## 4 Discussion  
Neuroinflammatory is widely observed in neurodegenerative diseases, which might have been affected by glia cells activation and cytokines secretion. Fangfeng may play a neuroprotective function by reducing the process of neuroinflammation, though the detailed mechanisms are not clear. In our study, metabolite-profiling uncovered levodopa, docosahexaenoic acid, gamma hydroxybutyric acid, 5-hydroxy-L-tryptophan, alpha-tocopherol, etc., metabolites were upregulated by FFE in LPS-induced BV-2 cells. Levodopa is the precursor of dopamine, while dopamine is a neurotransmitter which has been shown to have anti-inflammatory effects [22; 23]. In the peripheral nervous system, dopamine receptor activation can control tissue inflammation and injury [24]. In our study, docosahexaenoic acid and gamma hydroxybutyric acid were also significantly upregulated upon FFE treatment compared with the LPS group. Research has shown that docosahexaenoic acid inhibits neuroinflammation via its conversion to specialized pro-resolving mediators and decreased oxidative stress and microglial pro-inflammatory activation [25; 26], while gamma hydroxybutyric acid, which is derived from \(\gamma\)-aminobutyric acid (GABA) [27], may act a neuromodulator in the CNS and has several neuronal mechanisms such as activation of the GABA receptor. Multiple studies revealed that alpha-tocopherol has an important role in antioxidant protection of neurodegenerative diseases and dietarytocopherol intake could improve PD-related parameters [28]. The results of metabolic pathway enrichment analysis indicated that the main upregulated metabolic pathways were alanine, aspartate and glutamate metabolism, purine metabolism, tyrosine metabolism, butanoate metabolism, primary bile acid biosynthesis, taurine and hypotaurine metabolism, tryptophan metabolism, and sphingolipid metabolism, etc. These results indicate that the differential metabolites of BV2 cells treated with FFE can effectively inhibit cell inflammation through amino acid and fatty acid metabolism and neurotransmitter and neuro-protective metabolites derived from them. The tyrosine and tryptophan metabolisms were demonstrated as crucial sources of neurotransmitters tightly associated with neuropsychiatric symptoms [29]. Besides, multiple amino acid-derived intermediates were critical for the [PERSON]'s disease pathological progression through immune cells infiltration-derived neuroinflammation [30]. At the same time, metabolites like folate, arachidonic acid, Prostaglandins, 12(S)-HPETE, 3-ketosphinganine, Galacoslysphingosine, and etc. were downregulated by FFE pre-protection when compared with LPS treatment. The downregulated pathways include: one carbon pool by folate, biosynthesis of unsaturated fatty acids, sphingolipid metabolism,  
Figure 5: Enrichment maps of downregulated metabolic pathways in negative and positive modes. Negative ion mode: (**A**) LPS vs. QF 0.1; (**B**) LPS vs. QF 1; (**C**) LPS vs. QF 10; (**H**) Control vs. LPS. Positive ion mode: (**D**) LPS vs. QF 0.1; (**E**) LPS vs. QF 1; (**F**) LPS vs. QF 10; (**G**) Control vs. LPS. Data point descriptions are in Table 2.  
and steroid biosynthesis. The impaired one-carbon metabolism and lower folate levels tightly correlated with elevated tau phosphorylation levels in neurodegenerative diseases and disturbed neuronal homeostasis [31]. However, recent research on the genetic and supplementation-manipulated C. elegans indicated that specific folate and one carbon metabolism reduction could be beneficial for longevity and proteoprotection [32]. These disparate observations probably reflect multi-layering of FFE effects on LPS-induced BV-2 cells since it could effectively decrease pro-inflammatory factor expression and downregulate folate metabolisms. Additionally, unsaturated fatty acids, sphingolipid, and steroid metabolism alteration in FFE-protected microglia cells reflected that fatty acids, especially long chain unsaturated fatty acids, were critical for FFE-derived neuroprotective effects.