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Rosmarinic Acid Alleviates Ischemic Stroke by Targeting BAG3 to Modulate Autophagy via the P62-Keap1-Nrf2 Pathway
Introduction
Globally, ischemic stroke stands as a critical neurological emergency, notable for its widespread occurrence and severe repercussions, including elevated mortality and persistent functional deficits(Jolugbo and Ariens, 2021, Wang et al., 2024b). The increasing prevalence of ischemic stroke is largely driven by an aging global population and the rise in lifestyle-related risk factors, with more than 13 million new cases reported annually(Saini et al., 2021). Current clinical interventions primarily aim to restore cerebral blood flow, chiefly involving intravenous recombinant tissue plasminogen activator (rt-PA)-mediated thrombolysis or endovascular clot retrieval to reinstate cerebral perfusion(Tsivgoulis et al., 2023, Cui et al., 2024). Despite its exclusive FDA approval status among thrombolytics, rt-PA therapy faces dual constraints: narrow therapeutic window (≤4.5 hours post-onset) and significant risks including cerebral hemorrhage and reperfusion-related damage(Arkelius et al., 2024). Neuroprotective agents, including non-competitive NMDA receptor antagonists (e.g., memantine) and free radical scavengers (e.g., Ebselen), have demonstrated limited clinical success due to adverse effects, inadequate blood-brain barrier penetration, narrow therapeutic window, and off-target effects(Zhang et al., 2024, Narayan et al., 2021). These challenges highlight the pressing need for safer, more effective therapeutic strategies for ischemic stroke.
The development of cerebral ischemia triggers an intricate sequence of molecular and cellular perturbations, including ionic imbalance, energy depletion, excitotoxicity, oxidative stress, inflammatory responses, blood-brain barrier disruption, and autophagy dysregulation(Qin et al., 2022). Emerging research implicates autophagic processes as key modulators of ischemic stroke pathogenesis(Shi et al., 2023). This primordial self-renewal pathway, executed through lysosomal processing of cellular constituents, forms an indispensable biological infrastructure for sustaining functional stability. Recent studies have shown that ischemic stress dynamically modulates autophagic flux, with moderate activation of autophagy exerting cytoprotective effects by clearing damaged organelles and misfolded proteins during the early phase of ischemia(Hou et al., 2019). However, prolonged energy deprivation can convert this adaptive process into a detrimental one, wherein excessive autophagosome accumulation overwhelms lysosomal degradation capacity, ultimately leading to cellular damage(Hou et al., 2019, Mo et al., 2020). Studies indicate that moderate autophagy participates in reparative processes such as neurogenesis and synaptic plasticity, whereas excessive autophagy can lead to neuronal death(Ajoolabady et al., 2021). Therefore, targeting autophagy regulation holds promise not only for "life-saving" effects in the acute phase but also for long-term "repair promotion." Modulating autophagy shows promise in protecting neurons and potentially extending the therapeutic window. Currently, pharmacological interventions targeting autophagy pathways (e.g., Rapamycin, 3-MA, NCOA4 inhibitors) and natural compounds (e.g., Ginkgolide B, HSYA) have demonstrated therapeutic potential by fine-tuning autophagic activity(Wu et al., 2025). Clinical studies have reported elevated LC3-II/LC3-I ratios and decreased P62 levels in the serum of stroke patients, correlating with infarct size and unfavorable clinical outcomes(Wang et al., 2024a). Furthermore, experimental studies demonstrate that neuronal death and brain injury are alleviated in both cellular and animal models of ischemic stroke through pharmacological autophagy inhibition or genetic knockdown of critical autophagy mediators such as Beclin1 and ATG7. Additionally, triolein has been shown to mitigate ischemic injury by suppressing autophagy and inflammation via activation of the AKT/mTOR signaling pathway(Wang et al., 2024a). Collectively, these observations position the autophagy-lysosomal system as a viable therapeutic target for stroke intervention.
Traditional Chinese Medicine (TCM) and polyphenolic natural products represent a largely underexplored reservoir of neuroprotective agents for ischemic stroke, offering multi-target mechanisms that align well with the disease’s complex pathophysiology(Abdelsalam et al., 2023). Polyphenols, including epigallocatechin gallate (EGCG) and salvianolic acid B (Sal B), exhibit neuroprotective effects in cerebral ischemia by targeting multiple pathological pathways, such as attenuating ROS-mediated damage, suppressing neuroinflammatory cascades, and restoring mitochondrial bioenergetics(Park et al., 2024, Guo et al., 2022). Among these, rosmarinic acid (RosA), abundantly present in Salvia rosmarinus Spenn., has attracted significant attention due to its pleiotropic biological activities. A network pharmacology study has identified seven active compounds (RosA, chlorogenic acid, ferulic acid, Sal B, caffeic acid, (Z)-ligustilide, and tanshinone IIA), five core molecular targets (TNF-α, IL-1β, AKT, BCL2, and CASP3), and four key signaling pathways involved in the treatment of ischemic stroke(Pang et al., 2024). RosA has been reported to alleviate post-stroke depression in rats following transient focal cerebral ischemia by enhancing antioxidant defenses(Wang et al., 2021). In addition, RosA exerts neuroprotective effects through activation of the Nrf2 and HO-1 signaling pathways(Xu et al., 2021). Mechanistic studies further show that RosA attenuates cerebral injury and cognitive deficits in permanent MCAO-induced ischemic stroke, primarily through enhancing neuronal synaptic transmission, reducing inflammatory processes, and boosting brain-derived neurotrophic factor (BDNF) levels(Xu et al., 2021). Studies have confirmed that RosA exerts multiple effects in autophagy regulation. For instance, it can enhance autophagy by targeting to hepatocyte nuclear factor 4α (HNF4α)(Chen et al., 2025), activate the JAK2/STAT3/CTSC pathway to restore autophagic flux and lysosomal function(Luo et al., 2024), and regulate the phosphorylation level of ERK1/2 to modulate autophagy(Sengelen and Onay-Ucar, 2024). However, most current studies focus on its downstream signaling pathways or indirect target molecules, and the direct molecular targets of RosA in ischemic stroke remain unclear.
In this study, we demonstrated that RosA exerted neuroprotective effects following ischemic stroke in both in vitro and in vivo models. Utilizing a chemoproteomics approach, we designed a clickable RosA probe (RosA-p) and identified Bcl-2-associated athanogene 3 (BAG3) as a direct covalent target of RosA through activity-based protein profiling (ABPP) and cellular thermal shift assays (CETSA). BAG3 is a crucial autophagic adapter protein(Liu et al., 2023b, Sweeney et al., 2024). However, it remains unclear whether RosA can affect the autophagic process by binding to BAG3, regulating the expression or activity of BAG3. Elucidating this interaction represents a significant innovative aspect of this study. Our findings revealed that RosA covalently bound to cysteine 378 (Cys378) of BAG3 via Michael addition, thereby disrupting its interaction with the autophagy receptor P62. This interaction activated the P62/Keap1/Nrf2 signaling axis, leading to the suppression of autophagy and a reduction of cerebral infarction in ischemic stroke mice. Importantly, neuron-specific knockdown of BAG3 abolished the therapeutic effects of RosA, confirming BAG3 as a key mediator of RosA’s neuroprotective activity. These findings not only supported the therapeutic potential of RosA as a novel neuroprotectant but also validated BAG3 as a druggable target for ischemic stroke, offering a promising strategy for modulating autophagy in cerebrovascular disease.
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