*2.6. Wnt-Dependent mRNA Expression of LE Against Ischemic Reperfusion Injury*

XAV939 injection inhibited Wnt activity in mRNA levels to reverse neuroprotection. in the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and XAV939+MCAO+LE 20% group, Wnt1 mRNA expression levels were approximately 0.5 folds and 0.6 folds compared to the DMSO+Sham+Veh group, respectively. Expression of Wnt1 signals in the DMSO+MCAO+LE 20% was upregulated by approximately 2.2 folds compared to the DMSO+Sham+Veh group (*p* < 0.01, one-way ANOVA followed by Tukey's multiple comparison test) and 4.2 folds compared to the DMSO+MCAO+Veh group (*p* < 0.001, one-way ANOVA followed by Tukey's multiple comparison test). There was a significant decrease in Wnt1 expression in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+LE 20% group (*p* < 0.01, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 6a). Wnt3 expression was attenuated in all MCAO-injured groups compared to the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% group's Wnt3 expression was significantly decreased compared to the DMSO+MCAO+Veh group (*p*<0.001, one-way ANOVA followed by Tukey's multiple comparison test). The XAV939+MCAO+LE 20% group was also showed significant decreased in Wnt3 expression level compared to the DMSO+Sham+Veh group (*p* < 0.01, one-way ANOVA followed by Tukey's multiple comparison test) but displayed marked increase when compared to the DMSO+MCAO+LE

20% group (*p* < 0.001, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 6b). Mki67 levels increased in all MCAO-injured groups compared to the DMSO+Sham+Veh group. in the DMSO+MCAO+LE 20% group, Mki67 expression level increased significantly compared to the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test); while in the XAV939+MCAO+LE 20% group, it was significantly downregulated compared to the DMSO+MCAO+LE 20% group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 6c). Porcn decreased significantly in the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) and XAV939+MCAO+LE 20% group compared to the DMSO+Sham+Veh group. Significant increase of Porcn was observed in the DMSO+MCAO+LE 20% group when 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 decreased in Porcn compared to the DMSO+Sham+Veh group but did not differ significantly. The XAV939+MCAO+LE 20% group expression levels of Porcn were significantly decreased compared to the DMSO+MCAO+LE 20% group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test) (Figure 6d). Inflammatory markers, IL-1β, IL-6, IL-8 and TNF-α, significantly increased in MCAO-injured groups compared to the DMSO+Sham+Veh group. Significantly lower inflammatory mRNA markers were observed in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group (*p* < 0.05, one-way ANOVA). XAV939 injection significantly reversed attenuated levels of inflammation by LE 20% (Figure 6e–h).

#### **3. Discussion**

Our results suggest that LE provides neuroprotection against ischemic reperfusion injury in a dosage-dependent manner. Intra-arterial injection of 20% LE during reperfusion after MCAO was able to reduce infarction significantly. The decreases in infarction volumes have been reflected in the improved performance in the neurological deficit assessment. Rats injected with LE 20% possessed better control over their paralyzed limb. Increased levels of Wnt1 signaling in both protein and mRNA levels in 20% LE-treated rats were observed, which in turn enhanced the resistance to reperfusion injury following ischemia. Especially, a notable increase in Wnt1 mRNA expression was observed, which may have led to increased Wnt1 protein expression. The elevated levels of Wnt1 are consistent with previous studies that have indirectly stimulated Wnt/β-catenin signaling pathway for cell survival after ischemic injury [20,36]. The expression levels of Wnt3 decreased in all MCAO groups which may be due to a decline in cell populations as a result of ischemic reperfusion injury [37]. Although Wnt3 is also known as a canonical Wnt signal, not all canonical Wnt signals seem to be involved in the protection process. As in our previous study [31], Wnt3 did not seem to be involved in protection process in response to the administration of LE. In addition, the phosphorylation of GSK-3β increased due to 20% LE injection, which inactivated the β-catenin destruction complex. β-catenin was preserved to promote cell survival, which resulted in enhanced resistance to the ischemic reperfusion damage. Elevation of PORCN expression may have been due to the increase in demand for Wnt1 to resist damage; however, a clear link between PORCN and LE cannot be formulated at this stage. A previous study on the ischemic reperfusion injury in the heart has reported that palmitic acid of LE affects the lipid-modification of Wnt ligands for protection [38]. Clear distinctions between cardiac cells and neurons exist but Wnt modifications seem to occur regardless of their metabolic factors. Inflammatory markers, IL-1β, IL-6, IL-8 and TNF-α, are known to increase after ischemic reperfusion injury [34,35]. Inflammation by oxidative stress was significantly decreased in the 20% LE-treated group, indicating an anti-inflammatory action of LE in the central nervous system. The preservation of β-catenin through the activation of the Wnt signaling pathway induces the transcription of T cell factor/lymphoid enhancer factor, which results in decreased inflammatory activity [39]. Therefore, the expressions of inflammatory cytokines, including IL-1β, IL-6 and TNF-α, might be reduced. The decrease in the expression of IL-8 is assumed to be reduced by an overall decline in inflammatory activity.

