BEHAVIOURAL BRAIN RESEARCH
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2024
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IF 2.6
Chenjie Ge,Shiliang Wang,Xuqi Wu,Lilei Lei
Background Depression is a common psychiatric disorder with limited effective treatments. Research suggests that depression involves apoptosis mechanisms. Quercetin (QUE) has been reported to have anti-apoptotic activities. In this study, we aimed to investigate the effects and mechanisms of QUE in chronic unpredictable mild stress (CUMS)-induced depression. Methods After establishing mouse models of CUMS-induced depression, the mice were randomly assigned into four groups: control, CUMS, CUMS+QUE, and CUMS+Fluoxetine (FLX). The body weight of the mice was measured during the study. Then, depression-associated behaviors were evaluated using the sucrose preference test (SPT), novelty suppressed feeding test (NSFT), forced swim test (FST) and tail suspension test (TST). Apoptosis in the hippocampus and prefrontal cortex was determined using flow cytometry. Bcl-2 and Nrf2 protein expressions in the hippocampus and prefrontal cortex were also detected. Furthermore, Western blot was used to measure the protein levels of p-ERK, ERK, p-CREB, CREB, and Nrf2 in brain tissues. Results QUE or FLX administration increased the body weight of the CUMS mice. Behavioral tests indicated that CUMS mice developed a state of depression, but QUE or FLX treatment improved their depression-associated behaviors. Meanwhile, QUE or FLX treatment decreased apoptosis in the hippocampus and prefrontal cortex. Furthermore, the decreased Nrf2 protein expression, ERK and CREB phosphorylation in CUMS group were enhanced by QUE or FLX administration. Conclusion QUE could attenuate brain apoptosis in mice with CUMS-induced depression, and the mechanism may be related to the ERK/Nrf2 pathway, indicating that QUE could be a potential treatment for depression.
研究领域:
神经科学
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药理学
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Cell Death & Disease
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2025
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IF 9.6
Ren Yuanyuan,Cui Yue,Wang Zhen,Luo Yizhi,Jin Junchang,Yuan Yiyi,Li Xuan,Zhang Yaning,Cao Nan,Li Xiaofang,Yu Yi,Xiong Yuyan
Gastric cancer (GC) is a prevalent gastrointestinal malignancy in which ferroptosis, mitochondrial dysfunction, and macrophage reprogramming remarkably contribute to disease progression. However, the molecular interplay among these processes in contributing to GC remains poorly understood. In this study, we identified ferroptosis- and mitochondrial dysfunction-related genes (FMDRGs) implicated in GC through bioinformatics analyses. Among them, aldehyde dehydrogenase 3 family member A2 (ALDH3A2) was identified as a key FMDRG significantly downregulated in GC tissues and cell lines. Functional assays revealed that ALDH3A2 overexpression in GC cell lines suppressed proliferation, migration, and invasion while enhancing ferroptosis, effects that were reversed by GPX4 overexpression. ALDH3A2 also impaired the mitochondrial unfolded protein response (UPR mt ) and induced mitochondrial dysfunction. Restoration of UPR mt ameliorated ALDH3A2-induced mitochondrial dysfunction and ferroptosis. Mechanistically, ALDH3A2 impaired UPR mt by downregulating SLC47A1 through blockade of NRF2 nuclear translocation, leading to mitochondrial dysfunction, GPX4 downregulation, lipid peroxidation, and subsequent ferroptosis. Synergistically, ALDH3A2-induced ferroptosis promoted IL-6 release, which drove macrophage polarization toward the M1 phenotype with elevated IL-1β production. This macrophage reprogramming, in turn, inhibited GC cell progression by downregulating PD-L1 expression. Therapeutically, both genistein treatment and ALDH3A2 overexpression significantly attenuated GC progression in vitro and in vivo. These findings elucidate ALDH3A2 as a dual regulator of tumor-intrinsic ferroptosis and tumor-extrinsic immune remodeling in contributing to GC pathogenesis, highlighting its potential as a promising therapeutic target in GC.
