## 2 Results and Discussion
### Evaluation of the Anti-Neuroinflammatory Efficacy of Spilanthol  
Chronic neuroinflammationflammation is a pathogenic hallmark in NDDs and affects neuronal plasticity, impairs memory, induces secondary neurotoxicity, and sustains neurodegeneration [45]. Microglia in the CNS are predominantly responsible for sustained neuroinflammation. LPS is a well-known endotoxin produced by gram-negative bacteria that elicits a strong inflammatory response in microglia via the TLR4/NF-kB signaling pathway [46]. Therefore, the potential of spilanthol to attenuate neuroinflammation was assessed in LPS-induced BV-2 cells by measuring NO, TNF-\(\alpha\), and IL-6 concentration in the culture supernatant. Briefly, the cells were pretreated with spilanthol (25, 50, and 100 uM) for 2 h, followed by induction with LPS (1 ug/mL) for 22 h. The NO expression in the cell culture supernatant was then measured using the Griess reaction. The results demonstrated that the LPS-induced cells accounted for significantly high NO levels (13.58 \(\pm\) 0.99 uM) in comparison with the untreated cells (1.07 \(\pm\) 1.13 uM). Pretreatment with spilanthol produced a concentration-dependent decline in NO levels. In comparison with the LPS-only group, treatment with spilanthol at 100 and 50 uM significantly decreased the concentrations of nitrite in the cell culture supernatants by 38.59% (8.43 \(\pm\) 1.39 uM) and 31.3% (9.33 \(\pm\) 1.22 uM), respectively. however, treatment with spilanthol alone had no significant effects on NO production in BV-2 cells (Figure 6B). In addition, treatment with spilanthol exhibited no noticeable effect on cell viability (Figure 6A), indicating that the reduction of NO expression is not due to the decrease in cell viability or proliferation. Figure 6: Anti-neuroinflammatory efficacy of spilanthol on LPS-treated BV-2 cells. (**A**) Impact of the treatment on cell viability. (**B**) Impact of spilanthol on NO release. Influence of spilanthol on proinflammatory cytokine expression: (**C**) TNF-\(\alpha\) and (**D**) IL-6 levels. Data show the mean \(\pm\) SD values of three independent experiments. \({}^{\#\#}\)_p_ < 0.001 compared with the control group, \({}^{*}\)_p_ < 0.05, \({}^{**}\)_p_ < 0.01, and \({}^{***}\)_p_ < 0.001 compared with the LPS-control group, ANOVA followed by [PERSON]’s post hoc test. ELISA was utilized to assess the potential of spilanthol to attenuate the release of proinflammatory cytokines, specifically TNF-\(\alpha\) and IL-6, in LPS-induced BV-2 cells. The treatment of BV-2 cells with LPS has demonstrated significantly high levels of both TNF-\(\alpha\) and IL-6, attaining concentrations of 399.5 \(\pm\) 5.07 and 467.5 \(\pm\) 60.58 pg/mL, respectively, compared with the control (34.36 \(\pm\) 4.48 and 66.87 \(\pm\) 6.11 pg/mL, respectively) (Figure 6C,D). However, treatment with spilanthol induced a concentration-dependent reduction in the LPS-induced production of TNF-\(\alpha\) and IL-6, and the inhibition was statistically significant for the treatments using 50 \(\upmu\)M and 100 \(\upmu\)M. Treatment with spilanthol at 100 and 50 \(\upmu\)M significantly reduces the release of TNF-\(\alpha\) by 33.64% (265.1 \(\pm\) 28.23 pg/mL) and 18.42% (325.9 \(\pm\) 35.52 pg/mL), respectively, with respect to the LPS-induced group. Similarly, IL-6 expression was also significantly inhibited by the treatment of spilanthol. At 100 \(\upmu\)M, IL-6 expression was reduced by 49.75% (234.9 \(\pm\) 20.13 pg/mL), and at 50 \(\upmu\)M, IL-6 expression was reduced by 37.82% (290 \(\pm\) 14.00 pg/mL). Moreover, treatment of spilanthol alone did not elicit any significant alterations in the levels of proinflammatory cytokines. [PERSON] and co-workers evaluated the anti-inflammatory effect of spilanthol in BV-2 cells and revealed that treatment of spilanthol at a concentration of 10 \(\upmu\)M significantly inhibits the expression of IL-1\(\beta\). However, expression of TNF-\(\alpha\), iNOS, and COX-2 is significantly inhibited at 10 \(\upmu\)M concentration [47].