20、40 mg·mL^-1植物乳植杆菌HCS03-001的抗细菌性炎症功效。通过SwissTargetPrediction、GeneCards和DisGeNET数据库获取植物乳植杆菌代谢产物的主要成分和炎症靶点。靶点取交集后通过STRING数据库构建蛋白质-蛋白质相互作用（the protein-protein interaction，PPI）网络；使用Cytoscape 3.9.0构建菌株代谢产物“成分-靶点-炎症”网络；利用DAVID平台工具对共有靶点进行GO富集分析及KEGG通路富集分析。斑马鱼实验结果显示，不同剂量的HCS03-001均能降低斑马鱼卵黄囊中性粒细胞数量，其中高剂量（40 mg·mL^-1）的效果与模型组相比达到显著水平（P<0.05）。网络药理学结果发现，植物乳植杆菌代谢产物缓解炎症的过程中，作用较强的活性成分主要是异吡啶硫胺素、吲哚乙醛和麦角硫因等，可能作用于甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase，GAPDH）、环氧合酶2（prostaglandin endoperoxide synthase 2，Ptgs2）、过氧化物酶体增生激活受体γ（peroxisome proliferator-activated receptor gamma，PPARγ）等核心靶点，富集结果显示影响最显著的生物学过程是对LPS的反应，涉及的细胞组分为细胞膜，影响最显著的通路为癌症通路。结果表明，植物乳植杆菌HCS03-001具有显著的抗细菌性炎症功效，其具体机制可能是代谢成分异吡啶硫胺素、吲哚乙醛和麦角硫因等通过抑制细菌LPS与机体细胞膜的结合，以及通过调控GAPDH、Ptgs2、PPARγ等关键靶点影响癌症相关通路实现的。

关键词: 乳酸菌, 脂多糖, 抗炎作用, 网络药理学, 斑马鱼

Abstract:

Excessive inflammation is seriously harmful to human health. The anti-inflammatory effect of Lactiplantibacillus plantarum HCS03-001 was verified and the anti-inflammatory mechanism was explored. Using transgenic neutrophil green fluorescent zebrafish as model， bacterial LPS was used to induce inflammation， and indomethacin was used as positive control to explore the anti-bacterial inflammatory effect of Lactiplantibacillus plantarum HCS03-001 of 10， 20 and 40 mg·mL^-1. The main components and inflammatory targets of the metabolites of Lactiplantibacillus plantarum were obtained by SwissTargetPrediction， GeneCards and DisGeNET database. After the targets were intersected， the PPI network was constructed by STRING database， the metabolic product “component-target-inflammation” network was constructed by Cytoscape3.9.0， and the common targets were analyzed by GO enrichment analysis and KEGG pathway enrichment analysis using DAVID platform tools. The results of zebrafish experiment showed that different doses of HCS03-001 could reduce the number of neutrophils in zebrafish yolk sac， and the effect of high dose （40 mg·mL^-1） was significantly higher than that of the model group. The results of network pharmacology showed that in the process of relieving inflammation， the main active components of Lactobacillus plantarum metabolites were isopyridine thiamine， indole acetaldehyde and ergothione， which may act on core targets such as GAPDH， Ptgs2 and PPARγ. The enrichment results showed that the most significant biological process was the response to LPS， the cell components involved were cell membranes， and the most significant pathway was cancer pathway. The results showed that Lactiplantibacillus plantarum HCS03-001 had a significant anti-bacterial inflammatory effect， and the specific mechanism may be that metabolic components such as isopyridine thiamine， indole acetaldehyde and ergothione affect cancer-related pathways by inhibiting the binding of bacterial LPS to the plasma membrane and regulating key targets such as GAPDH， Ptgs2 and PPARγ.

Key words: Lactobacillus, lipopolysaccharide, anti-inflammatory effect, network pharmacology, zebrafish

中图分类号:

Q815

陈雪娇, 余萍, 赵迪, 宋佳, 闵祥博. 基于斑马鱼模型及网络药理学研究植物乳植杆菌HCS03-001抗细菌炎症的作用[J]. 生物技术进展, 2024, 14(2): 295-303.