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FOOD AND CHEMICAL TOXICOLOGY
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2026
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IF 3.5
Chenghao He,Jiaxin Jiang,Shuguang Hou,Runchun Xu,Qinwan Huang
Perfluorohexanesulfonic acid (PFHxS), a prevalent short-chain per- and polyfluoroalkyl substance, has been implicated in hepatocellular carcinoma (HCC), but mechanisms remain unclear. We integrated transcriptomic analyses of The Cancer Genome Atlas (TCGA) liver cancer cohort and an HCC single-cell dataset with molecular docking and molecular dynamics simulations and in vitro assays to examine a PFHxS-relevant hypothesis involving KEAP1–NRF2 signaling. Across clinical datasets, higher NQO1, an NRF2-associated gene, was linked to adverse clinicopathologic features; NQO1-high tumor cells showed elevated NRF2-activity signatures and computationally inferred increased MIF signaling toward macrophages. Because exposure information is unavailable, these observations indicate association and define a PFHxS-relevant vulnerability axis rather than PFHxS-driven tumor states. Docking/dynamics suggested PFHxS can bind the KEAP1 Kelch domain near the NRF2-binding site. In HepG2 cells, PFHxS modestly increased viability/DNA-synthesis readouts and enhanced NRF2 nuclear localization, NQO1 protein abundance, and MIF secretion; pharmacologic NRF2 inhibition partially attenuated NRF2/NQO1 readouts and reduced MIF secretion. Together, the data support the hypothesis that PFHxS may engage a KEAP1–NRF2-related vulnerability axis, accompanied by NRF2/NQO1 pathway readouts and increased MIF secretion, motivating exposure-characterized and genetic studies to establish causality.
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LIFE SCIENCES
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2026
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IF 5.1
Wei Hua,Yilei Sun,Mingze Sun,Yiying Liu,Hongxue Sun,Huinan Chen,Shuainan Ma
Aims Neuronal ferroptosis affects the pathogenesis of diabetes-associated cognitive decline (DACD). Exogenous hydrogen sulphide (H 2 S) treatment ameliorates cognitive deficits in diabetic mice. This study aimed to explore whether H 2 S exerts neuroprotective effects by suppressing ferroptosis through activation of Nrf2/GPX4 signalling pathway. Materials and methods STZ-induced diabetic mice received NaHS or NaHS combined with the Nrf2 inhibitor ML385, after which cognitive functions were evaluated using the Morris water maze test. Hippocampal neuronal damage and mitochondrial morphological changes were identified using transmission electron microscopy, haematoxylin and eosin (H&E) staining, Nissl staining, and immunofluorescence staining. The expression levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and iron were determined using assay kits. Western blotting was performed to evaluate the expression of the Nrf2/GPX4 axis, iron transport-related proteins, and GSH synthesis enzymes. Key findings Exogenous H 2 S treatment ameliorated cognitive deficits by mitigating hippocampal neuronal loss and improving synaptic ultrastructural integrity. The levels of ROS, MDA, and iron were reduced, whereas SOD, GSH, and Nrf2/GPX4 expression were increased following NaHS administration. Exogenous H 2 S also ameliorated mitochondrial impairment and suppressed ferroptosis in the hippocampal neurones. However, these neuroprotective effects were abolished by ML385. Diabetic mice cotreated with ML385 and NaHS exhibited prolonged escape latency and reduced time in the target quadrant. Additionally, ML385 co-administration with NaHS exacerbated neuronal damage, oxidative stress, synaptic ultrastructural abnormalities, and iron dysregulation, while downregulating Nrf2/GPX4 expression and inhibiting ferroptosis suppression. Significance Exogenous H 2 S treatment alleviates DACD by suppressing ferroptosis via activation of the Nrf2/GPX4 signalling pathway.
