In-Vitro Evaluation of the Anti-Inflammatory Potential of Empagliflozin Via Nf-Κb Pathway Inhibition

Background: Empagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, exhibits profound
cardioprotective and renoprotective benefits that extend beyond its primary role in glycemic control. Emerging
evidence suggests these pleiotropic effects are driven by intrinsic immune-modulating properties. This study aimed to
systematically evaluate the in vitro anti-inflammatory efficacy and the underlying molecular mechanisms of
empagliflozin in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.
Methods: Macrophages were pre-treated with safe, non-cytotoxic concentrations of empagliflozin (10, 25, and 50
μM), determined via CCK-8 assay, prior to stimulation with LPS (1 μg/mL). Extracellular nitric oxide (NO)
production was quantified using the Griess reagent, and the secretion of primary pro-inflammatory cytokines (TNF-
α, IL-6, and IL-1β) was measured via highly sensitive enzyme-linked immunosorbent assays (ELISA).
Compartmentalized protein extraction and Western blotting were executed to evaluate the modulation of the nuclear
factor-kappa B (NF-κB) signaling axis.
Results: Empagliflozin maintained macrophage viability (>96%) at all tested doses. Upon LPS stimulation,
empagliflozin dose-dependently and significantly (p < 0.001) attenuated the hyper-secretion of NO, TNF-α, IL-6, and
IL-1β. Mechanistic evaluations revealed that empagliflozin robustly rescued cytosolic IκBα from degradation and
simultaneously suppressed the phosphorylation of the p65 subunit (p-p65). Consequently, the subsequent nuclear
translocation of p65 was dramatically abrogated.
Conclusion: Empagliflozin exerts potent, direct anti-inflammatory effects by systematically stabilizing IκBα and
paralyzing the NF-κB signaling cascade. These findings provide critical mechanistic insights into its pleiotropic
properties, further substantiating its therapeutic utility in mitigating chronic inflammation associated with severe
cardio-metabolic disorders.