**Figure 6.** Effects of LE or vehicle on mRNA expression on the MCAO and reperfusion injury after the administration of DMSO or XAV939. (**a**,**b**) Wnt expressions in experimental groups. The Wnt1 mRNA expression of the DMSO+MCAO+Veh group was significantly decreased compared to the DMSO+Sham+Veh group. Significantly increased expression of Wnt1 was expressed Wnt1 decreased significantly in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+LE 20% group. There was no significant difference in Wnt1 expression in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group. Wnt3 expressions were significantly lower in MCAO-injury groups compared to the DMSO+Sham+Veh group. Significantly decreased Wnt3 expressions were observed in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups; (**c**) Mki67 expression was increased in all MCAO-injury groups compared to the DMSO+Sham+Veh group. Significant increase in Mki67 expression was observed in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh. The XAV939+MCAO+LE 20% group had a significant decrease in Mki67 expression compared to the DMSO+MCAO+LE 20% group; (**d**) Porcn expression was significantly decreased in the DMSO+MCAO+Veh group compared to the DMSO+Sham+Veh group. Significant increase of Porcn was observed in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh. The XAV939+MCAO+LE 20% group had a significant decrease in Porcn expression compared to the DMSO+MCAO+LE 20% group. There was no significant difference in Porcn expression in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group; (**e**–**h**) The mRNA expression of inflammatory markers. MCAO injury groups expressed significantly increased levels of inflammatory markers compared to the DMSO+Sham+Veh group. Significantly decreased inflammatory expression levels were observed in the DMSO+MCAO+LE 20% group compared to DMSO+MCAO+Veh group. Significantly increased levels of inflammatory markers were observed in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+LE 20% group and DMSO+Sham+Veh group but there was no significant difference compared to the DMSO+MCAO+Veh group. Data are presented as mean ± standard error of mean (SEM); *n* = 8 for each group; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 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. Wnt subfamily mRNA expressions are shown in Supplementary Materials S1.

The Wnt/β-catenin signaling pathway has been considered a potential therapeutic target in preventing cell death for an extensive amount of time [18,40,41]. The down-regulation of Wnt ligands and increased antagonistic activity have been observed in neurodegenerative and excitotoxic disorders [42–44]. In order to verify the protective mechanism of LE, which increased Wnt1, we were required to interrupt the protective process. XAV939, a Wnt/β-catenin signaling pathway inhibitor, has been commonly utilized to inhibit Wnt activity in many studies [45,46]. in the present study, the i.p. injection of XAV939 successfully inhibited Wnt activity and reversed the protective effects induced by LE. The XAV939+MCAO+LE 20% group exhibited infarction volumes nearly as arge as our control, the DMSO+MCAO+Veh group. Akin to earlier studies, our results show that

