Cerebroprotective action of butylphthalide in acute ischemic stroke: Potential role of Nrf2/HO-1 signaling pathway

Stroke remains a significant health issue globally, and the limitations of current treatments underscore the urgent need for innovative therapeutic strategies. While systemic thrombolysis and mechanical thrombectomy are critical for restoring blood flow, their narrow treatment windows and potential complications highlight the importance of exploring alternative approaches [1]. Advances in stroke research suggest that several mechanisms that exacerbate ischemic injury include oxidative stress, thrombosis, NETosis, inflammation, and apoptosis [2,3].

Recently, ferroptosis has emerged as a significant topic of interest in stroke research. This iron-dependent form of non-apoptotic cell death is characterized by lipid peroxidation and has been implicated in neuronal damage during ischemia and reperfusion [4,5]. Understanding the role of ferroptosis in stroke could pave the way for new therapeutic strategies aimed at mitigating brain damage and improving outcomes. By targeting the pathways involved in ferroptosis, it may be possible to develop interventions that not only protect neurons but also enhance recovery after an ischemic event. This approach, alongside existing therapies, could help address the unmet need for effective stroke treatments.

Butylphthalide (3-n-butylphthalide or NBP), derived from celery seeds, has been shown to provide cerebroprotection in an animal model of ischemic stroke. It can ameliorate various pathological processes of ischemic stroke-induced brain injury and significantly improve local microcirculation blood flow in the ischemic area [6]. Previous studies have demonstrated that NBP may reduce acute brain injury by modulating key ferroptosis regulators, such as glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) [7,8].

In the September 2024 issue of Neurotherapeutics, Sun et al. found a new mechanism by which NBP may reduce acute brain injury. The authors provided evidence that the neuroprotective effect of NBP is due to the inhibition of ferroptosis, most likely related to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway [9]. The authors further demonstrated that NBP exerts cerebroprotective effects in cerebral ischemia-reperfusion injury, reducing lipid peroxidation, iron accumulation, and mitochondrial damage. Furthermore, they showed that NBP reduced markers of ferroptosis, including glutathione (GSH), nitric oxide (NO), and GPX4, with concomitant upregulation of Nrf-2 and HO-1 [9].

Nrf2 is one key regulator for the antioxidant response and plays a vital role in maintaining redox homeostasis. Upon activation, Nrf2 translocates to the nucleus, induces the expression of various antioxidants such as HO-1 and GPX4, which neutralize reactive oxygen species (ROS) and lipid peroxides, and protects cells from oxidative stress and ferroptosis. The activation of the Nrf2/HO-1 signaling pathway has been demonstrated to reduce injury following ischemic stroke [10]. While deletion of Nrf2 is associated with worsened stroke outcomes [11], Sun et al. used a pharmacological inhibitor of Nrf-2 (ML385) with NBP in a filament model of ischemic stroke in rats. They found that NBP treatment enhances the nuclear translocation of Nrf2 in the cerebral cortex with reduced cytosolic Nrf2 [9]. This translocation led to increased expression of HO-1 and helped to mitigate oxidative stress and ferroptosis. Treatment with an Nrf2 inhibitor significantly reversed the protective effects of NBP on markers of ferroptosis-lipid peroxidation, iron accumulation, and mitochondrial damage. The reduced infarct and improved neurological functional outcomes with NBP were reversed by the Nrf2 inhibitor, suggesting that Nrf2 may mediate cerebroprotective effect of NBP in acute ischemic stroke. Although the authors' findings suggest that the beneficial effect of NBP is via the Nrf2/HO-1 pathway, the possibility that ML-385 may have off-target effects cannot be ruled out. Further studies using an Nrf2 mutant strain are warranted to dissect the exact mechanism by which NBP exerts cerebroprotective effect in acute ischemic stroke.

Despite the promising findings of this study, there are several limitations that define directions for future investigations. While Nrf2 inhibition attenuates the cerebroprotective effects of NBP, the possibility that NBP may exert effects through alternative pathways other than Nrf2 cannot be ruled out. For example, it is known that Nrf2 activation results in inhibition of NF-ĸB pathways and suppression of pro-inflammatory cytokines production [12]. Ischemia-reperfusion injury can lead to necrosis, apoptosis, and autophagy [13]. NBP might exert cerebroprotection by its potential to inhibit inflammation or other cellular death pathways. Another limitation of the study is that the authors show the beneficial effect of NBP at 24 ​h following the onset of acute ischemic stroke but not long-term functional outcomes. In order to assess the translational potential of these preclinical results, it is essential to evaluate long-term functional outcomes and test the cerebroprotective effect of NBP in models including sex, age, and traditional comorbidities such as hypertension and obesity, which are risk factors of human stroke. The authors can also evaluate the cerebroprotective effect of NBP in a multicenter pre-clinical platform like the Stroke Preclinical Assessment Network (SPAN) [14,15].

In conclusion, the findings by Sun et al. suggest that the cerebroprotective effect of NBP in acute ischemic stroke may be related to the activation of the Nrf2/HO1 signaling pathway. These findings further support NBP as a promising therapeutic agent that may warrant future investigation in clinical trials of patients who have undergone mechanical thrombectomy to improve functional outcomes following acute ischemic stroke.

Authors Contributions

Dr. Patel: Wrote original draft of the manuscript.

Dr Chauhan: Reviewed and edited the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.