鼠曲草总黄酮通过激活Nrf2/SLC7A11/GPX-4信号通路抑制肝细胞铁死亡缓解对乙酰氨基酚诱导的小鼠急性肝损伤

doi:10.12122/j.issn.1673-4254.2024.11.17

摘要/Abstract

摘要：

目的基于网络药理学和实验验证探讨鼠曲草总黄酮对对乙酰氨基酚（APAP）诱导的急性肝损伤（ALI）的保护作用及其机制。方法通过文献检索获取鼠曲草总黄酮的主要化学成分，使用多重数据库分析预测鼠曲草总黄酮的药理机制。动物实验验证：将36只SPF级小鼠分为空白对照组，APAP模型组，鼠曲草总黄酮低剂量组（100 mg/kg）、中剂量（200 mg/kg）、高剂量（400 mg/kg）组，阳性药物联苯双酯组（150 mg/kg），每组6只。通过大体标本与生化检查、HE染色、普鲁士蓝染色及分子生物学检查，评估鼠曲草总黄酮干预APAP诱导急性肝损伤的药效及药理机制。结果网络药理学结果显示，鼠曲草总黄酮调控APAP肝损伤的主要活性成分为槲皮素、木樨草素、卡脲酸、山奈酚，GO功能富集分析得到GO条目632个，其中生物过程472个，细胞组成42个，分子功能条目118个。KEGG富集分析显示，鼠曲草总黄酮调节APAP肝损伤的通路主要涉及铁死亡相关信号通路。动物验证实验显示，鼠曲草总黄酮治疗组的谷丙转氨酶、谷草转氨酶均降低（ P<0.05），肝脏组织病理和炎症浸润均有所改善。鼠曲草总黄酮可减轻肝组织内铁沉积（ P<0.05），改善脂质过氧化相关指标（ P<0.05）；促进Nrf2、HO-1、SLC7A11、GPX-4蛋白表达，抑制keap1蛋白表达（ P<0.05）。结论通过网络药理学和动物实验可推断鼠曲草总黄酮激活Nrf2/SLC7A11/GPX-4信号通路调控肝细胞铁死亡对APAP肝损伤起到预防和治疗的作用。

关键词: 鼠曲草总黄酮, Nrf2/SLC7A11/GPX-4, 铁死亡, 网络药理学, 急性肝损伤

Abstract:

Objective To investigate the protective effect of total flavonoids of Salvia divinorum extract against acetaminophen (APAP)-induced acute liver injury (ALI) and its molecular mechanism. Methods The main chemical constituents of total flavonoids of Salvia divinorum were obtained through literature search, and their pharmacological mechanisms were predicted using bioinformatics analysis. In a mouse model of APAP-induced ALI, the protective effects of 100, 200 and 400 mg/kg total flavonoids of Salvia miltiorrhiza and 150 mg/kg bifidus were evaluated by observing changes in blood biochemistry and liver histopathology and detecting expressions of the key proteins in the Nrf2/HO-1 signaling pathway. Results Network pharmacology analysis suggested that the main active components in total flavonoids of Salvia divinorum for regulating APAP-induced liver injury included quercetin, lignocerol, caruric acid, and kaempferol, for which GO function enrichment analysis yielded 632 GO entries, including 472 involving biological processes, 42 involving cellular composition, and 118 involving molecular function. KEGG enrichment analysis showed that the total flavonoids of Salvia divinorum regulated APAP-induced liver injury mainly through ferroptosis-related signaling pathway. In mice with APAP-induced ALI, treatment with the total flavonoids significantly lowered ALT and AST levels, improved liver histopathology and inflammatory cell infiltration, reduced iron deposition in liver tissues, improved lipid peroxidation-related indexes, promoted the expressions of Nrf2, HO-1, SLC7A11, and GPX-4 proteins, and inhibited the expression of keap1 protein. Conclusion The total flavonoids of Salvia divinorum alleviate APAP-induced ALI in mice possibly by suppressing hepatocyte ferroptosis via activating the Nrf2/SLC7A11/GPX-4 signaling pathway.

Key words: total flavonoids of Salvia divinorum, Nrf2/HO-1, ferroptosis, network pharmacology, acute liver injury

蔡华俊, 陈致岐, 胡文婷, 谭伟, 吴昊, 王超. 鼠曲草总黄酮通过激活Nrf2/SLC7A11/GPX-4信号通路抑制肝细胞铁死亡缓解对乙酰氨基酚诱导的小鼠急性肝损伤[J]. 南方医科大学学报, 2024, 44(11): 2201-2208.

Huajun CAI, Zhiqi CHEN, Wenting HU, Wei TAN, Hao WU, Chao WANG. Total flavonoids of Salvia miltiorrhiza alleviate acetaminophen-induced acute liver injury in mice by suppressing hepatocyte ferroptosis via activating the Nrf2/HO-1 signaling pathway[J]. Journal of Southern Medical University, 2024, 44(11): 2201-2208.

图/表 8

图1 网络药理学结果

Fig.1 Result of network pharmacology analysis. A: Intersection target Wayne plot of the ethanol extract of Herba Desmodii Styracifolii and liver injury caused by acetaminophen. B: Protein-protein interaction (PPI) network. C: Drug-active ingredient-target-disease network.

图2 GO分析( A)和KEGG富集分析图( B)

Fig.2 GO analysis ( A) and KEGG enrichment analysis diagram ( B).

图3 TFM对血清中ALT、AST水平的影响

Fig.3 Effect of total flavonoids of Salvia divinorum extract (TFM) on serum ALT and AST levels in mice with APAP-induced ALI ( Mean±SD, n=6). ** P<0.01 vs control group; #P<0.05, ##P<0.01 vs APAP group.

图4 TFM减轻APAP诱导的肝损伤(HE染色)

Fig.4 Pathological changes in the liver tissues of mice with APAP-induced ALI treated with TFM (HE staining).

图5 TFM对小鼠血清炎症因子TNF-α、IL-6的影响

Fig.5 Effect of TFM on serum inflammatory factor TNF- α and IL-6 in the mice. ** P<0.01 vs control group; #P<0.05, ##P<0.01 vs APAP group.

图6 TFM对肝组织中4-HNE( A)、MDA( B)、ROS( C)、GSH( D)含量的影响

Fig.6 Effects of TFM on the contents of 4-HNE ( A), MDA ( B), ROS ( C), and GSH( D) in the liver tissues of the mice. ** P<0.01 vs control group; #P<0.05, ##P<0.01 vs APAP group.

图7 TFM减少了肝组织中的铁沉积

Fig.7 TFM reduces iron deposition in the liver tissues of the mice. A: Perls staining; B: Quantitative results ofPerls staining. ** P<0.01 vs control group; #P<0.05, ##P<0.01 vs APAP group.

图8 TFM对APAP肝损伤的铁死亡相关蛋白的表达的影响

Fig.8 　Expression levels of Nrf2, HO-1, keap1, GPX-4 and SLC7A11 proteins in the liver tissues of the mice detected by Western blotting. **P<0.01vs control group; #P<0.05, ##P<0.01vs APAP group.

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