Mechanisms of total flavonoids from Sophora flavescens for the treatment of non-alcoholic fatty liver disease and experimental validation in zebrafish

doi:10.12307/2025.612

Abstract

Abstract: BACKGROUND: Total flavonoids from Sophora flavescens have a variety of pharmacological effects, including anti-inflammatory, immunomodulatory, antioxidant, and anti-hepatic injury, but the therapeutic effects and mechanisms in non-alcoholic fatty liver disease are not clear.
OBJECTIVE: To reveal the mechanism of total flavonoids from Sophora flavescens in the treatment of non-alcoholic fatty liver disease using bioinformatics, network pharmacology and zebrafish experimental validation.
METHODS: A zebrafish model of non-alcoholic fatty liver disease was constructed to observe lipid accumulation, pathomorphologic changes, and expression of inflammatory genes in the liver of zebrafish after treatment with total flavonoids from Sophora flavescens. The active ingredients of total flavonoids from Sophora flavescens and non-alcoholic fatty liver disease-related targets were obtained from TCMSP, Swiss Target Prediction, and Bat-man databases. STRING was used to perform protein-protein interaction network analysis, GO functional enrichment and KEGG pathway enrichment analysis. Based on the GSE33814 dataset, the differentially expressed genes of total flavonoids from Sophora flavescens and non-alcoholic fatty liver disease intersection targets were screened out. Correlation analysis and receiver operating characteristic curve were performed using R4.3.2 software. Core genes were verified by the validation set GSE89632. RT-qPCR and western blot assays were performed to verify the expression of core pathway-related genes and proteins.
RESULTS AND CONCLUSION: (1) Total flavonoids from Sophora flavescens could improve lipid accumulation in the liver of zebrafish with non-alcoholic fatty liver disease, significantly inhibited the elevation of lipid and aminotransferase levels in zebrafish (P < 0.05), and regulated the expression of genes related to inflammation and lipid metabolism. (2) A total of 168 common targets were obtained using the network pharmacology, and top 10 core genes, identified by cytoscape topology analysis, were HSP90AA1, STAT3, PIK3R1, MAPK1, AKT1, RXRA, PIK3CA, EGFR, JAK2, and ESR1. GO and KEGG analysis pathways mainly included insulin resistance, lipids, and atherosclerosis. There were a total of 59 differentially expressed genes after intersection of total flavonoids from Sophora flavescens and non-alcoholic fatty liver disease targets. The receiver operating characteristic curve and validation set analyses yielded six core targets that were significantly different between healthy individuals and patients with non-alcoholic fatty liver disease (P < 0.01). (3) RT-PCR and western blot results verified that total flavonoids from Sophora flavescens inhibited the activation of the JAK2/STAT3 signaling pathway in zebrafish. To conclude, total flavonoids from Sophora flavescens may alleviate the inflammatory response through the JAK2/STAT3 signaling pathway, thus inhibiting lipid accumulation and improving non-alcoholic fatty liver disease.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: flavonoids from Sophora flavescens, non-alcoholic fatty liver disease, zebrafish, network pharmacology, bioinformatics, JAK2, STAT3

CLC Number:

Gu Yufeng, Deng Bingying, Li Niren, Zeng Yixuan, Lu Sifan, Zhu Chen, Chen Lei, Liu Yi. Mechanisms of total flavonoids from Sophora flavescens for the treatment of non-alcoholic fatty liver disease and experimental validation in zebrafish [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(14): 2969-2978.

