# Neuroprotective Effect of the Mixture of _Gastrodiae elata_ and _Glycyrrhiziae__urlensis_ In Vitro
## 4 Discussion  
The present study demonstrated that GGW inhibited cell death by A\(\beta\)-treatment in the neuronal cell lines of SH-SY-5Y cell and SK-N-SH cell. Additionally, GGW55 and GGW73 modulated the AChE pathway and MAPKs to prove the neuroprotective effect in SH-SY-5Y cell. Moreover, GGW55 and GGW73 regulated apoptotic factors such as BCI-2 and Bax. GU has been shown to prevent oxidative damage and cognitive impairment in A\(\beta\)-treated mice [35]. In addition, memory deficiencies in mice, induced by diaazepam and scopolamine, were improved by the administration of GU water extract [36]. GE has been reported to exert neuroprotective effects by inhibiting A\(\beta\)-induced PC12 cell death and ameliorating acute hypomnesia [19]. In addition, GE water extract recovered memory impairment in the Morris water maze test and passive avoidance test in a chronic restraint stress-stimulated mice model [37]. Gastrodin, a major compound of GE, decreased protein kinase/eukaryotic initiation factor-2 alpha and BACE-1 expression in an AD mouse model [38]. These studies suggest that GE and GU are effective in improving cognitive function disorders, degenerative neurological disease, and that better effects could be expected using a combination of GE and GU. Among GGWs, GE, and GU treatments, GGW55, GGW73, GEW, and GRW did not show cytotoxicity in the neuronal cell line SH-SY-5Y cell (Figure 1A). Therefore, we chose GGW55 and GGW73 to explore the mechanisms underlying their neuroprotective effects. In addition, GGW55 and GGW73 significantly reduced cell death following A\(\beta\) treatment in SH-SY-5Y cell (normal group; 100.00 \(\pm\) 6.67%, A\(\beta\) group; 67.52 \(\pm\) 9.19%; GGW55 100 group; 79.57 \(\pm\) 5.96%, GGW73 100 group; 87.04 \(\pm\) 9.19%) and SK-N-SH cell (normal group: 100 \(\pm\) 4.06%, GGW55 100 group; 87.72 \(\pm\) 4.81%, GGW73 100 group; 88.26 \(\pm\) 5.46%) (Figure 2). In particular, these results suggested that GGW73 treatment had more protective effect on neuronal damage than GGW55. Impairment of cholinergic function, reduced ACh levels, and a role in the pathogenesis of dementia have been mainly reported in degenerative diseases such as AD and cognitive function disorder [39]. A previous study suggested that AChE, which downregulates ACh, is a biomarker of neurotoxicity [40]. In addition, AChE is associated with neuronal mineralization and differentiation [41]. Attempts to treat cognitive function disorders have been made using AChE inhibitors such as donepezil, tacrine, and rivastigmine [42]. Both GGW55 and GGW73 attenuated AChE mRNA expression, and GGW73 at 100 \(\upmu\)g/mL significantly reduced AChE mRNA expression (Figure 3A). BDNF is primarily responsible for maintaining the central nervous system and is widely expressed in the brain [43]. However, neurological inflammation significantly  
Figure 6: The regulatory effect of GGW55 and GGW73 on H\({}_{2}\)O\({}_{2}\)-stimulated ROS expression on SK-N-SH cell and RAW264.7. The fluorescent staining was observed with fluorescence microscope (100\(\times\) magnification). reduces BDNF expression in the brain, leading to cognitive function disorder [44]. \(\beta\)-secretase (BACE-1), together with \(\gamma\)-secretase, sequentially cleave APP to produce A\(\beta\). Inhibiting the expression of BACE-1 to prevent degenerative disease is considered a strategy to remedy cognitive function disorders by lowering cerebral A\(\beta\) levels [45]. GGW55 and GGW73 increased BDNF expression upregulation by A\(\beta\) treatment (GGW55: normal group; 1855.60 \(\pm\) 494.11 pg/mL, A\(\beta\) group; 1219.65 \(\pm\) 119.52 pg/mL, GGW55 100 group; 1422.26 \(\pm\) 450.03 pg/mL, GGW73: normal group; 2041.90 \(\pm\) 213.48 pg/mL, A\(\beta\) group; 1019.04 \(\pm\) 152.24 pg/mL, GGW73 100 group; 1608.13 \(\pm\) 239.95 pg/mL) (Figure 3C) and decreased BACE-1 mRNA expression (Figure 3B). These results indicate that GGWs have a protective effect against the A\(\beta\)-induced neuronal damage.