the Wnt/β-catenin signaling pathway might be a potential therapeutic target for neuroprotection. The administration of XAV939 inhibited the mRNA expression level of Wnt1, indicating that Wnt1 may be dependent on the activity of GSK-3β. XAV939 is known to inhibit tankyrase 1, which prevents the degradation of axis inhibition protein 2 (AXIN2), allowing the accumulation of the GSK-3β destruction complex [47]. Therefore, pGSK-3β was attenuated in the XAV939+MCAO+LE 20% group, which reversed the protective effect of LE by the degradation of the downstream survival marker, β-catenin (Figure 4k). We can infer that LE has effects on the upstream signals of GSK-3β and not on the downstream signals of GSK-3β (Figure 7). If LE affected the GSK-3β downstream marker, β-catenin, XAV939 might not have affected the protection effect. In addition, if LE affected GSK-3β directly, a less effective or partial reversal of protection may have occurred. We observed that the infarction damage was not significantly different between the DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group, implying an effective reversal from the effects of 20% LE injection.

**Figure 7.** Schematic diagram of the protective mechanism of LE in ischemic reperfusion injury. LE increases the lipid modification of Wnt proteins by PORCN. Lipid-modified/active Wnt proteins are released to the extra-cellular space for the binding to Frizzled/LRP receptors. The activated receptors induce the phosphorylation of GSK-3β, which prevents the destruction complex from the degradation of β-catenin by proteasomes. Preserved levels of β-catenin lead to cell survival. The injection of XAV939 prevents the phosphorylation of GSK-3β, which allows β-catenin degradation for cell death. Abbreviations: PORCN—porcupine, Wnt—wingless integration, LRP—low-density lipoprotein receptor-related protein, GSK-3β—glycogen synthase kinase 3β, APC—adenomatous polyposis coli, AXIN—axis inhibition protein.

The protective roles of fatty acids have received much attention in studies regarding stroke. Different compositions of lipid emulsions, such as omega-3, have been reported to provide neuroprotection against ischemic stroke injuries [48]. Emulsions of n-3 fatty acids have been shown to effectively reduce infarction volume and inflammation in the ischemic brain in neonatal mice [49]. in the present study, we observed significant neuroprotective effects of LE against ischemic reperfusion injury but limitations exist. Although we were able to investigate protective properties of LE regarding the Wnt/β-catenin signaling pathway, we are yet to discover specific targets of LE. Further studies should consider other signals that are involved in the Wnt/β-catenin signaling pathway, such as frizzled-1, low-density lipoprotein receptor-related protein 5/6 or PORCN, that can be subjected for inhibition. Elucidation of specific signals may lead to more effective therapeutic targets in the brain. Through our study, we suggested that the neuroprotective mechanism of LE is dependent on the Wnt/β-catenin signaling pathway through the inhibition of GSK-3β phosphorylation. We assumed that lipid modification occurs through PORCN through the constituents of LE that interact with the protein. As a result, the expression levels of Wnt1 increased, which also affects its downstream signals. The scope for future studies is considerable: LE may be utilized with other lipophilic anti-inflammatory drugs to further extend its neuroprotective properties. Furthermore, the investigation of different routes of delivery may also be considered with regard to invasiveness and safety.

In conclusion, the intra-arterial administration of 20% LE alleviated ischemic reperfusion injury induced by MCAO and reperfusion. Infarction volumes and Bederson scores were attenuated in a dosage-dependent manner. Protein and mRNA expression levels of the Wnt/β-catenin signaling pathway were elevated and inflammatory markers decreased significantly in the 20% LE-treated group. Especially, GSK-3β was phosphorylated significantly, which in turn preserved β-catenin to promote cell survival. The protective actions were reversed by the administration of XAV939 indicating that the protection mechanism of LE has been induced through the Wnt/β-catenin signaling pathway. These findings regarding the anti-inflammatory and neuroprotective properties of LE may provide a foundation for further research regarding excitotoxic neural injuries and functional recovery.
