## 4 Discussion  
Astrocytes perform various crucial functions in the central nervous system (CNS) and reactive astrocytes are considered as therapeutic targets in neurodegenerative diseases. We have previously demonstrated that orally administered \(\beta\)-lactolin is delivered to the brain and improves memory function in in vivo models [20]. However, the effects of \(\beta\)-lactolin on astrocytes have not been investigated. This study showed that \(\beta\)-lactolin suppresses intracellular ROS production in LPS-treated astrocytes. Several studies have shown that excessive ROS production in astrocytes is a characteristic of reactive astrocytes, which  
Figure 3: The effects of \(\beta\)-lactolin on dopamine (DA) metabolism in astrocytes. Cells were treated with 0 or 10 \(\upmu\)M \(\beta\)-lactolin for 3 h, following treatment with DA for 2 h. The following monoamine levels in the cells (**A–D**) and in culture media (**E–H**) were measured using high-performance liquid chromatography (HPLC): DA (**A,E**), 3,4-dihydroxybenzylacetic acid (DOPAC) (**B,F**), and homovanillic acid (HVA) (**C,G**). The (DOPAC + HVA)/DA ratio (**D,H**) was also determined. Data represent the mean \(\pm\) SEM (\(n\) = 6). The \(p\) values were calculated using the Student’s \(t\)-test. * \(p\) < 0.05, **\(p\) < 0.01. DA, dopamine; HPLC, high-performance liquid chromatography; DOPAC, 3,4-dihydroxybenzylacetic acid; HVA, homovanillic acid; SEM, standard error of the mean. mediates the pathogenesis of neurodegenerative diseases, such as AD and PD [30, 31]. The increase in ROS production in astrocytes triggers the release of pro-inflammatory cytokines, leading to neuronal dysfunction in the CNS [32]. H2O2, in particular, is an ROS that has a high membrane permeability; thus, its toxicity affects not only the astrocytes producing it but also their neighboring neurons [33]. Previous in vivo studies showed that LPS induces neuronal apoptosis, which is rescued by treatment with some bioactive products that attenuate astrocyte reactivity, including ROS production [34, 35, 36]. Similar neuroprotective effects of chemicals and/or bioactive compounds that suppress the reactivity of LPS-treated astrocytes were observed in ex vivo studies conducted on neuron-astrocyte co-culture models or neurons cultured in astrocyte-conditioned medium [37, 38]. Hence, the present results indicate potential neuroprotective effects of \(\beta\)-lactolin on astrocytes. Therefore, further studies are warranted to determine the effects of \(\beta\)-lactolin on the crosstalk between LPS-treated astrocytes and neurons. Next, we evaluated the inhibitory effect of \(\beta\)-lactolin on MAO-B in astrocytes. Consistent with the results of our previous study using human recombinant MAO-B [20], \(\beta\)-lactolin inhibited MAO-B enzyme activity in astrocytes. We assume that \(\beta\)-lactolin directly inhibits the enzyme activity because \(\beta\)-lactolin and WY peptide showed MAO-B inhibitory effects, whereas tryptophan did not in the previous studies [20, 39]. Astrocytes highly express MAO-B, generating H2O2 as a result of its enzymatic reaction. Accumulating evidence has indicated that astrocytic MAO-B activity could regulate ROS production in astrocytes [18]. Indeed, recent studies have shown that some MAO-B inhibitors suppress intracellular ROS production in astrocytes [14, 19]. Moreover, some food constituents that attenuate MAO-B activity showed protective effects by suppressing ROS production in these cells [40, 41, 42]. Therefore, the inhibitory effect of \(\beta\)-lactolin on MAO-B might also suppress ROS production. It is notable that some MAO-B inhibitory food constituents have antioxidant activity [40, 41, 42]. Given that whey-derived peptides containing WY showed strong antioxidant activity [43, 44], the antioxidant activity of \(\beta\)-lactolin could be worth investigating. Furthermore, MAO-B mediates the synthesis of \(\gamma\)-aminobutyric acid (GABA) in astrocytes through the putrescine degradation pathway [45].