南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (8): 1131-1141.doi: 10.12122/j.issn.1673-4254.2021.08.02

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蒙药七味清肝散抗肝纤维化的作用机制：基于UHPLC-TOF-MS和网络药理学方法

张春艳，颜羽昕，高晓阳，马月宏   

  1. 内蒙古医科大学基础医学院，内蒙古自治区 呼和浩特 010059
  • 出版日期:2021-08-20 发布日期:2021-09-07

Therapeutic mechanism of the Mongolian medicine Qiwei Qinggan Powder against liver fibrosis based on UHPLC-TOF-MS combined with network pharmacological methods

ZHANG Chunyan, YAN Yuxin, GAO Xiaoyang, MA Yuehong   

  1. Department of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot 010059, China
  • Online:2021-08-20 Published:2021-09-07

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摘要：

目的 系统阐明蒙药七味清肝散化学成分组成，运用网络药理学探讨其关键靶点和相关通路，并通过实验进一步明确其抗肝纤维化的作用机制。方法 对蒙药七味清肝散进行UHPLC-TOF-MS测定及成分分析；在Swiss Target Prediction数据库筛选出蒙药七味清肝散相关靶点；在治疗靶点数据库 TTD 和 GeneCards 数据库筛选出肝纤维化相关靶点；通过Venny.2.1.0取交集获得蒙药七味清肝散抗肝纤维化靶点，通过STRING数据库进行蛋白互作分析，在Metascape平台进行GO功能和KEGG通路分析，运用AutoDock软件对核心靶点和活性成分进行分子对接验证；通过动物模型实验和离体细胞实验验证蒙药七味清肝散抗肝纤维化的作用机制。结果 共鉴定出化学成分45个，包括黄酮类、生物碱、香豆素、萜类、酚类、脂肪酸等。通过网络药理学分析，获得蒙药七味清肝散抗肝纤维化靶点62个，其中核心靶点10个。GO富集分析共涉及生物过程（BP）、细胞组分（CC）、分子功能（MF）三个方面。KEGG富集结果显示，PI3K/Akt、Rap1 、MAPK、AMPK、PPAR等是参与肝纤维化发生发展的通路。通过分子对接验证了活性成分 Quercetin、Luteolin、Kaempferol 分别与Akt1、PIK3R1、MAPK1紧密结合。在动物模型实验中，通过Masson染色观察出蒙药七味清肝散给药组可以使纤维组织增生减少，炎症细胞浸润减少，使纤维化的肝脏组织得到改善。Western blotting结果提示，蒙药七味清肝散可以使肝纤维化标志因子α-SMA、Collagen1（P<0.01）和PI3K、Akt蛋白表达量下降（P<0.01）；在离体细胞实验中，蒙药七味清肝散含药血清可以导致HSC-T6凋亡率增加。结论 蒙药清肝散通过多成分、多靶点、多通路的形式发挥抗肝纤维化的作用，作用机制可能参与调控PI3K、Akt等关键靶点，参与调控细胞凋亡和能量代谢。

关键词: 蒙药；七味清肝散；肝纤维化；UHPLC-TOF-MS；网络药理学；PI3K/Akt

Abstract: Objective To elucidate the chemical composition of the Mongolian medicine Qiwei Qinggan Powder and explore its key targets, related pathways and its therapeutic mechanism for liver fibrosis. Methods UHPLC-TOF-MS was used to analyze the composition of Qiwei Qinggan Powder. The therapeutic targets of Qiwei Qinggan Powder were screened in Swiss Target Prediction database, and liver fibrosis-related targets were screened in TTD and GeneCards databases to identify the anti-fibrosis targets of Qiwei Qinggan Powder by intersection using Venny.2.1.0. The protein interaction was analyzed using STRING database, the GO functions and KEGG pathways were analyzed on the Metascape platform, and the core targets and active components were verified by molecular docking using AutoDock software. The therapeutic mechanism of Qiwei Qinggan Powder against liver fibrosis was verified in rat models and cell experiment. Results We identified a total of 45 chemical constituents in Qiwei Qinggan Powder, including flavonoids, alkaloids, coumarins, terpenes, phenols and fatty acids. Network pharmacological analysis identified 62 targets of Qiwei Qinggan Powder, including 10 core targets. GO enrichment analysis suggested that the therapeutic effect of Qiwei Qinggan Powder was mediated by biological processes (BP), cell components (CC) and molecular functions (MF). KEGG enrichment results showed that PI3K/Akt, Rap1, MAPK, AMPK and PPAR were all pathways associated with liver fibrosis. Molecular docking showed that quercetin, luteolin and kaempferol could bind to Akt1, PIK3R1 and MAPK1, respectively. In rat models of liver fibrosis, treatment with Qiwei Qinggan Powder significantly suppressed proliferation of fibrous tissues and inflammatory cell infiltration to improve fibrosis in the liver tissue. Western blotting demonstrated that Qiwei Qinggan Powder significantly decreased the expressions of the Liver fibrosis markers including α-SMA, Collagen1, PI3K and Akt (P<0.01). In vitro cell experiment, Qiwei Qinggan Powder-containing serum obviously promoted apoptosis of HSC-T6 cells. Conclusion The therapeutic effect of Qiwei Qinggan Powder against liver fibrosis is mediated by multiple components, targets and channels, and its mechanism may involve the regulation of PI3K, Akt and other key targets and modulation of cell apoptosis and energy metabolism.

Key words: Mongolian medicine; Qiwei Qinggan Powder; liver fibrosis; UHPLC-TOF-MS; network pharmacology; PI3K/Akt