## 3 Results
### LA-1 Alleviates Brain Ischemia-Reverfusion Injury  
To investigate the effects of LA-1 on acute brain ischemia-reperfusion injury, we established a brain ischemia-reperfusion injury model in adult rats (Figure 5A). Following a 2 h induction with ischemia, we monitored cerebral blood flow dynamics using a laser speckle flow imaging system. The results showed a significant decrease in cerebral blood flow at the ischemic brain lesion in the MCAO model (Figure 5B), confirming the successful induction of the stroke model. After the occluding filament was removed, different treatments were administered to the MCAO rats via tail vein injection. Notably, treatment with LA-1 resulted in a marked increase in cerebral blood flow compared to the model group, highlighting the therapeutic effect of LA-1 in restoring blood flow. By performing magnetic resonance imaging analysis, we found that the brain damage was not prominent after 2 h of ischemia but was enhanced after 22 h of reperfusion (Figure 5F). Therefore, we evaluated the neurological function of MCAO model animals using the Zelonga score at 22 h post-treatment (Figure 5E). Most rats in the model group had a neurological score of 3, indicating worsening neurological deficits after MCAO surgery. The rats treated with 3 mg/kg of edaravone or 30 mg/kg loganin showed a reduction trend in neurological score but without statistically significant difference. In contrast, the treatment with 10 mg/kg of LA-1 reduced the scores significantly, suggesting that LA-1 may improve the neurological functions of rats after ischemia-reperfusion injury. TTC staining displayed a significant infarct area reduction in both edaravone and loganin treatment groups, consistent with the published data from other researchers [17; 37; 38]. In comparison with loganin, the infarct area in LA-1 group is more than 2-fold less than that of loganin group (7.743% vs. 16.843%), indicating that the neuroprotective effect of 10 mg/kg LA-1 is more potent than that of 30 mg/kg loganin. To evaluate the effects of LA-1 and Loganin on neuronal damage after cerebral ischemia-reperfusion, we performed H&E and Nissl staining (Figure 5G,H). In the H&E staining (Figure 5G), cortical neurons in the MCAO group showed significant damage, with disorganized arrangement and poor cellular structure compared to the sham group. LA-1 treatment improved these changes, restoring the regular arrangement and clearer structure of neurons. Loganin treatment also improved the morphology, though to a lesser extent than LA-1. Nissl staining (Figure 5H) revealed similar patterns, with the MCAO group displaying fewer neurons and weaker staining, indicating damage. LA-1 treatment significantly increased neuron count and restored Nissl substance staining, suggesting better neuroprotection. While Loganin also showed improvement, the recovery was less pronounced compared to LA-1. TUNEL staining (Figure 5I) assay displayed stronger green fluorescence in the MCAO group compared to the sham surgery group, indicating neuronal apoptosis was significantly induced by cerebral ischemia-reperfusion. When treated with LA-1, the green fluorescence-positive neurons significantly decreased from 34.0% to 11.4% (Figure 5J). The staining results suggest LA-1 effectively inhibits cerebral ischemia-reperfusion-induced neuron death, which is consistent with the reduction in neurological score. Taken together, LA-1 treatment may alleviate brain ischemia-reperfusion injury in the MCAO rat model. Even at one-third dose of loganin, the effectiveness of LA-1 is much stronger than that of loganin, indicating the modification with \(\alpha\)v\(\beta\)3 targeting dramatically improved the potency of loganin. Additionally, tissues from each group were also evaluated by performing H&E staining. No significant pathophysiological changes were observed in the heart, liver, spleen, and kidney tissue sections (Figure S7), indicating LA-1 may not lead to acute toxicity in vivo. Figure 5: Therapeutic effect of the LA-1 on a rat model of cerebral ischemia/reperfusion injury.