Abstract:
Objective: To investigate the protective effects of flavonomaside on HT22 mouse hippocampal neuronal cells by inhibiting the inflammatory response induced by lipopolysaccharide (LPS) in mouse microglia (BV2) and elucidate the underlying mechanisms. Methods: BV2 cells were categorized into control (Con), LPS model (1 μg/mL), flavonomaside low, medium, and high dose groups (50, 100, 200 μmol/L respectively), TLR4 inhibitor group (TAK, 10 μmol/L), and TAK+flavonomaside group (10 μmol/L+200 μmol/L). Nitric oxide (NO), tumor necrosis factor-alpha (TNF-
α), and interleukin-6 (IL-6) levels in supernatants and cell viability were quantified. Western blot assessed the protein levels of TLR4, Myd88, NF-
κB p65, and phosphorylated NF-
κB p65 in BV2 cells. The BV2 cell supernatant from each group acted as conditioned medium (CM) on HT22 cells to measure cell viability and observe morphological changes. Western blot also detected the protein levels of Bax, Bcl-2, cleaved Caspase-3, and Caspase-3 in HT22 cells. Results: No significant difference in BV2 cell viability was observed with varying flavonomaside concentrations compared to the Con group. The flavonomaside (200 μmol/L) group showed a significant reduction in NO, TNF-
α, and IL-6 secretion compared to the LPS group (
P<0.001). Western blot results indicated significant differences in TLR4, Myd88, NF-
κB p65, and phosphorylated NF-
κB p65 protein levels in the flavonomaside groups, respectively. Compared to the CM-Con group, HT22 cell viability significantly decreased in the CM-LPS group (
P<0.001), with cells exhibiting more shrinkage and rupture. HT22 cell viability in the CM-flavonomaside groups gradually improved in a dose-dependent manner, with cell morphology progressively normalizing to a spindle shape. Western blot analysis revealed increased expression of cleaved Caspase-3 and Bax and decreased Bcl-2 expression in the CM-flavonomaside groups compared to the CM-Con group, indicating flavonomaside's reversal of protein expression changes. In conclusion, flavonomaside can protect nerve cells by inhibiting neuroinflammation.