Preventive effect of the total polyphenols from Nymphaea candida in LPS-stimulated inflammation in RAW 264.7 cells

This study aims to investigate the anti-inflammatory mechanism of total polyphenols from Nymphaea candida (NCTP) by using LPS-induced RAW 264.7 cell inflammation model. In vitro, CCK-8 assay was used to test cell viability; the Griess reagent method was used to detect the content of nitric oxide (NO) in cell supernatant; ELISA methods were used to measure the levels of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the cell supernatant; immunofluorescence and flow cytometry were used to measure mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels; flow cytometry was used to test M1 polarization; western blot was utilized to determine the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), transforming growth factor β-activated kinase 1 (TAK1), inhibitor of κB kinase (IKK), inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB), extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), P38 mitogen-activated protein kinase (P38 MAPK), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1), heme oxygenase-1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2-related factor 2 (NRF2). The results showed that NCTP could inhibit the levels of NO, PGE2, COX-2, IL-1β, IL-6 and TNF-α (P<0.01); and prevented LPS-induced RAW 264.7 macrophages M1 polarization (P<0.01). NCTP also downregulated the expression of TLR4, MyD88, TAK1, IKK, p-IκB/IκB, p-NF-κB/NF-κB, p-JNK/JNK, p-P38 MAPK/P38 MAPK, NLRP3, ASC, Caspase-1, KEAP1 proteins, and upregulated the expression of NRF2 and HO-1 in RAW 264.7 cell (P<0.05, P<0.01). Meanwhile, NCTP elevated MMP and reduced ROS production (P<0.05, P<0.01). These results demonstrated that anti-inflammatory mechanism of NCTP might be closely related to antioxidation, ameliorating mitochondrial function, and regulating TLR4/MAPK/NF-κB, NLRP3, KEAP1/NRF2/HO-1 pathways.