护肝布祖热颗粒通过EGFR/Ras/PI3K/AKT信号通路改善免疫性肝损伤: 一项网络药理学研究和实验验证

doi:10.5246/jcps.2024.02.011

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (2): 123-141.DOI: 10.5246/jcps.2024.02.011

护肝布祖热颗粒通过EGFR/Ras/PI3K/AKT信号通路改善免疫性肝损伤: 一项网络药理学研究和实验验证

韩正茹¹, 宋婉慈¹, 罗旸¹, 肖敏¹, 王梦恒¹, 郑吴殷晓¹, 但汉雄¹, 尹强², 尹海龙², 尤朋涛¹^,^*()

^1. 湖北中医药大学湖北省中医药资源与化学重点实验室, 湖北武汉 430065
^2. 新疆维吾尔制药有限公司, 新疆乌鲁木齐 830001

收稿日期:2023-08-24 修回日期:2023-09-17 接受日期:2023-11-12 出版日期:2024-03-03 发布日期:2024-03-03
通讯作者: 尤朋涛

Hugan Buzure granule ameliorates immune liver injury through the EGFR/Ras/PI3K/AKT signaling pathway: A network pharmacology study and experimental validation

Zhengru Han¹, Wanci Song¹, Yang Luo¹, Min Xiao¹, Mengheng Wang¹, Wuyinxiao Zheng¹, Hanxiong Dan¹, Qiang Yin², Hailong Yin², Pengtao You¹^,^*()

¹ Hubei Key Laboratory of Resources and Chemistry of Chinese Medicine, Hubei University of Chinese Medicine, Wuhan 430065, Hubei, China
² Xinjiang Uygur Pharmaceutical Co., Ltd., Wulumuqi 830001, Xinjiang, China

Received:2023-08-24 Revised:2023-09-17 Accepted:2023-11-12 Online:2024-03-03 Published:2024-03-03
Contact: Pengtao You
Supported by:
Natural Science Foundation of Hubei Province (Grant No. 2018CFB657).

摘要/Abstract

摘要：

护肝布祖热颗粒是维吾尔族传统的护肝方。作者在先前的一项研究中通过调节免疫平衡和抑制IRF1/JNK信号诱导的细胞凋亡, 探讨了HBG抗免疫性肝损伤(ILI)的机制。但不是所有的机制都得到了探讨。本研究通过体内实验及网络药理学进一步研究了HBG降低小鼠刀豆球蛋白A(ConA)诱导的ILI的机制。首先, TCMSP、PharmMapper和GeneCards数据库确定了HBG治疗ILI的活性化合物和关键靶点。通过网络药理学和分子对接对确定的关键靶点和相关信号通路进行筛选。ConA诱导的小鼠ILI模型探讨了HBG对抗ILI的疗效和机制; 蛋白质印迹分析检测关键蛋白的表达水平; 网络药理学鉴定了16个化合物和53个靶标。成分-靶向通路(C-T-P)网络和分子对接表明, EGFR、HRAS、AKT1和PIK3R1是HBG对抗ILI的关键靶点。此外, 分子对接显示, 前八种活性化合物的对接得分高于阈值。与ConA组相比, HBG显著上调p-EGFR、Ras、p-AKT、p-PI3K、p-BAD和Bcl-2的表达, 并下调Bax、裂解caspase 9和3的蛋白水平。HBG通过EGFR/Ras/PI3K/AKT信号通路抑制细胞凋亡, 从而减轻ILI。

关键词: 护肝布祖热颗粒, 免疫性肝损伤, 分子对接, 细胞凋亡, 通路

Abstract:

