Luteolin: A Phytochemical to Mitigate S. Typhimurium Flagellin-Induced Inflammation in a Chicken In Vitro Hepatic Model

**Background:** Antimicrobial resistance is a growing global threat, and the overuse of antibiotics in livestock, including poultry, contributes to this problem. Natural feed additives such as flavonoids are being explored as alternatives to maintain animal health and productivity. Luteolin, a flavonoid found in many vegetables and herbs, has demonstrated anti-inflammatory and antioxidant properties in mammalian models. The liver is a primary target of Salmonella enterica serovar Typhimurium infection in chickens, and flagellin—the protein monomer of bacterial flagella—activates Toll-like receptor 5 (TLR5), triggering proinflammatory cascades via NF-κB. This study aimed to establish an in vitro model of S. Typhimurium-induced hepatic inflammation using chicken primary hepatocyte–non-parenchymal cell co-cultures and to evaluate the protective effects of luteolin.

**Methods:** Primary hepatic cells were isolated from three-week-old male Ross-308 broiler chickens using a three-step in situ perfusion technique. Hepatocyte and non-parenchymal cell fractions were mixed at a 6:1 ratio and cultured for 24 hours. Cells were then treated for 24 hours with: control medium, flagellin (250 ng/mL), luteolin (4 or 16 µg/mL), or flagellin (250 ng/mL) combined with luteolin (4 or 16 µg/mL). Metabolic activity was assessed using the CCK-8 assay. Cell membrane integrity was evaluated by measuring extracellular lactate dehydrogenase (LDH) activity. Cytokine concentrations (IFN-α, IFN-γ, IL-10) were measured using a multiplex bead-based assay (Milliplex), while IL-6 and IL-8 were measured by chicken-specific ELISA. Extracellular hydrogen peroxide (H₂O₂) was measured using the Amplex Red assay, and intracellular malondialdehyde (MDA) was determined via thiobarbituric acid reactive substances assay. All measurements were performed with n=6 replicates per group. Statistical analysis used the Wilcoxon signed-rank test, with significance set at p ≤ 0.05.

**Key Results:** Luteolin at 16 µg/mL significantly reduced metabolic activity (p = 0.002 alone, p = 0.004 with flagellin) and LDH activity (p = 0.009 alone, p = 0.030 with flagellin), indicating cytotoxicity; therefore, the 16 µg/mL groups were excluded from further analyses. Flagellin (250 ng/mL) alone significantly increased IL-8 concentration (p = 0.016) and the IFN-γ/IL-10 ratio (p = 0.025), while decreasing IL-10 concentration (p = 0.015) compared to control. Luteolin at 4 µg/mL significantly reduced the flagellin-induced IL-8 elevation (p = 0.016). Luteolin alone and in combination with flagellin significantly decreased IFN-α (p = 0.002 and p = 0.041, respectively). Luteolin co-treatment restored IL-10 levels to values not significantly different from control. Luteolin significantly reduced extracellular H₂O₂ both alone and with flagellin (both p = 0.002). MDA concentration was significantly decreased in the combined flagellin–luteolin group compared to flagellin alone (p = 0.016). No significant changes in IL-6 or IFN-γ were observed across groups.

**Clinical Implications:** This study provides initial evidence that luteolin at 4 µg/mL has anti-inflammatory and antioxidant effects in a chicken hepatic cell culture model of S. Typhimurium flagellin-induced inflammation. Luteolin reduced the proinflammatory cytokine IL-8, restored anti-inflammatory IL-10, normalized the IFN-γ/IL-10 ratio, and decreased markers of oxidative stress (H₂O₂ and MDA). These effects suggest luteolin may help maintain hepatic inflammatory and redox homeostasis during enteric bacterial infections. The findings support further investigation of luteolin as a natural feed additive in poultry nutrition to reduce antibiotic use. However, the authors emphasize that in vivo studies are needed to confirm these effects in living animals.