*2.3. LE Dosage-Dependent Alleviation of Ischemic Reperfusion Injury through the Wnt*/β*-Catenin Signaling Pathway and Reduction of Inflammatory Protein Markers*

The administration of LE affected the protein expression of survival and inflammation-related signals (Figure 3a). The expression of total Akt did not differ among the experimental groups (Figure 3b). The MCAO+Veh group showed significant decrease in phosphorylation of Akt levels (pAkt) compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The MCAO+LE 10% group also exhibited an increase in pAkt levels. However, such an increase was not statistically significant compared to the MCAO+Veh group (*p* > 0.05, one-way ANOVA). The significantly elevated level of pAkt in MCAO+LE 20% group compared to the MCAO+Veh group indicated the increased survival of neurons after ischemic reperfusion injury (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 3c). The ratio of pAkt and Akt was markedly decreased in the MCAO+Veh group compared to the Sham+Veh group (*p* < 0.05, one-way

ANOVA followed by Tukey's multiple comparison test). pAkt/Akt increased in expression in a dosage dependent manner in MCAO+LE 10% and MCAO+LE 20% groups; but only the MCAO+LE 20% group was significantly different from the MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 3d).

**Figure 2.** Neuroprotective effects of LE or vehicle on the MCAO and reperfusion injury after the administration of DMSO or XAV939. (**a**) TTC-stained brain slices for infarction measurement. Decrease in infarction volume was observed in the DMSO+MCAO+LE 20% group but not in the XAV939+MCAO+LE 20% group. Sham group (DMSO+Sham+Veh) did not experience ischemic reperfusion injury; (**b**) Measurement of infarction volume by TTC staining. The DMSO+MCAO+Veh group exhibited significant increase in infarction volume compared to the DMSO+Sham+Veh group. DMSO+MCAO+LE 20% group decreased significantly with respect to infarction volume when compared to the DMSO+MCAO+Veh. The XAV939+MCAO+LE 20% group had significantly increased infraction volume compared to the DMSO+MCAO+LE 20% group; (**c**) Bederson scores of experimental groups. The DMSO+MCAO+Veh group significantly increased in Bederson scores compared to the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% group showed significant decrease in Bederson scores compared to the DMSO+MCAO+Veh groups. The XAV939+MCAO+LE 20% group significantly increased in Bederscon scores compared to the DMSO+MCAO+LE 20% group. Data are presented as mean ± standard error of the mean (SEM); *n* = 16 for each group; \*\*\* *p* < 0.001 vs. DMSO+Sham+Veh, # *p* < 0.05 vs. DMSO+MCAO+Veh, † *p* < 0.05 vs. DMSO+MCAO+LE 20%. Statistical analysis for the measurement of infarction volume was performed using one-way ANOVA followed by Tukey's multiple comparison test. Statistical analysis for Bederson scores was performed using Kruskal-Wallis non-parametric test followed by Dunn's post hoc test.

**Figure 3.** Effects of LE or vehicle on protein expressions after MCAO and reperfusion injury. (**a**) Representative Western blots indicating the expression of specific proteins in the penumbra region of the left hemisphere; (**b**–**d**) Expression and phosphorylation of Akt in the experimental groups. pAkt levels in MCAO+Veh group decreased significantly compared to the Sham+Veh group. pAkt was significantly increased in the MCAO+LE 20% group compared to the MCAO+Veh group; (**e**–**g**) Expression and phosphorylation levels of GSK-3β (pGSK-3β) in the experimental groups. pGSK-3β and GSK-3β levels in the MCAO+Veh group was significantly lower than the Sham+Veh group. The MCAO+LE 20% had significantly increased pGSK-3β and GSK-3β levels compared to the MCAO+Veh group; (**h**–**m**) Wnt signal-related protein expressions of experimental groups. Decreased levels of Wnt1, Wnt3, PORCN and β-catenin were observed in the MCAO+Veh group compared to the Sham+Veh group. The MCAO+LE 20% group had significantly increased protein levels of Wnt1, PORCN and β-catenin compared to MCAO+Veh group. Increased levels of pβ-catenin and tankyrase 1 were observed in the MCAO+Veh group compared to the Sham+Veh group. The MCAO+LE 20% group had significantly decreased levels of pβ-catenin and tankyrase 1 compared to the MCAO+Veh group; (**n**–**q**) Inflammatory protein expressions of experimental groups. Significantly increased expressions of inflammatory markers were observed in MCAO-injured groups compared to the Sham+Veh group. The MCAO+LE 20% group had significantly decreased inflammatory protein expression levels 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 vs. Sham+Veh, # *p* < 0.05, ### *p* < 0.001 vs. MCAO+Veh, one-way ANOVA followed by Tukey's multiple comparison test.

