金银花提取物对小鼠阿霉素肝脏损伤的保护作用

doi:10.12122/j.issn.1673-4254.2024.08.16

摘要/Abstract

摘要：

目的通过网络药理学与分子对接技术探讨金银花对阿霉素（DOX）肝脏损伤的保护作用和机制，并运用DOX诱导小鼠肝脏损伤实验进行验证。方法通过网络药理学方法获得金银花靶点与疾病靶点之间的交集基因。利用STRING数据库构建交集基因PPI网络，利用Cytoscape软件进行分析，筛选核心靶点。采用DAVID数据库进行生物信息学分析，分子对接技术对核心成分和核心靶点进行验证。运用DOX诱导小鼠肝脏损伤验证网络药理学的预测结果。检测小鼠血清ALT、AST水平和肝脏组织HYP、ROS水平，HE染色和Masson染色观察肝脏组织病理变化，ELISA检测肝脏组织TNF-α、IL-6、COL-IV水平，Western blotting检测肝脏组织P53蛋白表达水平。结果从交集的43个基因中筛选出12个核心靶点，涉及癌症通路、IL-17信号通路、TNF等信号通路。分子对接结果显示，10个核心成分可以与不同的核心靶点结合。小鼠实验显示，与Sham组相比，DOX组血清AST和ALT水平升高（P<0.001）；HE和Masson染色显示肝脏损伤和肝脏纤维化，ROS、TNF-α、IL-6、HYP、COL-IV和P53蛋白水平升高（P<0.001）。与DOX组相比，金银花处理组血清AST和ALT水平降低（P<0.001），肝脏损伤和肝脏纤维化改善，肝脏组织ROS、TNF-α、IL-6、HYP和COL-IV水平和P53蛋白表达降低（P<0.001），肝脏组织氧化应激、炎症和纤维化均减轻。结论金银花可通过作用于Trp53、TNF、IL-6靶点减轻肝脏氧化应激、炎症和纤维化程度，减轻DOX诱导的肝脏损伤。

关键词: 网络药理学, 金银花, 阿霉素, 肝脏损伤, 氧化应激, 肝脏纤维化, 炎症

Abstract:

Objective To explore the mechanism underlying the protective effect of Lonicerae japonicae flos (LJF) extract against doxorubicin (DOX)-induced liver injury in mice. Methods Network pharmacology methods were used to obtain the intersection genes between LJF targets and disease targets, based on which the protein-protein interaction (PPI) network was constructed using STRING database for screening the core targets using Cytoscape software. DAVID database was used for bioinformatics analysis, and the core components and core targets were verified using molecular docking study. In a mouse model of DOX-induced liver injury, the effect of LJF extract on liver pathologies, serum levels of ALT and AST, and hepatic expressions of HYP, ROS, TNF-α, IL-6, COL-IV and P53 proteins were evaluated using HE and Masson staining, ELISA, and Western blotting. Results We identified 12 core targets from 43 intersection genes involving cancer pathway, IL-17 signaling pathway, and TNF signaling pathways. Molecular docking study suggested that 10 core components of LJF could bind to different core targets. The mice with DOX-induced liver injury showed elevated serum AST and ALT levels with obvious liver injury and fibrosis, increased ROS content, and enhanced expressions of TNF-α, IL-6, HYP, COL-IV and P53 proteins in the liver tissue. All these changes in the mouse models were significantly alleviated by treatment with LJF extract, suggesting obviously lowered levels of oxidative stress, inflammation and fibrosis in the liver tissues. Conclusion LJF extract is capable of alleviating DOX-induced liver injury in mice by downregulating Trp53, TNF and IL-6 to reduce liver oxidative stress, inflammation and fibrosis.

Key words: network pharmacology, Lonicerae japonicae flos, doxorubicin, liver damage, oxidative stress, liver fibrosis, inflammation

张钰明, 夏士程, 张淋淋, 陈梦茜, 刘晓婧, 高琴, 叶红伟. 金银花提取物对小鼠阿霉素肝脏损伤的保护作用[J]. 南方医科大学学报, 2024, 44(8): 1571-1581.

Yuming ZHANG, Shicheng XIA, Linlin ZHANG, Mengxi CHEN, Xiaojing LIU, Qin GAO, Hongwei YE. Protective effect of Lonicerae japonicae flos extract against doxorubicin-induced liver injury in mice[J]. Journal of Southern Medical University, 2024, 44(8): 1571-1581.

图/表 12

图1 金银花靶点、阿霉素肝脏损伤和肝脏纤维化相关靶点维恩图

Fig.1 Venn diagram of Lonicerae japonicae flos, Adriamycin-induced liver injury and hepatic fibrosis-related targets.

