Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates both oxidative stress and mitochondrial biogenesis. Our previous study reported the cardioprotection of calycosin against triptolide toxicity through promoting mitochondrial biogenesis by activating nuclear respiratory factor 1 (NRF1), a coregulatory effect contributed by Nrf2 was not fully elucidated. This work aimed at investigating the involvement of Nrf2 in mitochondrial protection and elucidating Nrf2/NRF1 signaling crosstalk on amplifying the detoxification of calycosin. Results indicated that calycosin inhibited cardiomyocytes apoptosis and F-actin depolymerization following triptolide exposure. Cardiac contraction was improved by calycosin through increasing both fractional shortening (FS%) and ejection fraction (EF%). This enhanced contractile capacity of heart was benefited from mitochondrial protection reflected by ultrastructure improvement, augment in mitochondrial mass and ATP production. NRF1 overexpression in cardiomyocytes increased mitochondrial mass and DNA copy number, whereas NRF1 knockdown mitigated calycosin-mediated enhancement in mitochondrial mass. For nuclear Nrf2, it was upregulated by calycosin in a way of disrupting Nrf2-Keap1 (Kelch-like ECH associated protein 1) interaction, followed by inhibiting ubiquitination and degradation. The involvement of Nrf2 in mitochondrial protection was validated by the results that both Nrf2 knockdown and Nrf2 inhibitor blocked the calycosin effects on mitochondrial biogenesis and respiration. In the case of calycosin treatment, its effect on NRF1 and Nrf2 upregulations were respectively blocked by PGC alpha/Nrf2 and NRF1 knockdown, indicative of the mutual regulation between Nrf2 and NRF1. Accordingly, calycosin activated Nrf2/NRF1 and the signaling crosstalk, leading to mitochondrial biogenesis amplification, which would become a novel mechanism of calycosin against triptolide-induced cardiotoxicity.

Background: Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms. Methods: The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID₅₀), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman’s algorithm. Results: This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis. Conclusion: This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication. Graphical

Background: According to traditional Chinese medicine (TCM), drugs supplementing the vital energy, Qi, can eliminate tumors by restoring host immunity. The objective of this study is to investigate the underlying immune mechanisms of anti‐tumor activity associated with Qi‐supplementing herbs, specifically the paired use of Huangqi and Danggui.Methods: Analysis of compatibility regularity was conducted to screen the combination of Qi‐supplementing TCMs. Using the MTT assay and a transplanted tumor mice model, the anti‐tumor effects of combination TCMs were investigated in vitro and in vivo. High content analysis and flow cytometry were then used to evaluate cellular immunity, followed by network pharmacology and molecular docking to dissect the significant active compounds and potential mechanisms. Finally, the anti‐tumor activity and the mechanism of the active ingredients were verified by molecular experiments.Results: There is an optimal combination of Huangqi and Danggui that, administered as an aqueous extract, can activate immunity to suppress tumor and is more effective than each drug on its own in vitro and in vivo. Based on network pharmacology analysis, PIK3R1 is the core target for the anti‐tumor immunity activity of combined Huangqi and Danggui. Molecular docking analysis shows 6 components of the combined Danggui and Huangqi extract (quercetin, jaranol, isorhamnetin, kaempferol, calycosin, and suchilactone) that bind to PIK3R1. Jaranol is the most important component against breast cancer. The suchilactone/jaranol combination and, especially, the suchilactone/kaempferol combination are key for immunity enhancement and the anti‐tumor effects of the extract.Conclusions: The combination of Huangqi and Danggui can activate immunity to suppress breast cancer and is more effective than the individual drugs alone.