*2.4. Wnt*/β*-Catenin-Dependent Alleviation of Ischemic Reperfusion Injury and Reversal of Protection-Related Proteins*

The administration of XAV939 inhibited the activity of the Wnt/β-catenin signaling pathway, which was reflected in the protein expression (Figure 4a) of distinct genes. The expression of total Akt did not differ among the experimental groups (Figure 4b). The DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group significantly showed substantial decrease in pAkt levels compared to the DMSO+Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). There was significant elevation in the level of pAkt in DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and XAV939+MCAO+LE 20% group (*p* < 0.01, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4c). The pAkt/Akt expression was decreased significantly in the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups compared to the DMSO+Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The expression of pAkt/Akt in the DMSO+MCAO+LE 20% group was significantly increased compared to DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4d).

Significant decrease in total GSK-3β expression was observed in the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups when compared to the DMSO+Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). Total GSK-3β expression of the DMSO+MCAO+LE 20% group was also significantly increased compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4e). The level of pGSK-3β was significantly decreased in the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups compared to the DMSO+Sham+Veh group (*p* < 0.01, one-way ANOVA followed by Tukey's multiple comparison test), which might be indicative of a compromised Wnt activity. The phosphorylation of GSK-3β was significantly increased in the DMSO+MCAO+LE 20% group compared to the DMSO+Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). There was also a significant increase in the pGSK-3β levels in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups (*p* < 0.001, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4f). The pGSK-3β/GSK-3β activity was significantly decreased in the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups when compared to the DMSO+Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and significantly increased in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The administration of XAV939 effectively decreased the phosphorylation GSK-3β induced by LE (Figure 4g).

Wnt1 was significantly decreased in the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups compared to the DMSO+Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and significantly increased in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4h). All experimental groups, excluding the DMSO+Sham+Veh group, showed steep decrease (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) in Wnt3 (Figure 4i). PORCN decreased significantly in the DMSO+MCAO+Veh group (*p* < 0.01, one-way ANOVA followed by Tukey's multiple comparison test) and XAV939+MCAO+LE 20% group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) compared to the DMSO+Sham+Veh group. PORCN expression increased in expression in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4j). β-catenin was decreased in all MCAO injury groups compared to the DMSO+Sham+Veh group due to infarction. Significantly attenuated level of β-catenin was also

observed in the XAV939+MCAO+LE 20% group, while DMSO+MCAO+LE 20% group were preserved significantly compared to DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). Average β-catenin expression decreased in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+LE 20% group but not to a significant level (*p* > 0.05 one-way ANOVA) (Figure 4k). pβ-Catenin was increased in the DMSO+MCAO+Veh group compared to the DMSO+Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test), indicating lowered cellular survival. The level of pβ-catenin was significantly decreased in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The pβ-catenin expression level was significantly increased in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+LE 20% group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4l). The accumulation of tankyrase 1 was significant in the DMSO+MCAO+Veh compared to DMSO+Sham+Veh (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The tankyrase 1 expression of DMSO+MCAO+LE 20% group significantly decreased compared to the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The XAV939+MCAO+LE 20% group had significantly higher levels of tankyrase 1 expression levels compared to the DMSO+MCAO+LE 20% group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The administration of XAV939 successfully inhibited tankyrase 1 activity which increased degradation of β-catenin (Figure 4m).

Inflammatory protein markers of ischemic reperfusion damage, IL-1β, IL-6, IL-8 and TNF-α, also significantly increased for all MCAO-injured groups compared to the DMSO+Sham+Veh group. Attenuated levels of inflammatory markers were observed in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The expression levels of the XAV939+MCAO+LE 20% group were not significantly different from the DMSO+MCAO+Veh group (*p* > 0.05, one-way ANOVA) The TNF-α expression level of the XAV939+MCAO+LE 20% group increased significantly compared to the DMSO+MCAO+LE 20% group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 4n–q).

### *2.5. LE Dosage-Dependent mRNA Expression Against Ischemic Reperfusion Injury*

According to the results of qPCR, Wnt1 can be implicated as one of the main regulators for neuroprotection. in the MCAO+Veh group, Wnt1 expression level was approximately 0.4 folds compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The mRNA expression of Wnt1 signals of MCAO+LE 20% was upregulated by approximately 2.4 folds compared to the Sham+Veh group (*p* < 0.01, one-way ANOVA followed by Tukey's multiple comparison test) and 4 folds compared to the MCAO+Veh group (*p* < 0.001, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 5a). Wnt3 expression was attenuated in all MCAO-injured groups compared to the Sham+Veh group. The MCAO+LE 10% (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and MCAO+LE 20% groups' (*p* < 0.001, one-way ANOVA followed by Tukey's multiple comparison test) Wnt3 expression levels were significantly decreased compared to the MCAO+Veh group (Figure 5b). Mki67, a cell proliferation marker, increased in all MCAO-injured groups compared to the Sham+Veh group. The Mki67 expression level increased significantly in the MCAO+LE 20% group compared to the MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test), which may have been affected by the elevated expression level of Wnt1 (Figure 5c). The Wnt regulator, Porcn, decreased significantly in the MCAO+Veh group compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). Significant increase of Porcn was observed in the MCAO+LE 20% group compared to the MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test), which may be induced by the increase in Wnt1 activity (Figure 5d). Inflammatory markers, IL-1β, IL-6, IL-8 and TNF-α, significantly increased in MCAO-injured groups compared to the Sham+Veh group. Significantly lower inflammatory mRNA markers were observed in the MCAO+LE 20% group compared to the MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 5e–h).

**Figure 5.** Effects of LE or vehicle on mRNA expression after the MCAO and reperfusion injury. (**a**,**b**) Wnt expressions in experimental groups. The Wnt1 mRNA expression of the MCAO+Veh group was significantly decreased compared to the Sham+Veh group. Significantly increased expression of Wnt1 was expressed in the MCAO+LE 10% and MCAO+LE 20% groups compared to the MCAO+Veh group. Wnt3 expressions were significantly lower in MCAO injury groups compared to the Sham+Veh group. Significantly decreased Wnt3 expressions were observed in the MCAO+LE 10% and MCAO+LE 20% groups compared to the MCAO+Veh group; (**c**) Mki67 expression was increased in all MCAO-injury groups compared to the Sham+Veh group. Significant increase in Mki67 expression was observed in the MCAO+LE 20% group compared to the MCAO+Veh group; (**d**) Porcn expression was significantly decreased in the MCAO+Veh group compared to the Sham+Veh group. Significant increase of Porcn was observed in the MCAO+LE 20% group compared to the MCAO+Veh group; (**e**–**h**) mRNA expression of inflammatory markers. MCAO-injury groups expressed significantly increased levels of inflammatory markers compared to the Sham+Veh group. Significantly decreased inflammatory expression levels were observed in the MCAO+LE 20% group compared to the MCAO+Veh group. Data are presented as mean ± standard error of the mean (SEM); *n* = 8 for each group; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. Sham+Veh, # *p* < 0.05, ### *p* < 0.001 vs. MCAO+Veh, one-way ANOVA followed by Tukey's multiple comparison test. Wnt subfamily mRNA expressions are shown in Supplementary Materials S1.