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BIOLOGICAL & PHARMACEUTICAL BULLETIN
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2026
·Vol. 49
·期 7
·1108-1118
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IF 1.8
Binjing Zhao,Xinyi Jiang,Xiaojing Wei,Lunli Lan,Rui Wang,Yuanyuan Li
Prolonged or high-dose administration of nonsteroidal anti-inflammatory drugs (NSAIDs) can induce gastric mucosal injury (GMI), potentially progressing to gastric ulcer (GU). Studies have shown that oxidative stress and necroptosis were closely associated with GU. Vladimiria souliei, a traditional Chinese herb, has been used to treat digestive disorders, particularly GU, but its pharmacological mechanisms remain unidentified. This study aimed to investigate the pharmacological mechanisms of GMI treatment in this herb. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to analyze blood-enterable components. Aspirin (ASP) was orally administered to induce GMI in mice. After intervention with Vladimiria souliei extract (VSE), the morphology of gastric tissues was observed, and the levels of pepsin and prostaglandin E2 (PGE2) were detected. Further therapeutic evaluation of VSE in GMI was performed using hematoxylin and eosin (H&E) and periodic acid-schif (PAS) staining. Serum levels of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were measured, while immunohistochemistry and Western blot were used to assess nuclear factor erythroid 2-related factor 2 (Nrf2) and receptor interacting protein kinase 1 (RIP1)-related signaling. Six prototype components were identified in the drug-containing serum of the VSE. Moreover, VSE mitigated ASP-induced GMI with shrunken areas of ulcers and promoted gastric mucin secretion, increased the levels of SOD and PGE2, and decreased the levels of pepsin, MDA, IL-1β, IL-6, and TNF-α. Pharmacological studies revealed that VSE upregulated the expression of Nrf2, NAD (P) H quinone dehydrogenase 1 (NQO1), and heme oxygenase-1 (HO-1) and down-regulated the expression of kelch-like ECH-associated protein-1 (Keap1), RIP1, RIP3, mixed lineage kinase domain-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), and dynamin-related protein 1 (Drp1) in the gastric mucosa. Therefore, VSE alleviated oxidative stress and necroptosis by regulating Nrf2 and RIP1 signaling in ASP-induced GMI mice, providing a theoretical basis for GU treatment and for the development of medicinal resources with this herb.
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Biology Direct
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2026
·Vol. 21
·期 1
·113
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IF 5.5
Wang Wei,Hu Yuanyuan,Xu Yan,Ding Ning,Wei Jiping,Li Cairong,Chen Yong
Background Doxorubicin (DOX) is a widely used broad-spectrum chemotherapy drug, but its severe organ toxic and side effects limit its clinical application. Tetrahydropalmatine (THP) protects against DOX-induced renal injury, yet its underlying mechanism remains unclear. This study aimed to verify THP’s protective effect and explore if it acts via the SIRT3-mediated Nrf2/HO-1 signaling pathway. Methods This study adopted an integrated in vivo and in vitro research system to explore the relevant mechanisms. In vivo, a DOX-induced mouse renal injury model was established with THP intervention; renal function was evaluated by detecting serum creatinine (Scr) and blood urea nitrogen (BUN), while renal injury severity was assessed via urine microalbumin (mALB) and urine albumin/creatinine ratio (UACR). Renal histopathological changes including glomerular injury and tubulointerstitial fibrosis were observed, and subcellular structures such as podocyte integrity and mitochondrial morphology were analyzed by transmission electron microscopy (TEM); additionally, the expression of pathway-related proteins was detected. In vitro, a DOX-induced cell injury model was constructed using mouse podocytes (MPC-5), and Western blot and immunofluorescence techniques were employed to determine the expression of key molecules in the Sirt3-Nrf2/HO-1 pathway, combined with in vivo and in vitro experiments to clarify the underlying mechanism. Results THP significantly attenuated DOX-induced renal injury, improved renal function (reduced Scr/BUN), mitigated histopathological/mitochondrial abnormalities, and preserved podocyte integrity. Molecularly, THP upregulates podocyte markers Nephrin/Podocin, enhances antioxidant capacity (increased GSH, SOD, and CAT, decreased MDA), inhibits inflammation (downregulated IL-6, IL-1β, and TNF-α), and suppresses apoptosis (downregulated Bax/caspase-3, upregulated Bcl-2). Notably, both in vivo and in vitro data link THP’s protection to SIRT3-mediated Nrf2/HO-1 pathway activation. Conclusion THP protects against DOX-induced renal injury in mice. Mechanistically, this may involve activating the SIRT3-mediated Nrf2/HO-1 pathway, improving podocyte function, and inhibiting oxidative stress, inflammation, and apoptosis.