Hugan Buzure granule (HBG) is a traditional prescription in Uygur medicine that is known for its hepatoprotective properties. In a previous study, the authors have investigated the mechanism through which HBG protects against immune liver injury (ILI) by regulating immune balance and inhibiting apoptosis mediated by the IRF1/JNK signaling pathway. However, not all mechanisms are thoroughly explored. To address this issue, the present study further investigated the mechanism by which HBG reduced concanavalin A (ConA)-induced ILI in mice using network pharmacology and in vivo experiments. Initially, the livers of the mice were examined through pathological sections, which prompted further screening. The TCMSP, PharmMapper, and GeneCards databases were employed to identify the active compounds and key targets of HBG in the treatment of ILI. Subsequently, the key targets and related signaling pathways were screened using network pharmacology and molecular docking. The efficacy and mechanism of HBG against ILI were explored in a ConA-induced mouse model. Key proteins were analyzed, and their expression levels were detected using Western blotting analysis. The network pharmacology analysis revealed 16 compounds and 53 targets associated with HBG. The component-target-pathway (C-T-P) network and molecular docking indicated that EGFR, HRAS, AKT1, and PIK3R1 were the key targets of HBG in the context of ILI. TUNEL staining results demonstrated that HBG significantly reduced apoptosis in mice with ILI. Moreover, HBG markedly upregulated the expression of p-EGFR, Ras, p-AKT, p-PI3K, p-BAD, and Bcl-2, while down-regulating the levels of Bax, cleaved-caspase 9, and cleaved-caspase 3 proteins, as compared to the ConA group. These findings suggested that HBG alleviated ILI by inhibiting apoptosis through the EGFR/Ras/PI3K/AKT signaling pathway.

Key words: HBG, Immune liver injury, Molecular docking, Apoptosis, Pathway

Supporting: /attached/file/20240308/20240308224323_768.pdf

韩正茹, 宋婉慈, 罗旸, 肖敏, 王梦恒, 郑吴殷晓, 但汉雄, 尹强, 尹海龙, 尤朋涛. 护肝布祖热颗粒通过EGFR/Ras/PI3K/AKT信号通路改善免疫性肝损伤: 一项网络药理学研究和实验验证[J]. 中国药学(英文版), 2024, 33(2): 123-141.

Zhengru Han, Wanci Song, Yang Luo, Min Xiao, Mengheng Wang, Wuyinxiao Zheng, Hanxiong Dan, Qiang Yin, Hailong Yin, Pengtao You. Hugan Buzure granule ameliorates immune liver injury through the EGFR/Ras/PI3K/AKT signaling pathway: A network pharmacology study and experimental validation[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(2): 123-141.

图/表 11

Figure 1. Study flowchart.

Table 1. Identified compounds with high intensity (mzCloud score > 80) from HBG by LC-ESI-QTOF/MS.

Figure 2. Chemical structures of the 16 active components.

Table 2. The top eight targets and top eight compounds according to the degree value.

Figure 3. HBG and ILI-related network and enrichment analysis (A) Venn diagram of HBG and ILI-related targets; (B) The top 30 significantly enriched GO terms; (C) The top 30 significantly enriched KEGG pathways; (D) C-T-P network. Purple represents compounds, blue represents targets, and orange represents pathways; (E) PPI network. The larger the node and the darker the color, the greater the degree value.

Figure 4. Results of LibDock. The larger the circle, the greater the LibDock score.

Figure 5. Molecular docking of the top four targets with cynaroside (A), astragalin (B), esculin (C), and diosmetin (D).

Table 3. Dock binding free energies (∆Gb) and bonds of the docked compounds against proteins.

Figure 6. HBG activates EGFR/Ras/AKT/PI3K signaling pathway. (A) H&E staining of mouse liver tissue (× 200); (B) Western blotting analysis determined the expression of EGFR, p-EGFR, PI3K, p-PI3K, AKT, p-AKT, and Ras. Gray values were calculated. Data are expressed as mean ± SD. ##P < 0.01, vs. the control group. *P < 0.05 and **P < 0.01 vs. the ConA group.

Figure 7. HBG inhibits hepatocyte apoptosis. (A) TUNEL staining (× 200) showing apoptotic cells in five groups after ConA injection; (B) The expression of BAD, p-BAD, Bax, Bcl-2, cleaved-caspase 9, and cleaved-caspase 3 determined by Western blotting analysis. Gray values were calculated. Data are expressed as mean ± SD. ##P < 0.01, vs. the control group. *P < 0.05 and **P < 0.01 vs. the ConA group.

Figure 8. Schematic illustration of the HBG protective mechanism against ILI in mice.

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护肝布祖热颗粒通过EGFR/Ras/PI3K/AKT信号通路改善免疫性肝损伤: 一项网络药理学研究和实验验证

Hugan Buzure granule ameliorates immune liver injury through the EGFR/Ras/PI3K/AKT signaling pathway: A network pharmacology study and experimental validation

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