Yin Guoliang, Liang Hongyi, Sun Wenxiu, Zhang Shizhao, Feng Yanan, Liang Pengpeng, Chen Suwen, Liu Xiangyi, Pan Wenchao, Zhang Fengxia
The First Clinical Medical School, Shandong University of Traditional Chinese Medicine, Jinan, China.
Department of Nursing, Taishan Vocational College of Nursing, Taian, China.
Front Pharmacol. 2022 Nov 23;13:1016745. doi: 10.3389/fphar.2022.1016745. eCollection 2022.
Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. Our previous studies have found that Shuangyu Tiaozhi Decoction (SYTZD) could produce an improvement in NAFLD-related indicators, but the underlying mechanism associated with this improvement remains unclear. The study aimed to investigate the potential mechanism of SYTZD against NAFLD through network pharmacology and experimental verification. The components of SYTZD and SYTZD drug containing serum were analyzed using ultra-performance liquid chromatography to quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF-MS). Active components and targets of SYTZD were screened by the traditional Chinese medical systems pharmacology (TCMSP) and encyclopedia of traditional Chinese medicine (ETCM) databases. NAFLD-related targets were collected from the GeneCards and DisGeNET databases. The component-disease targets were mapped to identify the common targets of SYTZD against NAFLD. Protein-protein interaction (PPI) network of the common targets was constructed for selecting the core targets. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the core targets was performed using the database for annotation, visualization, and integrated discovery (DAVID) database. Furthermore, animal and cell models were constructed for validating the predictions of network pharmacology. Lipid accumulation, liver histopathology, insulin resistance, and core gene expression were measured by oil red O staining, hematoxylin and eosin staining, insulin tolerance test, real-time quantitative polymerase chain reaction, and Western blotting, respectively. Two components and 22 targets of SYTZD against NAFLD were identified by UPLC-Q/TOF-MS and relevant databases. PPI analysis found that ESR1, FASN, mTOR, HIF-1α, VEGFA, and GSK-3β might be the core targets of SYTZD against NAFLD, which were mainly enriched in the thyroid hormone pathway, insulin resistance pathway, HIF-1 pathway, mTOR pathway, and AMPK pathway. Experimental results revealed that SYTZD might exert multiple anti-NAFLD mechanisms, including improvements in lipid deposition, inflammation, and insulin resistance. SYTZD treatment led to decreases in the lipid profiles, hepatic enzyme levels, inflammatory cytokines, and homeostatic model assessment for insulin resistance (HOMA-IR). SYTZD treatment affected relative mRNA and protein levels associated with various pathways. Our findings reveal that SYTZD could alleviate NAFLD through a multi-component, multi-target, and multi-pathway mechanism of action.
非酒精性脂肪性肝病（NAFLD）是全球最常见的慢性肝病之一。我们之前的研究发现，双郁调脂汤（SYTZD）可使NAFLD相关指标得到改善，但其改善的潜在机制仍不清楚。本研究旨在通过网络药理学和实验验证来探究SYTZD抗NAFLD的潜在机制。采用超高效液相色谱-四极杆/飞行时间质谱联用仪（UPLC-Q/TOF-MS）分析SYTZD及其含药血清的成分。通过中药系统药理学（TCMSP）数据库和《中药大辞典》（ETCM）筛选SYTZD的活性成分和靶点。从GeneCards和DisGeNET数据库收集NAFLD相关靶点。将成分-疾病靶点进行映射，以确定SYTZD抗NAFLD的共同靶点。构建共同靶点的蛋白质-蛋白质相互作用（PPI）网络以筛选核心靶点。使用注释、可视化和综合发现数据库（DAVID）对核心靶点进行京都基因与基因组百科全书（KEGG）通路分析。此外，构建动物和细胞模型以验证网络药理学的预测结果。分别通过油红O染色、苏木精-伊红染色、胰岛素耐量试验、实时定量聚合酶链反应和蛋白质免疫印迹法检测脂质蓄积、肝脏组织病理学、胰岛素抵抗和核心基因表达。通过UPLC-Q/TOF-MS和相关数据库鉴定出SYTZD抗NAFLD的两种成分和22个靶点。PPI分析发现，雌激素受体1（ESR1）、脂肪酸合酶（FASN）、哺乳动物雷帕霉素靶蛋白（mTOR）、缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子A（VEGFA）和糖原合成酶激酶-3β（GSK-3β）可能是SYTZD抗NAFLD的核心靶点，这些靶点主要富集于甲状腺激素通路、胰岛素抵抗通路、HIF-1通路、mTOR通路和AMPK通路。实验结果表明，SYTZD可能发挥多种抗NAFLD机制，包括改善脂质沉积、炎症和胰岛素抵抗。SYTZD治疗可降低血脂水平、肝酶水平、炎性细胞因子以及胰岛素抵抗稳态模型评估（HOMA-IR）。SYTZD治疗影响与各种通路相关的相对mRNA和蛋白质水平。我们的研究结果表明，SYTZD可通过多成分、多靶点和多通路的作用机制减轻NAFLD。