Xuejiao CHEN, Ping YU, Di ZHAO, Jia SONG, Xiangbo MIN. Study on the Anti-inflammatory Effect of Lactiplantibacillus plantarum HCS03-001 Based on Zebrafish Model and Network Pharmacology[J]. Current Biotechnology, 2024, 14(2): 295-303.

图/表 9

表1 植物乳植杆菌HCS03-001的MTC实验结果（n=30）

Table 1 MTC results of Lactiplantibacillus plantarum HCS03-001（n=30）

组别	浓度/(mg·mL^-1)	益生菌计数/（CFU·mL^-1）	表型
正常对照组	-	-	未见明显异常
模型对照组	-	-	未见明显异常
植物乳植杆菌HCS03-001	5.00	0.50×10⁹	与模型对照组状态相似
	10.0	1.00×10⁹	与模型对照组状态相似
	20.0	2.00×10⁹	与模型对照组状态相似
	40.0	4.00×10⁹	与模型对照组状态相似
	80.0	8.00×10⁹	较模型对照组状态差

图1 植物乳植杆菌HCS03-001处理后斑马鱼卵黄囊中性粒细胞数量注：*，***表示与模型对照组相比较，处理组在P<0.05、P<0.001水平上具有统计学意义。

Fig. 1 The number of neutrophils in yolk sac of zebrafish treated with Lactiplantibacillus plantarum HCS03-001

图2 植物乳植杆菌HCS03-001处理后斑马鱼卵黄囊中性粒细胞典型图注：黄色虚线框为分析部位，绿色荧光点为中性粒细胞，图中标尺为100 μm。

Fig. 2 Typical map of neutrophils in yolk sac of zebrafish treated with Lactiplantibacillus plantarum HCS03-001

表2 植物乳植杆菌代谢产物主要活性成分信息

Table 2 Information of main active components of metabolites from Lactiplantibacillus plantarum

中文名称	分子质量/(g·mol^-1)	脂水分配系数	H供体	H受体	可旋转键数量
2-酮丁酸	102.09	0.1	1	3	2
γ-氨基丁酸	103.12	-3.2	2	3	3
哌啶-2-羧酸酯	129.16	-2.3	2	3	1
氧化脯氨酸	129.12	-0.8	2	3	1
吲哚酚	133.15	2.2	2	1	0
黄嘌呤	152.11	-0.7	3	3	0
吲哚乙醛	159.18	1.3	1	1	2
麦角硫因	229.30	0.3	2	3	3
哌啶	85.15	0.8	1	1	0
5-羟基吲哚乙酸	191.18	1.1	3	3	2
多巴胺	153.18	-1.0	3	3	2
异吡啶硫胺素	201.25	0.9	1	3	2
吲哚丙酮酸酯	203.19	1.3	2	3	3

图3 炎症靶点与植物乳植杆菌代谢产物靶点韦恩图

Fig. 3 Venn diagram of inflammatory targets and metabolites of Lactiplantibacillus plantarum

图4 “成分-靶点-炎症”网络注：图中蓝色圆形的图标代表交集靶点，黄色方块图标代表植物乳植杆菌代谢产物的活性成分，红色菱形图标代表植物乳植杆菌代谢产物。

Fig. 4 Network diagram of effective “ingredients -targets-inflammation”

图5 植物乳植杆菌代谢产物缓解炎症的PPI网络图

Fig. 5 PPI network diagram of Lactiplantibacillus plantarum metabolites in the relieve inflammation

图6 核心靶点GO功能富集分析

Fig. 6 GO function enrichment analysis

图7 KEGG通路富集分析气泡图

Fig. 7 Bubble diagram of KEGG enrichment analysis

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基于斑马鱼模型及网络药理学研究植物乳植杆菌HCS03-001抗细菌炎症的作用

Study on the Anti-inflammatory Effect of Lactiplantibacillus plantarum HCS03-001 Based on Zebrafish Model and Network Pharmacology

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