Figures/Tables 12

2.1 苦参总黄酮用药质量浓度筛选如图1所示，在一定范围内随着苦参总黄酮暴露质量浓度的升高斑马鱼死亡率呈剂量依赖性升高。斑马鱼在药物质量浓度为12 μg/mL 时，死亡率为100%，在质量浓度不高于6 μg/mL时，斑马鱼状态良好，无异常情况，根据CNAS-15088体系标准，未超过10%的斑马鱼出现死亡、畸形等毒性表型，确定高剂量为6 μg/mL，低剂量为3 μg/mL。 2.2 各组斑马鱼表观形态及病理形态比较给药72 h后，与模型组相比，苦参总黄酮组体长均有所下降，这表明苦参总黄酮可以抑制斑马鱼肥胖，见图2A。与空白组相比，模型组幼鱼肝脏组织质地无定形，结构紊乱，透明度降低，面积变大，表示肝脏退化，苦参总黄酮组则明显改善，见图2B。在Tg(fabp10:eGFP)品系斑马鱼中，模型组肝组织绿色荧光面积明显增大，说明其肝脏肿胀，给药后荧光强度减弱，见图2C。如图3所示，空白组斑马鱼肝细胞排列紧密，肝组织形态正常；模型组斑马鱼肝脏有大量的空泡，肝细胞不规则排列，肝组织中脂滴沉积；苦参总黄酮组肝细胞间隙的脂滴和空泡减少，排列趋于规律。苦参总黄酮组治疗效果佳，病理切片与空白组相似。油红O染色结果显示，模型组斑马鱼幼鱼肝组织染色呈深红色，轮廓不清晰，说明模型组斑马鱼中的脂质蓄积水平较高；苦参总黄酮组肝脏部位红染程度减轻，见图4A。尼罗红染色结果显示，空白组肝脏中几乎无红色荧光，模型组有红色强烈荧光，提示模型组脂肪沉积，苦参总黄酮组荧光强度减弱，提示苦参总黄酮能够缓解斑马鱼脂质代谢紊乱水平，见图4B。 2.3 各组斑马鱼脂质、转氨酶含量比较与空白组相比，模型组三酰甘油和总胆固醇含量显著增加(P < 0.01)；与模型组相比，苦参总黄酮低、高剂量组三酰甘油和总胆固醇含量显著降低(P < 0.01)。与空白组相比，模型组谷丙转氨酶、谷草转氨酶含量均显著升高(P < 0.01)；与模型组相比，苦参总黄酮低、高剂量组谷丙转氨酶、谷草转氨酶含量显著降低，见图5，以上结果说明苦参总黄酮对非酒精性脂肪肝具有一定的治疗作用。 2.4 各组斑马鱼脂代谢、炎症相关基因表达比较与空白组相比，模型组斑马鱼脂代谢相关基因apoa1a mRNA表达显著下降(P < 0.01)，fasn、srebf1 mRNA表达显著升高(P < 0.01)；与模型组相比，苦参总黄酮低、高剂量组apoa1a mRNA表达显著下降(P < 0.01)，fasn、srebf1 mRNA表达显著升高(P < 0.01)。与空白组相比，模型组斑马鱼炎症因子肿瘤坏死因子α、白细胞介素1b、转化生长因子β1 mRNA表达显著升高(P < 0.01)；与模型组相比，苦参总黄酮低、高剂量组肿瘤坏死因子α、白细胞介素1b、转化生长因子β1基因mRNA表达显著下降(P < 0.01)，见图6。 2.5 苦参总黄酮成分与非酒精性脂肪肝疾病靶点获取根据数据库以及文献整理，在Swiss ADME平台中进行类药性筛选，结合文献中明确的有效成分作为补充，共从苦参中获得76个黄酮活性成分，对应的作用靶点有624个。通过Gene Cards数据库、OMIM数据库、DIsGe Net数据库综合检索获取非酒精性脂肪肝相关靶点，化合物靶点与疾病相关靶标蛋白取交集，绘制韦恩图，获得168个共同靶点，见图7。 2.6 有效成分-疾病靶点蛋白互作网络构建利用STRING和Cytoscape 3.9.1两个平台对苦参黄酮成分-非酒精性脂肪肝病的168个共同作用靶点进行蛋白互作网络分析，网络中共有143个节点，866条边，见图8。图中节点大小和颜色与度的大小呈正相关，节点越大表示度越大，节点的颜色越深表示度越大。利用Cytoscape 3.9.1中插件CytoNCA对靶标进行网络拓扑分析，综合筛选出排名前10的核心基因，分别为HSP90AA1、STAT3、PIK3R1、MAPK1、AKT1、RXRA、PIK3CA、EGFR、JAK2、ESR1，见图9，表2，提示这些基因在蛋白互作网络中发挥了关键作用。 2.7 潜在靶点基因的GO 功能注释及 KEGG 通路富集分析由图10所示，GO分析共取得732个GO条目(P < 0.05)，按P值由小到大的顺序排序，将各组分排名前10的条目"

绘制成可视化条形图。生物学过程主要包括对外源性刺激的反应、对脂多糖的反应、蛋白质磷酸化、细胞凋亡过程的负调控等，主要与蛋白磷酸化以及细胞凋亡过程有关；细胞组分相关性大的主要包括细胞质膜、膜阀区、大分子复合物、细胞外泌体等；分子功能主要包括酶结合、RNA聚合酶Ⅱ转录因子活性、配体激活的序列特异性DNA结合、胰岛素受体底物结合等。苦参总黄酮治疗非酒精性脂肪肝的通路主要集中在胰岛素抵抗、脂质与动脉粥样硬化、糖基化终末产物-糖基化终末产物受体(AGE-RAGE)信号通路和化学致癌-受体激活等通路。 2.8 差异分析检索GEO数据库获得数据集GSE89632，包括健康对照组样本13例，非酒精性脂肪肝样本12例。将168个共同靶点根据|log2FC| > 1和FDR < 0.05对基因进行差异化筛选，筛选得到差异基因59个，其中上调基因28个，下调基因41个，见图11。 2.9 受试者工作特征曲线分析受试者工作特征曲线下面积用来量化分类，曲线下面积 > 0.6的基因被认为具有诊断性能，曲线下面积 > 0.8的基因被认为具有良好的诊断性能，受试者工作特征曲线分析发现，10个核心基因HSP90AA1、STAT3、PIK3R1、MAPK1、AKT1、RXRA、PIK3CA、EGFR、JAK2、ESR1的曲线下面积分别为0.760，0.724，0.904，0.635，0.590，0.788，0.808，0.962，0.740，0.923，除AKT1基因外，其余基因曲线下面积均大于0.6，在非酒精性脂肪肝诊断方面具有出色诊断性能，见图12。 2.10 核心基因二次验证使用验证集对10个核心基因进行验证，观察其在健康人和非酒精性脂肪肝患者中的表达，结果表明，HSP90AA1、STAT3、PIK3R1、RXRA、JAK2、ESR1基因在健康人和非酒精性脂肪肝患者之间出现差异表达，见图13，其余的MAPK1、AKT1、PIK3CA、EGFR基因在健康人和非酒精性脂肪肝患者之间表达无明显差异。经二次验证后发现6个基因HSP90AA1、STAT3、PIK3R1、RXRA、JAK2、ESR1在健康人和非酒精性脂肪肝患者之间出现差异表达，提示其可能发挥作用。"

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