Significant decrease in total GSK-3β expression was observed in the MCAO+Veh group compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The MCAO+LE 10% group showed substantial increase in GSK-3β levels but this increment was not statistically significant compared to the MCAO+Veh group (*p* > 0.05, one-way ANOVA). The total GSK-3β expression of the MCAO+LE 20% was also significantly increased compared to the MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 3e). The phosphorylation of GSK-3β (pGSK-3β) was significantly decreased in the MCAO+Veh group compared to the Sham+Veh group (*p* < 0.01, one-way ANOVA), which might be indicating a diminishedWnt activity. The phosphorylation of GSK-3β was significantly increased in theMCAO+LE 10% (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and MCAO+LE 20% (*p* < 0.001, one-way ANOVA followed by Tukey's multiple comparison test) group when compared to the MCAO+Veh group. There was also a significant increase in pGSK-3β in the MCAO+LE 20% group compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test), thereby indicating an increased activity of the Wnt/β-catenin signaling pathway (Figure 3f). The pGSK-3β/GSK-3β activity exhibited marked decrease in the MCAO+Veh group compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The pGSK-3β/GSK-3β expression for MCAO+LE 10% did not differ significantly compared to the MCAO+Veh group (*p*>0.05, one-way ANOVA). pGSK-3β/GSK-3βactivity significantly increased 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 3g).

Wnt1, a canonical Wnt signal and upstream marker of GSK-3β, was significantly decreased in the MCAO+Veh group compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and significantly increased 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 3h). All experimental groups (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test), excluding the Sham+Veh group, displayed robust decrease in the neurogenesis marker, Wnt3. Although Wnt3 is one of canonical Wnt signals, it does not seem to affect the Wnt/β-catenin signaling pathway induced by LE (Figure 3i). Porcupine (PORCN), a key regulator of Wnt proteins [33], 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). PORCN expression increased in the MCAO+LE 20% group compared to the MCAO+Veh group, implying elevated activity in the modulation of Wnt proteins (Figure 3j). The downstream survival marker of GSK-3β, β-catenin, was decreased in all MCAO injury groups compared to the Sham+Veh group due to infarction. However, β-catenin was significantly preserved 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 3k). Phosphorylation of β-catenin (pβ-catenin) was increased 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), indicating an elevated level of β-catenin degradation. The MCAO+LE 20% group had a significantly lower expression level of pβ-catenin compared to the MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test), which supported the survival of cells (Figure 3l). The expression level of tankyrase 1 was significantly increased in the MCAO+Veh group compared to the Sham+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test), indicating the accumulation of tankyrase 1 through increased axis inhibition protein (AXIN) stabilization for β-catenin degradation. The MCAO+LE 20% group had a significantly lower expression level of tankyrase 1 compared to the MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 3m).

Inflammatory protein markers of ischemic reperfusion damage, IL-1β, IL-6, IL-8 and TNF-α [34,35], increased significantly for all MCAO-injured groups when compared to the Sham+Veh group. However, attenuated levels of inflammatory 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), indicating the reduction of ischemic reperfusion injury (Figure 3n–q).

**Figure 4.** Effects of LE or vehicle on protein expression on the MCAO and reperfusion injury after the administration of DMSO or XAV939. (**a**) Representative Western blots of proteins in the penumbra region of the left hemisphere; (**b**–**d**) Levels of Akt and pAkt in the experimental groups. The DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group decreased significantly in pAkt level compared to DMSO+Sham+Veh group. pAkt was significantly increased in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group; (**e**–**g**) Levels of GSK-3β and pGSK-3β in the experimental groups. GSK-3β and pGSK-3β levels of DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group were significantly lower than the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% had significantly increased pGSK-3β and GSK-3β levels compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups; (**h**–**m**) Wnt signal-related protein expressions of experimental groups. Significantly decreased levels of Wnt1, Wnt3, PORCN and β-catenin were observed in the DMSO+MCAO+Veh group compared to the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% group had significantly increased protein levels of Wnt1 and PORCN compared to the DMSO+MCAO+Veh group. The XAV939+MCAO+LE 20% group had decreased expression levels of Wnt1 and PORCN compared to the DMSO+MCAO+LE 20% group. The β–catenin expression level in the DMSO+MCAO+LE 20% and XAV939+MCAO+LE 20% groups did not differ significantly. There was a significant increase in pβ–catenin and tankyrase 1 in the DMSO+MCAO+Veh compared to the DMSO+Sham+Veh group. Pβ–catenin and tankyrase1 was significantly decreased in the DMSO+MCAO+LE 20% compared to the DMSO+MCAO+Veh group. The XAV939+MCAO+LE 20% group had significantly increased pβ–catenin and tankyrase 1 expression levels compared to the DMSO+MCAO+LE 20% group; (**n**–**q**) Inflammatory protein expressions in the experimental groups. Significantly increased expressions of inflammatory markers were observed in the MCAO-injured groups compared to the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% group had significantly decreased inflammatory protein expression levels compared to the DMSO+MCAO+Veh group. Significant decrease in inflammatory protein expressions were observed in the XAV939+MCAO+LE 20% group compared to the DMSO+Sham+Veh group. TNF-α expression level was increased significantly in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+LE 20% group. Data are presented as mean ± standard error of the mean (SEM); *n* = 8 for each group; \* *p* < 0.05, \*\* *p* < 0.01 vs. DMSO+Sham+Veh, # *p* < 0.05, ### *p* < 0.001 vs. DMSO+MCAO+Veh, † *p* < 0.05, ††† *p* < 0.001 vs. DMSO+MCAO+LE 20%, one-way ANOVA followed by Tukey's multiple comparison test.
