## 4 Discussion  
Stem cells have several favorable characteristics, such as ease of harvest for autologous transplantation, high proliferation rate, and ability to differentiate into multiple cell types including neural cells; therefore, they are used for treating several diseases [20,21]. Numerous bioactive molecules secreted by stem cells, such as cytokines, chemokines, and growth factors, regulate several physiological processes [22]. In addition, many studies have reported the neuroprotective effect of stem cells that exist in various organs and tissues, such as bone marrow, blood, and adipose tissue, owing to their antioxidant, anti-inflammatory, and anti-apoptotic properties [23, 24, 25]. Among various tissues, adipose tissue-derived stem cells caused the degradation of A\(\beta\), thereby making them a useful agent for the treatment of AD [26]. However, stem cells do not survive for a long duration in the body and can cause potential side effects, including immune rejection, allergic reactions, and damage to healthy organs [22, 27]. In particular, stem cell membrane contains human leukocyte antigens, major histocompatibility complex (MHC) I, and MHC II, which induce immune rejection during stem cell therapy due to their highly variable characteristics [28]. Therefore, to overcome the limitations of stem cells, MFSCE was prepared by removing the membranes of adipose tissue-derived stem cells. The purpose of this study was to evaluate the neuroprotective effects of MFSCE on neurotoxicity in SH-SYSY cells-treated with A\(\beta_{25\text{-}35}\). Figure 6: Effect of membrane-free stem cell extract (MFSCE) on expression of amyloidogenic pathway-related proteins in SH-SYSY cells treated with A\(\beta_{25\text{-}35}\). \(\beta\)-actin was used as a loading control. Values are presented as mean \(\pm\) SD (\(n\) = 6). The different letters (a–e) among groups represent significant differences determined by ANOVA and Duncan’s multiple range test (\(p\) < 0.05). \({}^{*}\)\(p\) < 0.05 vs. A\(\beta_{25\text{-}35}\)-treated cells. A\(\beta_{25-35}\) is a divided fragment obtained from A\(\beta_{1-40}\), a highly toxic fragment that causes neurotoxicity in AD [29]. The SH-SYSY treated with A\(\beta_{25-35}\) cell model is widely used to evaluate the neuroprotective ability of various materials, including stem cells [30; 31]. LDH is an intracellular enzyme, and the release of LDH is an indicator of cell death in SH-SYSY neuronal cells treated with A\(\beta_{25-35}\)[32]. A\(\beta_{25-35}\) concentration to induce neurotoxicity in SH-SYSY cells has been carried out by previous studies. The study was conducted from 5 to 50 uM of A\(\beta_{25-35}\). The treatment of 25 uM A\(\beta_{25-35}\) significantly decreased cell viability by about 60%, which was the proper cell viability to the evaluate protective effect of samples from neurotoxicity [33; 34; 35]. In this study, treatment with A\(\beta_{25-35}\) inhibited cell viability and increased LDH activity, compared with the control cells, indicating A\(\beta_{25-35}\)-induced neurotoxicity. However, treatment with MFSCE attenuated neurotoxicity by increasing cell viability and inhibiting LDH-release activity. A previous study reported that adipose tissue-derived stem cells exhibit a neuroprotective effect by restoring the viability of oxidative stress-induced SH-SYSY cells [36]. In addition, conditioned medium of adipose tissue-derived stem cells inhibited LDH-release activity in cortical neuronal cells treated with glutamate, which is suggestive of a neuroprotective effect [37]. Our results also indicated that the neuroprotective effect of MFSCE on neurotoxicity led to an increase in cell viability and inhibition of LDH activity in neuronal cells. Oxidative stress is caused by an imbalance between the ROS level and the antioxidant system [38]. A\(\beta_{25-35}\) causes oxidative stress by increasing the ROS level in the brain, resulting in lipid peroxidation, neuroinflammation, and cognitive impairment [38; 39; 40].