Integrating UHPLC-HRMS with SSA-BP neural network identifies optimal components of Astragali Radix‑Salviae Miltiorrhizae combination for mitigating cerebral ischemia-reperfusion injury via mitophagy and ferroptosis pathways.

Authors

Chao Guo, Dong Xu, Zhongying Ma, Kai Gao, Wei Zhang, Yang Du, Meina Zhao, Xingru Tao, Yi Ding, Jingwen Wang

Published in

Phytomedicine : international journal of phytotherapy and phytopharmacology. Volume 160. Pages 158658. Jul 28, 2026. Epub Jul 28, 2026.

Abstract

Astragali Radix (Huangqi) and Salvia miltiorrhiza (Danshen) represent a frequently paired herbal combination in traditional Chinese medicine for tonifying Qi and promoting blood circulation. Previous pharmacological investigations have shown that phytochemicals derived from these herbs exhibit neuroprotective properties against cerebral ischemia-reperfusion (CI/R) injury. Nevertheless, the underlying principles governing the combined application of Huangqi and Danshen (QD) and the molecular pathways involved in their therapeutic efficacy remain unexplored in ischemic stroke management.
This study sought to systematically investigate the bioactive components in the QD formulation and elucidate their mechanisms of action against cerebral ischemic injury.
The therapeutic effects of QD were evaluated in a mouse model of middle cerebral artery occlusion (MCAO). UHPLCHRMS was used to identify QD-derived components in blood and brain tissues. An SSA-BP neural network was constructed to predict the optimal combination of active ingredients. 4D label-free proteomics, coupled with GO and KEGG enrichment analyses, was performed to identify key pathways. Molecular docking and molecular dynamics simulations were used to validate candidate targets. Mitochondrial function and iron homeostasis were assessed by measuring ROS, mitochondrial membrane potential (ΔΨm), ATP, complex I activity, Fe²⁺, and the GSH/GSSG ratio. Western blotting and immunofluorescence were used to detect FUNDC1, Nrf2, SLC7A11, NCOA4, p62, FTH1, UQCRC2, and GPX4. The autophagy inhibitor 3-MA was used for mechanistic validation. For cellular studies, HT22 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), and QD was added during reoxygenation. Cell viability and cytotoxicity were assessed using CCK-8 and LDH assays, respectively. ROS, ΔΨm, Fe²⁺, and GSH were measured using commercial kits. Western blotting was used to analyze FUNDC1, UQCRC2, LC3-II/I, p62, GPX4, SLC7A11, NCOA4, Nrf2, and FTH1. siFUNDC1 was transfected 48 h before OGD/R to assess FUNDC1 dependency.
Among the tested ratios (1:1, 2:1, 3:1, 3:2, and 2:3), the 3:2 QD combination was the most effective, significantly improving neurological function, reducing infarct size, and alleviating neuronal damage in MCAO mice. UHPLCHRMS identified 21 compounds absorbed into the bloodstream, seven of which were detected in brain tissue. Astragaloside IV, astragaloside II, lithospermic acid, tanshinone IIA, and calycosin were identified as key active components. Their combination, predicted by the SSA-BP neural network, exerted significant neuroprotection in vivo. Proteomics revealed 124 differentially expressed proteins, with GO and KEGG enrichment analyses identifying mitophagy and ferroptosis as the primary therapeutic pathways. Molecular docking and dynamics analyses revealed high-affinity binding interactions between QD constituents and key targets, including FUNDC1, UQCRC2, GPX4, and SLC7A11. QD upregulated FUNDC1, UQCRC2, GPX4, SLC7A11, Nrf2, and FTH1 and downregulated p62 and NCOA4; these effects were partially reversed by 3-MA. In HT22 cells, QD improved cell viability, reduced LDH release, restored ΔΨm and GSH levels, and attenuated ROS and Fe²⁺ accumulation following OGD/R. The protein expression changes were consistent with those observed in vivo. Knockdown of FUNDC1 largely blocked the protective effects of QD, confirming that FUNDC1 is essential for QD-mediated neuroprotection.
QD ameliorates MCAO-induced cerebral ischemic injury via mitophagy and ferroptosis pathways, offering a novel therapeutic perspective for treating ischemic stroke with traditional Chinese medicine that tonifies Qi and promotes blood circulation.

PMID:
42542064
Bibliographic data and abstract were imported from PubMed on 02 Aug 2026.

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