He Shi-Qi, Yang Si-Yan, Li Yuan, Ding Rong, Zhang Jie-Lin, Zhong Shi-Hong, Gu Rui
School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, China.
School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, China.
J Ethnopharmacol. 2026 Jan 30;355(Pt A):120612. doi: 10.1016/j.jep.2025.120612. Epub 2025 Sep 14.
Shilajit, a natural product utilized in traditional Tibetan medicine and known in China as ZhaXun (ZX), has been historically used for treating liver disorders. Recent studies have indicated that bioactive components in ZX, such as humic acid, fulvic acid, and isorhamnetin, exhibit anti-inflammatory, antioxidant, and immunomodulatory activities. These properties suggest its potential therapeutic relevance in alleviating acetaminophen (APAP)-induced acute liver injury (ALI).
This study aims to investigate the hepatoprotective effects of ZX against APAP-induced ALI.
The potential targets of ZX for ALI treatment were screened and then used to construct the protein-protein interaction (PPI) network. Core targets were identified using Cytoscape software, followed by GO and KEGG analyses. The effects of ZX on APAP-stimulated HepG2 cells and mice were determined by evaluating oxidative stress, inflammation and apoptosis. The potential mechanisms of ZX against ALI were verified by immunohistochemistry (IHC), immunofluorescence (IF), Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Western blotting (WB) methods.
Pretreatment with ZX at doses of 0.4, 0.8, 1.6 g/kg (administration by gavage) effectively attenuated APAP-induced liver injury (300 mg/kg), as confirmed by a decrease in plasma ALT and AST levels (75.0 % and 69.8 %, respectively). Network pharmacology analysis predicted ZX exerts a comprehensive protective effect against APAP-induced ALI by regulating oxidative stress, inflammatory response and apoptotic pathways. The results suggested that ZX not only ameliorated APAP-induced liver oxidative damage by regulating oxidative stress markers (SOD, MDA, CAT, GSH), but also diminished liver inflammation by inhibiting the levels of related inflammatory factors (TNF-α, IL-6, IL-1β). In addition, hepatocyte apoptosis was quantified by TUNEL staining. Finally, WB and RT-qPCR results further verified that ZX attenuated APAP-induced liver inflammation through PI3K/AKT and TLR4/NF-κB pathways. ZX inhibited hepatocyte apoptosis by balancing Bax/Bcl-2 expression and reducing Caspase-3 activation.
ZX alleviates APAP-induced ALI by attenuating inflammatory response and hepatocyte apoptosis. Both in vivo and in vitro experiments have demonstrated that ZX alleviates APAP-induced ALI through NF-κB/AKT/Caspase-3 pathways.
希拉季特是一种在传统藏药中使用的天然产物，在中国被称为查训（ZX），历史上一直用于治疗肝脏疾病。最近的研究表明，ZX中的生物活性成分，如腐殖酸、富里酸和异鼠李素，具有抗炎、抗氧化和免疫调节活性。这些特性表明其在减轻对乙酰氨基酚（APAP）诱导的急性肝损伤（ALI）方面具有潜在的治疗意义。
本研究旨在探讨ZX对APAP诱导的ALI的肝保护作用。
筛选ZX治疗ALI的潜在靶点，然后用于构建蛋白质-蛋白质相互作用（PPI）网络。使用Cytoscape软件鉴定核心靶点，随后进行基因本体（GO）和京都基因与基因组百科全书（KEGG）分析。通过评估氧化应激、炎症和细胞凋亡来确定ZX对APAP刺激的HepG2细胞和小鼠的影响。通过免疫组织化学（IHC）、免疫荧光（IF）、逆转录定量聚合酶链反应（RT-qPCR）和蛋白质印迹（WB）方法验证ZX抗ALI的潜在机制。
以0.4、0.8、1.6 g/kg的剂量（灌胃给药）用ZX预处理可有效减轻APAP诱导的肝损伤（300 mg/kg），血浆丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平降低（分别为75.0%和69.8%）证实了这一点。网络药理学分析预测，ZX通过调节氧化应激、炎症反应和凋亡途径对APAP诱导的ALI发挥全面的保护作用。结果表明，ZX不仅通过调节氧化应激标志物（超氧化物歧化酶（SOD）、丙二醛（MDA）、过氧化氢酶（CAT）、谷胱甘肽（GSH））改善APAP诱导的肝脏氧化损伤，还通过抑制相关炎症因子（肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β））的水平减轻肝脏炎症。此外，通过末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色对肝细胞凋亡进行定量。最后，WB和RT-qPCR结果进一步证实，ZX通过磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（AKT）和Toll样受体4（TLR4）/核因子κB（NF-κB）途径减轻APAP诱导的肝脏炎症。ZX通过平衡Bax/Bcl-2表达和降低半胱天冬酶-3（Caspase-3）激活来抑制肝细胞凋亡。
ZX通过减轻炎症反应和肝细胞凋亡来减轻APAP诱导的ALI。体内和体外实验均表明，ZX通过NF-κB/AKT/Caspase-3途径减轻APAP诱导的ALI。
