## 3 Results
### Effect of MFSCE on Apoptosis in SH-SYSY Cells Treated with \(A\beta_{25-35}\)  
To investigate the effect of MFSCE on neuronal apoptosis, protein levels of Bax and Bcl-2 were measured. The expression of the pro-apoptotic protein, Bax, was upregulated in A\(\beta_{25-35}\)-treated cells compared with that in the control cells (Figure 5). However, treatment with MFSCE inhibited Bax protein expression. In addition, the expression of Bcl-2, an anti-apoptotic factor, was significantly inhibited in A\(\beta_{25-35}\)-treated cells, while MFSCE-treated cells upregulated the expression of Bcl-2. Furthermore, MFSCE-treated groups showed a dose-dependent decrease in the Bax/Bcl-2 ratio, compared with that of A\(\beta_{25-35}\)-treated cells. In particular, the group treated with 5 \(\mu\)g/mL of MFSCE showed a higher extent of decrease in the Bax/Bcl-2 ratio than the groups treated with other concentrations. Therefore, this result demonstrates that MFSCE exerts protective effects against neuronal apoptosis via the downregulation of Bax and upregulation of Bcl-2 protein expressions.  
Figure 3: Effect of membrane-free stem cell extract (MFSCE) on production of reactive oxygen species in SH-SYSY cells treated with A\(\beta_{25-35}\). (**A**) Changes in intensity of ROS fluorescence during 60 min. (**B**) The production of ROS in cells treated with different concentrations of MFSCE at 60 min. Values are presented as mean \(\pm\) SD (\(n=6\)). The different letters (a–d) among groups represent significant differences determined by ANOVA and Duncan’s multiple range test (\(p<0.05\)). * \(p<0.05\) vs. A\(\beta_{25-35}\)-treated cells.  
Figure 4: Effect of membrane-free stem cell extract (MFSCE) on neuroinflammation-related protein expression in SH-SY5Y cells treated with A\({}_{\beta 25\text{-}35}\). Values are presented as mean \(\pm\) SD (\(n=6\)). The different letters (a–f) 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. \(\beta\)-actin was used as a loading control.  
Figure 5: Effect of membrane-free stem cell extract (MFSCE) on neuronal apoptosis-related protein expression in SH-SY5Y cells treated with A\({}_{\beta 25\text{-}35}\). 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. \(\beta\)-actin was used as a loading control.  
Effect of MFSCE on Amyloidogenic Pathway in SH-SYSY Cells Treated with A\(\beta_{25\text{-}35}\)  
To demonstrate the effect of MFSCE on amyloidogenesis, the levels of proteins related to the amyloidogenic pathway were measured. The expression of amyloidogenic proteins, namely, APP, BACE, PS1, and PS2, was significantly upregulated in A\(\beta_{25\text{-}35}\)-treated cells, compared with that in the control cells (Figure 6). However, treatment with MFSCE inhibited the protein expression of APP, BACE, PS1, and PS2. Treatment with MFSCE at all concentrations (1, 2.5, and 5 ug/mL) significantly inhibited APP protein expression, while MFSCE at a concentration of 5 ug/mL significantly reduced BACE and PS1 expression, compared to other concentrations. With respect to PS2, the MFSCE-treated group exhibited significant downregulation at all concentrations, and this inhibitory activity was the highest among all amyloidogenic proteins. Therefore, this result demonstrated that MFSCE has a protective effect against A\(\beta_{25\text{-}35}\)-induced amyloidogenesis via the downregulation of APP, BACE, PS1, and PS2 protein expression in SH-SYSY cells.