## 4 Discussion
On the other hand, after crossing the blood-brain barrier, LA-1 acts directly on neurons by scavenging intracellular ROS and restoring the expression of antioxidant enzymes such as SOD, CAT, and GSH, ultimately inhibiting ROS-induced neuronal apoptosis. This dual-action mechanism explains the neuroprotective effects of LA-1 in ischemia-reperfusion injury. It not only demonstrates LA-1's capacity to clear ROS in endothelial cells but also emphasizes its direct antioxidant and anti-apoptotic effects on neurons. Overall, the neuroprotective effects of LA-1 are achieved by reducing oxidative stress, suppressing inflammatory responses, and preventing neuronal death through multiple pathways. During cerebral ischemia and reperfusion, the increased release of arachidonic acid results in an accumulation of lipid peroxides [44], while the intracellular calcium overload eliminates mitochondrial oxidative phosphorylation, reduces mitochondrial membrane potential, and causes excessive reactive oxygen species (ROS) generation [45]. The excessive production of lipid peroxides and ROS exacerbates oxidative stress responses, which in turn induce neuronal apoptosis [46]. Endogenous antioxidant mechanisms play a balancing role, and it is essential to scavenge ROS by upregulating antioxidant enzymes [47]. Nrf is considered a major regulator of redox homeostasis [48], regulating antioxidant and anti-inflammatory responses [49]. Under oxidative stress, activated Nrf binds to antioxidant response elements (AREs) to promote detoxification and the production of antioxidant enzymes such as HO-1, NQO1, and GSH [50]. Previous studies have shown that overexpression of HO-1 significantly reduces cerebral infarction size in permanent MCAO experiments [51]. We detected significant decreases in antioxidant enzymes, such as SOD, CAT, and GSH, and the reduced activity of the Nrf/HO-1 signaling pathway in MCAO rat brains. After treatment with LA-1, the levels of those dysregulated proteins and the activity of the Nrf/HO-1 signaling pathway were partly restored, indicating the mechanism of neuroprotection of LA-1 may be associated with oxidative stress response pathways. Both the increased release of arachidonic acid and the intracellular calcium overload are associated with the reduced activity of WNT signaling pathway during cerebral ischemia-reperfusion injury [52] Inhibition of Wnt/\(\beta\)-catenin signaling was reported in ischemic stroke patients and corresponding mouse models, which is considered to contribute to the release of inflammatory factors TNF-\(\alpha\), IL-1, IL-6,and IL-8, and aggravates the inflammatory response [53]. Our study also observed increases of pro-inflammation cytokines TNF-\(\alpha\) and IL-1\(\beta\) in MCAO rat brains. Similar to antioxidant enzymes, the increases of pro-inflammation cytokines were reversed by LA-1 treatment. Therefore, we hypothesized that regulation of WNT signaling pathway may play an essential role in the neuroprotection of LA-1 against cerebral ischemia-reperfusion injury. By performing proteomic analysis, we found that PI3K associated pathways may be involved in cerebral ischemia-reperfusion injury. PI3K/Akt regulates pathological and physiological processes such as cell apoptosis [54], neuroinflammation, and oxidative stress [55]. Activation of the PI3K/Akt signaling pathway was reported to reduce apoptosis by enhancing Bcl-2 expression in brain ischemia-reperfusion injury [56]. Furthermore, the PI3K/Akt/Wnt signaling crosstalk was identified as a critical pathological process in brain ischemia-reperfusion injury. As the PI3K/AKT signaling pathway is positioned upstream of Wnt/\(\beta\)-catenin signaling, activation of PI3K/Akt signaling pathway simultaneously activates Wnt/\(\beta\)-catenin signaling [52]. To validate the mechanisms of neuroprotection of LA-1, we detected the activation of PI3K/Akt pathway by Western blot. Our results revealed that LA-1 restored the activation of PI3K/Akt pathway, whose functions were inhibited in ischemia-reperfusion injury.