# Protective Effects of _Allium sensecens_ Extract against 6-Hydroxydopamine in Neurons
## 4 Discussion  
This study documented the protective function of _A. senescence_ extract against modeled cells for oxidative neurodegeneration and an invertebrate animal: brine shrimp. Based  
Figure 6: Effects of the extract on oxidative stress in the CNS and VNS of brine shrimp. Images for TUNEL assay in the CNS and VNS of brine shrimp exposed to 6-hydroxyopamine (6-OHDA). Histograms showing fluorescence intensity of cells in the CNS. CNS, central nervous system; T, thoracic ganglia region; A, abdominal ganglia region; ns, not significant; TUNEL, terminal deoxynucleotideyl transferase dUTP nick end labeling; VNS, ventral nervous system (* \(p\) \(<\) 0.05, ** \(p\) \(<\) 0.01). Figure 7: Effects of the extract on mitochondrial apoptosis in the CNS and VC of brine shrimp. Images for whole-mount immunohistochemistry with APC-labeled anti-caspase-3 in the CNS and VC of brine shrimp under 6-hydroxyopamine (6-OHDA) treatment. Histograms show the staining intensity of the cells. The numbers in the posterior regions represent cell colonies. CNS, central nervous system; T, thorax; A, abdomen; ns, not significant; VC, ventral cells, Ext; _Allium senescens_ extract, 6-OHDA; 6-hydroxyopamine (* \(p\) \(<\) 0.05, ** \(p\) \(<\) 0.01). on the results of the cell viability analysis (Figure 1), the extract was not cytotoxic to neuronal cells. Non-cytotoxic phytoextracts are useful in the application of functional foods. Interestingly, this study demonstrated three protective functions against 6-OHDA-induced oxidative stress. First, the extract prevented mitochondrial apoptosis by decreasing ROS levels in neuroblasts. Although 6-OHDA inhibits mitochondrial activity, the extract maintains its activity in addition to decreasing ROS under 6-OHDA treatment. Moreover, the extract downregulates MAOB and Cas3 in the neuroblasts. Under ROS accumulation, the CNS triggers neurodegeneration and accelerates aging [26]. ROS are toxic to various molecules, including proteins, lipids, carbohydrates, and DNA [27]. Furthermore, accumulated ROS causes neurodegenerative disorders [28]. These results document the potential functions of the extract in the prevention of oxidative neurodegeneration. Second, the extract was found to drive the expression of apoptotic and anti-apoptotic genes. The extract activates the upregulation of anti-apoptotic genes but not of apoptotic genes. As shown in Figure 5, all concentrations of 6-OHDA decreased the apoptotic markers. However, the extract dramatically increased the levels of the apoptotic markers despite exposure to 6-OHDA. In particular, the protective effect of the extract for neurodegeneration increased dependently on the 6-OHDA concentrations (Figure 5). Increased ROS levels trigger the upregulation of apoptotic genes in the mitochondria [29]. In neurons, NF-kB affects neuronal survival and apoptosis under mitochondrial dysfunction [30]. Furthermore, with increased ROS levels, the activation of NF-kB without the upregulation of BCL-2 promote the activation of the apoptotic signaling pathway [30]. The activation of these two molecules indicates that the extract protects neuronal cells from apoptosis induced by 6-OHDA. Increased ROS levels inhibit the upregulation of _AKT_ and _BCL-2_ genes but not of _Bax_, which activates the upregulation of CytC and Cas3 [31]. Although 100 \(\upmu\)M 6-OHDA significantly downregulates the _AKT_ gene expression and upregulates _Bax_ and _CytC_ genes, the extract dramatically modulated _AKT_, _Bax_, and _CytC_ gene expression to promote the survival of neuroblasts following exposure to 6-OHDA (Figure 5 and Supplementary Material). These results suggest that the extract is effective in preventing mitochondrial apoptosis in neuroblasts. Third, the extract protected the CNS and VNS against oxidative apoptosis in vivo. In developing brine shrimp (Figure 6), the extract prevented the apoptosis of cells of the CNS and VNS and VC under the 6-OHDA treatment. Without the extract, the 6-OHDA treatment accelerated the apoptosis of the CNS and VNS and VC in brine shrimp.