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PHYTOMEDICINE
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2026
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IF 11.3
Ludong Tan,Xiaodong Bai,Peiyuan Yu,Tinghan Jiang,Zhe Jin
Artemisia capillaris extract alleviates acetaminophen-induced liver injury by inhibiting ferroptosis. • Capillarisin is identified as the primary bioactive constituent responsible for the hepatoprotection. • Capillarisin directly binds to and inhibits Glycogen Synthase Kinase 3β (GSK3β). • Inhibition of GSK3β by Capillarisin activates the Nrf2-mediated anti-ferroptotic pathway. • This study reveals a novel molecular mechanism for a traditional hepatoprotective herb. Background Acetaminophen (APAP) overdose is a leading cause of acute liver failure worldwide, characterized by oxidative stress-driven hepatocyte necrosis. Recent evidence implicates ferroptosis, an iron-dependent cell death, as a critical pathogenic event. Artemisia capillaris Thunb. (ACE) is a traditional hepatoprotective herb; however, its bioactive constituents and molecular targets regulating ferroptosis remain elusive. Purpose This study aimed to identify the bioactive compounds of ACE and elucidate the molecular mechanism underlying its protective effect against APAP-induced hepatotoxicity, with a specific focus on the ferroptosis pathway. Methods We integrated multi-omics, network pharmacology, and rigorous biophysical assays to decipher the protective mechanism. The ethanol extract of A. capillaris was chemically characterized using UPLC-LTQ-Orbitrap MS. An APAP-induced acute liver injury model in C57BL/6 mice and primary mouse hepatocytes were employed. Transcriptomics (RNA-seq) was utilized to identify key signaling pathways. The direct physical interaction between the active compound and its target was validated using Microscale Thermophoresis (MST), Cellular Thermal Shift Assay (CETSA), Drug Affinity Responsive Target Stability (DARTS), and molecular dynamics (MD) simulations. Functional validation was performed using gene overexpression and specific inhibitors. Results ACE pretreatment significantly attenuated APAP-induced liver injury, inflammation, and oxidative stress in mice. Transcriptomic profiling identified ferroptosis as a primary target pathway. Through chemical screening, we identified Capillarisin as the key bioactive coumarin responsible for this effect. Capillarisin treatment effectively suppressed lipid peroxidation, iron overload, and the expression of ferroptotic markers (COX2, ACSL4) while restoring GPX4 levels both in vivo and in vitro . Mechanistically, we demonstrated that Capillarisin acts as a direct inhibitor of Glycogen Synthase Kinase 3β (GSK3β). Capillarisin binds stably to the ATP-binding pocket of GSK3β ( K d = 0.87 µM), thereby inhibiting its kinase activity. This inhibition blocks the phosphorylation-dependent degradation of Nrf2, promoting its nuclear translocation and the subsequent transcriptional activation of anti-ferroptotic genes ( Gpx4, Slc7a11 ). Overexpression of GSK3β or pharmacological inhibition of Nrf2 abolished the protective effects of Capillarisin. Conclusion Our study reveals a novel mechanism whereby Capillarisin alleviates APAP-induced hepatotoxicity by directly targeting GSK3β to activate the Nrf2-mediated anti-ferroptosis defense. These findings provide a rigorous scientific basis for the ethnopharmacological use of Artemisia capillaris and highlight Capillarisin as a promising lead candidate for targeting GSK3β in the treatment of drug-induced liver injury. Download: Download high-res image (228KB) Download: Download full-size image
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TOXICON
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2026
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IF 2.8
Jing Li,Xiaoxiao Wang,Liya Tu,Yufeng Chen