图2 PPI网络图和核心靶点网络图

Fig.2 PPI network diagram (A) and core targets network diagram (B).

图3 GO和KEGG富集分析

Fig.3 GO (A) and KEGG (B) enrichment analysis.

表1 金银花核心成分

Tab.1 Core components of Lonicerae japonicae flos

Mol ID	Molecule	OB	DL
MOL000006	luteolin	36.16	0.25
MOL000098	quercetin	46.43	0.28
MOL000422	kaempferol	41.88	0.24
MOL002914	Eriodyctiol (flavanone)	41.35	0.24
MOL003006	(-)-(3R,8S,9R,9aS,10aS)-9-ethenyl-8-(beta-D-glucopyranosyloxy)-2,3,9,9a,10,10a-hexahydro-5-oxo-5H,8H-pyrano[4,3-d]oxazolo[3,2-a]pyridine-3-carboxylic acid_qt	87.47	0.23
MOL003014	secologanic dibutylacetal_qt	53.65	0.29
MOL003044	Chryseriol	35.85	0.27
MOL003095	5-hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)chromone	51.96	0.41
MOL003111	Centauroside_qt	55.79	0.5
MOL003117	Ioniceracetalides B_qt	61.19	0.19

表2 核心靶点与核心成分结合能

Tab.2 Binding energy of the core targets and core ingredients (kcal/mol)

Mol ID	TrP53 Binding energy	TNF Binding energy	IL-6 Binding energy
MOL000006	-4.49	-5.8	-3.93
MOL000098	-3.93	-5.6	-4.24
MOL000422	-4.85	-5.62	-4.59
MOL002914	-5.61	-5.09	-4.75
MOL003006	-4.7	-6.97	-5.31
MOL003014	-2.53	-2.96	-0.83
MOL003044	-5.02	-5.99	-5.1
MOL003095	-4.7	-4.9	-3.59
MOL003111	-2.85	-4.11	-3.23
MOL003117	-4.94	-4.79	-4.24

图4 分子对接可视化结果

Fig.4 Visualization of molecular docking results. A: Potential interaction between MOL002914 and Trp53. B: Potential interaction between MOL003044 and Trp53. C: Potential interaction between MOL003044 and TNF. D: Potential interaction between MOL003006 and TNF. E: Potential interaction between MOL003006 and IL-6. F: Potential interaction between MOL003044 and IL-6.

图5 各组小鼠血清AST和ALT水平

Fig.5 Serum levels of AST (A) and ALT (B) in mice in each group (Mean±SD, n=6). ***P<0.001 vs Sham; ###P<0.001 vs DOX; $P<0.05, $$P<0.01 vs DOX+LJF (0.2 g/kg); &&P<0.01 vs DOX+LJF (0.4 g/kg).

图6 各组小鼠肝脏HE染色结果

Fig.6 HE staining of mouse liver tissues in each group (Original magnification: ×40). Black arrows indicate erythrocyte exudates, red arrows indicate hepatocyte ballooning, and blue arrows indicate inflammatory cell infiltration.

图7 各组小鼠肝脏Masson染色结果

Fig.7 Masson staining of mouse liver tissue in each group (×120). The black arrows indicate fibrotic hyperplasia and the red arrows indicate hepatocyte balloons.

图9 各组小鼠肝脏组织中TNF-α和IL-6水平

Fig.9 TNF-α (A) and IL-6 (B) levels in mouse liver tissues in each group (Mean±SD, n=6). ***P<0.001 vs Sham; ###P<0.001 vs DOX; $$P<0.01, $$$P<0.001 vs DOX+LJF (0.2 g/kg) ; &&P<0.01 vs DOX+LJF (0.4 g/kg).

图10 各组小鼠肝脏组织中HYP和COL-Ⅳ水平

Fig.10 HYP (A) and COL-IV (B) levels in mouse liver tissues in each group (Mean±SD, n=6). ***P<0.001 vs Sham; ###P<0.001 vs DOX; $P<0.05, $$P<0.01, $$$P<0.001 vs DOX+LJF (0.2 g/kg) ; &P<0.05, &&P<0.01 vs DOX+LJF (0.4 g/kg).

图11 各组小鼠肝脏组织中P53蛋白表达水平

Fig.11 Protein expression level of P53 in mouse liver tissue in each group (Mean±SD, n=6). A: P53 protein expression detected by Western blotting in each group. B: Quantitative data of P53 expression. ***P<0.001 vs Sham; ###P<0.001, ##P<0.01, #P<0.05 vs DOX; &P<0.05 vs DOX+LJF (0.2 g/kg).

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