Paeoniflorin Attenuates Oxidized Low-Density Lipoprotein-Induced Dysfunction in RAW264.7 Cells with AMP-Activated...

This study describes a protocol integrating network pharmacology and in vitro experiments to evaluate the protective effects of Huangqin Tang (HQT) and one of its bioactive constituents, paeoniflorin (PF), against oxidized low-density lipoprotein (ox-LDL)-induced macrophage dysfunction and to investigate AMP-activated protein kinase (AMPK)-related signaling. Network pharmacology identified HQT targets and pathways associated with atherosclerosis (AS). An ox-LDL-induced RAW264.7 macrophage model was used to assess lipid accumulation, cholesterol efflux, adenosine triphosphate (ATP) levels, inflammatory responses, mitochondrial function, oxidative stress, apoptosis, and macrophage phenotypic markers. HQT and PF were compared at non-cytotoxic concentrations. AMPK involvement was evaluated using a cell thermal shift assay (CETSA), pharmacological inhibition, and small interfering RNA (siRNA)-mediated knockdown. ATP-binding cassette transporter A1 (ABCA1), liver X receptor alpha (LXRα), nuclear factor kappa B (NF-κB)-related proteins, and apoptosis-associated proteins were examined by western blotting. Network pharmacology identified 12 overlapping targets, including TNF, PPARG, and NOS3, enriched in pathways related to lipid metabolism, inflammation, AS, and AMPK signaling. Both PF and HQT increased AMPK phosphorylation, reduced ox-LDL-induced lipid accumulation and inflammatory cytokine secretion, and improved cholesterol efflux. PF restored ATP levels and mitochondrial membrane potential, reduced reactive oxygen species production and apoptosis, and increased ABCA1 expression. CETSA demonstrated enhanced AMPK thermal stability following PF treatment. Pharmacological inhibition or siRNA-mediated knockdown of AMPK attenuated PF-associated changes in ABCA1 expression, cholesterol efflux, lipid accumulation, LXRα activation, and NF-κB inhibition. PF reduced CD86⁺ macrophages and increased CD206⁺ macrophages, whereas AMPK silencing partially reversed these changes. NF-κB inhibition produced similar effects on cholesterol homeostasis and macrophage phenotypic markers. This protocol enables evaluation of PF- and HQT-mediated effects on ox-LDL-induced macrophage dysfunction and AMPK-related signaling. Under the tested conditions, PF protected RAW264.7 cells against ox-LDL-induced lipid metabolic dysfunction, inflammation, oxidative stress, mitochondrial dysfunction, and apoptosis, and PF and HQT produced directionally similar changes in macrophage-related endpoints.