## 3 Results
### Metabolomics Profiling and Differential Accumulated Metabolite (DAM) Analysis  
OPLS-DA analysis showed that the control group and LPS treatment group were far away from FFE-treated groups, meaning FFE and LPS could greatly disturb the metabolic profile of BV-2 cells. Drug-treated groups were effectively separated between different concentrations (Figure 2). In order to identify DAM, VIP value (>1.0), fold change (FC \(\geq\) 2 or FC \(\leq\) 0.5) and _t_-test \(p\) value (_p_ < 0.05) in volcano map (Figure 3) and OPLS-DA analysis were used as selection criteria. The DAM details can be found in supplementary file S1, such as levodopadopamine, docosahexaenoicenoic acid, gamma hydroxybutyric acid, 5-hydroxy-L-tryptophan, alpha-tocopherol, etc., which were upregulated by FFE pre-protection when compared with LPS treatment. MetaboAnalyst software was used for metabolomic pathway enrichment analysis. In the negative mode (Table 1), for the LPS group versus QF 0.1 group, alanine, aspartate and glutamate metabolism, purine metabolism, and pyrimidine metabolism were the top upregulated pathways (Figure 4A); for the LPS group  
Figure 1: Effects of Fangfeng extract on the production of pro-inflammatory cytokines (IL-6 and IL-1\(\beta\)) in LPS-induced BV-2 cells. (**A**) Effect on the production of proinflammatory cytokines IL-6, (**B**) Effect on the production of proinflammatory cytokines IL-1\(\beta\). All the experiments were repeated at least three times and similar results were observed. Values are mean \(\pm\) SE, (_n_ = 4 samples/group). #_p_ < 0.01 vs. control group, **_p_ < 0.01 vs. LPS-treated group. versus QF 1 group, purine metabolism, alanine, aspartate, and glutamate metabolism and aminoacyl-tRNA biosynthesis were the main upregulated pathways (Figure 4B); for the LPS group versus the QF 10 group, tyrosine metabolism, butanoate metabolism, and galactose metabolism were the main upregulated pathways (Figure 4C). In the positive mode, for the LPS group versus QF0.1 group, butanoate metabolism, arginine biosynthesis and starch, and sucrose metabolism were major upregulated pathways (Figure 4D); for the LPS group versus the QF1 group, primary bile acid biosynthesis, taurine and hypotaurine metabolism, and tryptophan metabolism were the most enriched upregulated pathways (Figure 4E); for the LPS group versus the QF10 group, tryptophan metabolism, sphingolipid metabolism, and tyrosine metabolism were the top enriched upregulated pathways (Figure 4F). Figure 4: _Cont_. Figure 3: Metabolite volcano map in negative ion mode: (**A**) LPS vs. QF 0.1; (**B**) LPS vs. QF 1; (**C**) LPS vs. QF 10; metabolite volcano map in positive ion mode: (**D**) LPS vs. QF 0.1; (**E**) LPS vs. QF 1; (**F**) LPS vs. QF 10. Figure 4: Enrichment maps of upregulated 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 1.  
Additionally, the downregulated DAM were also included in Supplementary File S1, such as folate, arachidonic acid, Prostaglandins, 12(S)-HPETE, 3-ketosphinganine, Galactosylsphingosine etc., and were downregulated by FFE pre-protection when compared with LPS treatment. Metabolomic pathway enrichment analysis were also performed and the results were as follows: in the negative mode (Table 2), for the LPS group versus the QF 0.1 group, thiamine metabolism, ether lipid metabolism, and galactose metabolism were the top downregulated pathways (Figure 5A); for the LPS group versus the QF 1 group, one carbon pool by folate, biosynthesis of unsaturated fatty acids, and Purine metabolism were the main downregulated pathways (Figure 5B); for the LPS group versus the QF 10 group, one carbon pool by folate, porphyrin and chlorophyll metabolism, and thiamine metabolism were the main upregulated pathways (Figure 5C).