TBN Protects Against Ischemic Brain Injury by Inhibiting RIPK1/RIPK3/MLKL-Mediated Neuronal Necroptosis

摘要

Objective: Necroptosis, identified as a regulated form of cell death in 2015, has been extensively studied and is now recognized as a key contributor to the pathophysiology of ischemic stroke at multiple stages of disease progression. In particular, it plays a significant role in shaping neurological recovery and neuroplasticity during the post-stroke recovery phase. Tetramethylpyrazine Nitrone(TBN), a multifunctional tetramethylpyrazine derivative containing a nitrone moiety, has previously demonstrated neuroprotective and neuroregenerative effects in various ischemic stroke models. This study aimed to investigate the therapeutic role of TBN in modulating necroptosis during the recovery stage of ischemic stroke and to elucidate the underlying molecular mechanisms. Methods: A transient middle cerebral artery occlusion(t-MCAO) model was established in C57 BL/6J mice. Following model induction, animals were randomly divided into six groups: Sham(n=24), Model(n=14), low-dose TBN(t-MCAO+TBN-L, n=10), medium-dose TBN(t-MCAO+TBN-M, n=19), highdose TBN(t-MCAO+TBN-H, n=15), and positive control edaravone group(t-MCAO+EdaPC, n=12). Drug administration was initiated 24 h after surgery and continued for six weeks. Neurological and behavioral functions were evaluated using the modified neurological severity score(mNSS), rotarod test(RT), pole test(PT), novel object recognition test(NOR), and Morris water maze(MWM) to comprehensively assess motor performance and cognitive capacity. Immunofluorescence(IF) staining was employed to quantify NEUN-, GFAP-, and Iba-1-positive cells in the cerebral cortex, thereby evaluating neuronal injury and neuroinflammatory responses. Transcriptomic alterations in the cortex were analyzed by RNA sequencing, and enrichment analyses were performed using KEGG and GO clustering to identify differentially expressed genes and signaling pathways relevant to disease progression and pharmacological intervention. Key transcriptomic findings were subsequently validated by RT-qPCR and Western blot assays. Results: Behavioral assessments demonstrated that TBN administration significantly improved motor deficits and neurological dysfunction in t-MCAO mice, indicating strong neuroprotective activity. TBN's efficacy was comparable to edaravone, with superior outcomes in memory restoration. Transcriptomic analysis revealed that TBN effectively corrected ischemia-induced dysregulation of several signaling pathways, including the NF-κB pathway. RT-qPCR analysis revealed that TBN significantly suppressed the post-stroke upregulation of Tnf, Ripk1, Ripk3, and Mlkl. Western blot further demonstrated that TBN significantly inhibited the phosphorylation of RIPK1 and MLKL, providing direct evidence that necroptosis is strongly activated in the postischemic recovery phase and that TBN treatment effectively attenuates this pathological process. Conclusion: Building upon prior research, this study provides the first demonstration that orally administered TBN exerts therapeutic efficacy during the recovery phase of ischemic stroke in a t-MCAO mouse model. Importantly, the findings reveal that TBN confers neuroprotection by suppressing RIPK1/RIPK3/MLKL-mediated neuronal necroptosis, thereby promoting functional recovery and reducing secondary injury. These results not only expand the current understanding of necroptosis as a therapeutic target but also provide novel mechanistic insights into the mode of action of TBN in ischemic stroke.

关键词

ischemic stroke / necroptosis / RIPK1/RIPK3/MLKL pathway / neuroprotection / post-stroke recovery phase