## 3 Results
### Effects of \(\beta\)-lactolin-Induced MAO-B Inhibition on DA Metabolism in Astrocytes  
To evaluate the involvement of \(\beta\)-lactolin in DA metabolism in astrocytes, the levels of DA and its metabolites were measured using an HPLC-electrochemical detection system. Using RT-qPCR, we confirmed the gene expression of the two main enzymes that metabolize DA, _Mao-b_ and _Comt_. The relative gene expression levels normalized to _Gapdh_ were 1.23 \(\pm\) 0.206 for _Mao-b_ and 1.04 \(\pm\) 8.4 \(\times\) 10\({}^{-3}\) for _Comt_ (_n_ = 3). Intracellular DA levels in astrocytes treated with \(\beta\)-lactolin were significantly higher than those in the control  
Figure 1: Reactive oxygen species (ROS) production in astrocytes treated with LPS and \(\beta\)-lactolin. (**A,C**) Representative images show intracellular ROS detected using CM-H2 DCFDA (green). The nuclei were stained with Hoechst 33,342 (blue). Scale bar is 100 \(\upmu\)m at \(\times\)20 magnification. (**A**) Murine astrocytes were treated with 0, 10, 50, or 100 ng/mL lipopolysaccharideaccharide (LPS) and 50 U/L IFN-Y for 24 h. (**C**) Cells were treated with PBS, 10 ng/mL LPS, and 50 U/L IFN-Y for 24 h, then treated with 0, 1, 5, or 10 \(\upmu\)M \(\beta\)-lactolin for 6 h prior to staining. (**B,D**) Quantification of ROS levels by measuring the relative CM-H2 DCFDA fluorescence intensity per nucleus. Data represent the mean \(\pm\) SEM (\(n=12\)). The \(p\) values were calculated using one-way analysis of variance (ANOVA) followed by [PERSON]’s post hoc test. **\(p<\) 0.01. ROS, reactive oxygen species; CM-H2 DCFDA, 5-(and-6)-chloromethyl-2,7-dichlorohydrofluorescein diacetate acetyl ester; LPS, lipopolysaccharideaccharide; IFN-Y, interferon gamma; PBS, phosphate-buffered saline; ANOVA, analysis of variance; SEM, standard error of the mean. Figure 2: The effect of \(\beta\)-lactolin on MAO-B activity in astrocyte mitochondria. Cells were treated with 0, 1, 5, or 10 \(\upmu\)M \(\beta\)-lactolin or 10 \(\upmu\)M pargyline for 5 h. MAO activity assay was performed on mitochondria isolated from astrocytes. Pargyline, a selective MAO inhibitor, was used as a positive control. Data represent the mean \(\pm\) SEM (\(n=3\)). The \(p\) values were calculated using one-way ANOVA followed by [PERSON]’s multiple comparison test. *\(p<\) 0.05, **\(p<\) 0.01. MAO-B, monoamine oxidase B; ANOVA, analysis of variance; SEM, standard error of the mean. group (1.97 \(\times\) 10\({}^{-2}\)\(\pm\) 8.59 \(\times\) 10\({}^{-4}\) vs. 3.10 \(\times\) 10\({}^{-2}\)\(\pm\) 4.01 \(\times\) 10\({}^{-3}\) ppm, \(p\) = 1.08 \(\times\) 10\({}^{-3}\); Figure 3A). Intracellular levels of 3,4-dihydroxybenzylacetic acid (DOPAC) after treatment with \(\beta\)-lactolin were significantly higher than those in the control group (9.54 \(\times\) 10\({}^{-2}\)\(\pm\) 5.01 \(\times\) 10\({}^{-3}\) vs. 0.104 \(\pm\) 4.14 \(\times\) 10\({}^{-3}\) ppm, \(p\) = 1.05 \(\times\) 10\({}^{-2}\); Figure 3B). Homovanillic acid (HVA) levels were not changed by \(\beta\)-lactolin treatment (Figure 3C). The turnover of intracellular DA ([DOPAC + HVA]/DA) in \(\beta\)-lactolin-treated cells was significantly lower than that in control cells (5.01 \(\pm\) 0.171 vs. 3.49 \(\pm\) 0.443 ppm, \(p\) = 2.27 \(\times\) 10\({}^{-4}\); Figure 3D). Extracellular DA levels in the culture supernatant of astrocytes were not changed by \(\beta\)-lactolin treatment (Figure 3E). However, extracellular DOPAC levels in the supernatant of cells treated with \(\beta\)-lactolin were significantly lower than those in the control (0.374 \(\pm\) 2.13 \(\times\) 10\({}^{-2}\) vs. 0.329 \(\pm\) 1.28 \(\times\) 10\({}^{-2}\) ppm, \(p\) = 2.20 \(\times\) 10\({}^{-3}\); Figure 3F). Furthermore, extracellular HVA levels in the supernatant of cells treated with \(\beta\)-lactolin were significantly lower than those in the control (5.40 \(\times\) 10\({}^{-2}\)\(\pm\) 2.53 \(\times\) 10\({}^{-3}\) vs. 4.67 \(\times\) 10\({}^{-2}\)\(\pm\) 1.03 \(\times\) 10\({}^{-3}\) ppm, \(p\) = 3.12 \(\times\) 10\({}^{-4}\); Figure 3G).