Ovarian cancer (OC) exhibits the highest mortality rate among gynecological cancers, and the development of chemotherapy resistance serves as the primary cause of patient recurrence and death. Natural products hold promise in reversing cisplatin (DDP) resistance. Trigonelline is a natural alkaloid possessing anti-tumor activity; however, it remains uncertain whether it can reverse DDP resistance in OC. In this study, DDP-resistant OC cell lines SKOV-3/DDP and A2780/DDP were successfully established. Cells were treated with non-toxic concentration of trigonelline in combination with DDP, and the molecular mechanism was investigated through the overexpression of Nrf2. Cell viability was detected to assess the sensitivity of the cells to DDP. Mitochondrial damage was evaluated by observing mitochondrial morphology, measuring mitochondrial membrane potential, ATP production, and ROS. Ferroptosis was assessed by detecting the levels of lipid peroxidation, MDA, SOD, Fe 2+ , and GSH/GSSG ratio in the cells. Additionally, the expression levels of ferroptosis-related proteins, namely Nrf2, HO-1, GPX4, and SLC7A11, were also detected. The results indicated that the utilization of low-concentration trigonelline exerted no toxic influence on OC drug-resistant cells, and instead potentiated the cytotoxicity of DDP against these cells. Furthermore, in comparison with the treatment using DDP alone, the combined treatment of trigonelline and DDP induced more severe mitochondrial damage and ferroptosis in drug-resistant cells. This led to the accumulation of ROS, notable increases in lipid peroxidation, MDA, and Fe 2+ levels within the cells, while decreasing SOD activity and the ratio of GSH/GSSG, and down-regulating the expression of Nrf2, HO-1, GPX4, and SLC7A11 proteins. Nevertheless, the overexpression of Nrf2 reversed the combined effect of trigonelline and DDP. These results indicate that trigonelline reverses DDP resistance in OC cells by promoting ferroptosis via the regulation of the Nrf2/GPX4 signaling pathway.
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Animals
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2026
·Vol. 16
·期 12
·1828
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IF 3.2
Junyu Wang,Min Li,Yaqi Zhou,Fuyin Fu,Feng Liu,Jinghe Tan,Mingjiu Luo,Shuai Gong
Simple SummaryPoor porcine oocyte and embryo quality limits pig breeding efficiency. This study tested metformin supplementation in culture media to improve oocyte maturation and embryo development. Low-dose metformin enhanced maturation and blastocyst formation by activating AMPK through a non-canonical pathway, reducing oxidative damage and improving mitochondrial function, while high doses impaired oocyte quality. Appropriate metformin use optimizes in vitro culture systems, supporting more efficient pig embryo production for agriculture and reproductive research.Metformin (MET) plays crucial regulatory roles in mammalian oocyte meiosis, yet the concentration-dependent biphasic impacts of MET on porcine oocyte in vitro maturation (IVM) and the related molecular mechanisms remain poorly clarified. This study aimed to explore the distinct effects and underlying pathways of low- and high-dose MET in porcine oocytes. Different concentrations of MET (0, 7.5, 15, 30, 150, and 300 μM) were supplemented during oocyte IVM, with phenotypic detection, untargeted metabolomic analysis, and Nrf2 inhibitor (ML385) intervention performed for mechanism exploration. Results showed that 15 μM low-dose MET facilitated oocyte maturation, mitochondrial function and redox balance, while 300 μM high-dose MET caused obvious developmental damage. Mechanistically, low-dose MET triggered noncanonical AMPK activation independent of the AMP/ATP ratio and enhanced AMPK–Nrf2 antioxidant signaling, whereas high-dose MET induced energy stress and oxidative injury via inhibiting mitochondrial complex I. Blockade of Nrf2 further abolished the protective effects of low-dose MET. Collectively, this finding illustrates the biphasic actions of MET on porcine oocytes and provides a theoretical reference for optimizing porcine in vitro embryo production.
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Reproductive Biology
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2026
·Vol. 26
·期 3
·101239
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IF 2.5
Jing Wu,Yu Zeng,Xianyan Xiong,Hongmei Zhu
Preeclampsia (PE, a pregnancy-specific hypertensive disorder) is characterized by placental hypoxia, oxidative stress, metabolic dysfunction, and trophoblast impairment, yet effective disease-modifying therapies remain limited. Although low-dose aspirin (Acetylsalicylic acid, ASA) is recommended for the prevention of PE and has shown protective effects in pregnancy, the underlying placental mechanisms remain incompletely understood. Nuclear factor erythroid 2–related factor 2 (NRF2), a central regulator of cellular antioxidant defense, plays a critical role in counteracting oxidative stress, but its role in placental pathology and its contribution to ASA-mediated protection in PE have not been fully elucidated. In this study, placental tissues were collected from normotensive pregnancies and from patients with mild and severe PE, and relevant maternal clinical characteristics were recorded to contextualize findings. In parallel, trophoblast cells were exposed to physiological hypoxia or hydrogen peroxide (H₂O₂)–induced oxidative stress to mimic PE-relevant placental stress conditions, followed by ASA treatment. Trophoblast function, oxidative stress, and apoptosis were evaluated, and the involvement of NRF2 was examined using shRNA-mediated knockdown. Placental tissues from PE pregnancies, particularly those from severe cases, exhibited increased oxidative stress accompanied by dysregulated NRF2-dependent antioxidant signaling. In vitro, both hypoxia and oxidative stress significantly impaired trophoblast proliferation and invasion while increasing reactive oxygen species (ROS) accumulation and apoptosis. ASA treatment markedly reduced oxidative stress and improved trophoblast function under both stress conditions. Notably, genetic silencing of NRF2 largely abolished the antioxidative and cytoprotective effects of ASA. These findings indicate that ASA alleviates placental oxidative injury and trophoblast dysfunction, at least in part, through an NRF2-dependent mechanism, providing mechanistic insight into the placental protective effects of ASA in PE and highlighting the potential clinical relevance of ASA for high-risk pregnancies.
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PHYTOMEDICINE
·
2026
·
IF 11.3
Bing Yu,Lina Zhang,Jing Lin,Qian Wang,Menghui Chi,Ziyi Wang,Wenyao Liu,Shan Wang,Zhipeng Wei,Cui Li,Guiqiu Zhao
Background Fungal keratitis is a vision-threatening corneal disease, in which uncontrolled inflammation and oxidative damage often result in poor clinical outcomes. Current antifungal drugs exert fungicidal activity but often fail to control inflammatory injury, limiting their therapeutic efficacy. Therapies capable of enhancing fungal clearance while modulating host inflammation are urgently needed. Farrerol (FA), a natural flavanone, has anti-inflammatory, antioxidant, and antimicrobial properties, yet its role in fungal keratitis remains unclear. Purpose To investigate the therapeutic effects and mechanisms of FA in Aspergillus fumigatus -induced keratitis. Methods RT-qPCR, Western Blot, Co-IP, ELISA, and immunofluorescence staining were performed to assess the effects of FA on inflammatory responses and oxidative stress in fungus-stimulated macrophages. MIC assay, Calcofluor White staining, and plate counting method were used to examine the antifungal activity. A murine fungal keratitis model was established to determine the therapeutic efficacy in vivo . Results FA significantly alleviated corneal inflammation and fungal burden in vivo . Mechanistically, FA enhanced autophagic flux and promoted autophagy-dependent degradation of Keap1, resulting in Nrf2 nuclear translocation. The activation of autophagy and Nrf2 feedback loop suppressed the production of pro-inflammatory cytokines and ROS while enhancing macrophage-mediated intracellular killing of conidia. Inhibition of autophagy abolished these protective effects. Conclusions FA protects against fungal keratitis by enhancing fungal clearance and limiting excessive inflammation through activation of autophagy and the Nrf2 pathway. These findings identify FA as a promising therapeutic candidate and highlight autophagy–Nrf2 signaling as a potential target in fungal infectious diseases.
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PHYTOMEDICINE
·
2026
·
IF 11.3
Xiaojue Xiang,Xu Lin,Qifang Wu,Xiaoming Zhang,Xueliang Fu,Linya Wu,Haibin Tong,Zhenyu Dai
Dendrobium officinale extract DOE ameliorates metabolic disorders and diabetic cardiomyopathy. • DOE reduced myocardial injury, fibrosis, and oxidative stress via activating NRF2 pathway. • Dendrobine was the principal cardioprotective alkaloid in DOE against diabetic cardiomyopathy . • Dendrobine directly targets KEAP1 Kelch domain to disrupt the KEAP1-NRF2 interaction . Background Dendrobium officinale Kimura et Migo is a prestigious traditional Chinese medicine historically used for treating "Xiao-Ke" (diabetes and its complications). However, the specific material basis and direct molecular targets responsible for its efficacy against diabetic cardiomyopathy (DCM) remain largely obscure. This study aimed to systematically investigate whether dendrobine, the characteristic active alkaloid of DO, serves as the principal cardioprotective component by directly targeting the KEAP1-NRF2 signaling axis. Methods The therapeutic effects of D. officinale extract (DOE) and dendrobine were evaluated in vivo using db/db mice and in vitro using high-glucose (HG)-challenged H9c2 cardiomyocytes. Cardiac pathological remodeling, lipid peroxidation, and systemic metabolic profiles were comprehensively assessed. The direct interaction between dendrobine and KEAP1 was definitively characterized using Co-Immunoprecipitation (Co-IP), Cellular Thermal Shift Assay (CETSA), Drug Affinity Responsive Target Stability (DARTS), MicroScale Thermophoresis (MST), and 100-ns Molecular Dynamics (MD) simulations. Reverse pharmacological validation was performed using NRF2-specific siRNA and the inhibitor ML385. Results In vivo , DOE significantly ameliorated systemic insulin resistance, hyperlipidemia, and myocardial pathological remodeling in db/db mice while robustly suppressing myocardial oxidative stress (MDA, 4-HNE) by upregulating NRF2-driven antioxidant enzymes (HO-1, NQO1, GPX4). In vitro , dendrobine dose-dependently replicated these cardioprotective and antioxidative efficacies. Mechanistically, dendrobine promoted NRF2 nuclear translocation and initiated the downstream antioxidant cascade. Crucially, MST and CETSA revealed that dendrobine binds directly to KEAP1 with high affinity ( K d = 1.18 μM), functioning as a competitive protein-protein interaction (PPI) inhibitor to disrupt the KEAP1-NRF2 complex. MD simulations and mutagenesis validation pinpointed the Kelch domain of KEAP1 (specifically residue Arg415) as the binding pocket. Finally, silencing or inhibiting NRF2 abolished the anti-hypertrophic and cytoprotective effects of dendrobine, and the in vivo administration of dendrobine successfully recapitulated the metabolic regulation and KEAP1/NRF2-mediated cardioprotection of DOE in db/db mice. Conclusion Dendrobine, the principal alkaloid of D. officinale , effectively ameliorates diabetic cardiomyopathy by functioning as a novel, direct competitive inhibitor of the KEAP1-NRF2 interaction. This targeted disruption liberates NRF2 to restore myocardial redox homeostasis, highlighting dendrobine as a promising lead compound for DCM therapy. Download: Download high-res image (395KB) Download: Download full-size image
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