Luteolin prevents TNF-α-induced NF-κB activation and ROS production in cultured human placental explants and endothelial cells

Abstract

Introduction: Preeclampsia (PE) is a serious hypertensive pregnancy disorder and a leading cause of maternal and perinatal morbidity and mortality. Despite the prevalence and complications, there are no approved therapeutics to relieve PE symptoms. Inflammation, oxidative stress, and angiogenic imbalance have been shown to contribute to the PE pathophysiology, though there is a lack of understanding in how best to target these pathways in PE. We recently demonstrated that the bioflavonoid luteolin is a potent inhibitor of the anti-angiogenic and pro-hypertensive soluble fms-like tyrosine kinase 1 (sFlt-1), and here we aimed to determine if luteolin was also capable of reducing inflammation and oxidative stress pathways.

Methods: Tumor necrosis factor (TNF)-α, which is upregulated in PE, was utilized to stimulate these pathways in human placental explants and endothelial cells. Endothelin-1 (ET-1) and interleukin (IL)-6 in the media from explants and cells were measured via ELISA, and NF-κB localization and reactive oxygen species were detected via fluorescence microscopy.

Results: Pretreatment with luteolin demonstrated significant reductions in NF-κB activation, reactive oxygen species, superoxide, and IL-6 and ET-1 expression in endothelial cells. We also saw a significant reduction in phosphorylation of NF-κB in human placental explants.

Discussion: These data demonstrate that luteolin inhibits pathways implicated in the development of PE and should be explored further for its potential as a PE therapeutic.

Keywords: ET-1; Inflammation; Luteolin; NF-κB p65; Preeclampsia; ROS.

Figure 1:. Luteolin inhibits phosphorylation of NF-κB subunit p65.

Placental explants exposed to TNF-α have a significant increase in p65 phosphorylation. Pre-treatment with luteolin prevents the increased phosphorylation, bringing levels down such that they are not different from control. Actin was not used for analysis, but to ensure equal loading. **p<0.01, ***p<0.001; N=7/ group.

Figure 2:. Effect of luteolin on TNF-α-induced upregulation of cytokines in placental explants.

Stimulation of placental explants with TNF-α led to significant upregulation of IL-6 (A). Luteolin pretreatment did not significantly decrease IL-6 expression. Stimulation of placental explants with TNF-α led to significant upregulation of vasoconstrictor proET-1. Pretreatment with luteolin resulted in a non-significant trend for decrease in proET-1 expression, and luteolin treatment alone had no effect on proET-1 expression (B). Samples were normalized to explant weight. **p<0.01, ***p<0.001; N=7/group.

Figure 3:. Luteolin prevents the nuclearization of NF-κB p65 subunit in HUVECs.

Cells exposed to TNF-α have significantly increased localization of the p65 subunit of NF-κB, with a lesser amount being present in the cytosol. When the cells are pretreated with luteolin, the translocation of p65 is inhibited, and is found in the cytosol, similar to control conditions. Cells were viewed under 60x magnification and scale bars represent 10μm *p<0.05, ****p<0.0001; N=12/ group.

Figure 4:. Luteolin prevents TNF-α-induced cytokine production in cultured endothelial cells.

HUVECs stimulated with TNF-α have significantly upregulated expression of inflammatory cytokine IL-6 (A) and vasoconstrictor ET-1 (B). Pretreatment with either luteolin or NF-κB inhibitor SN50 returned levels to normal, and treatment with luteolin or SN50 alone cause IL-6 levels to fall such that they were undetectable. For both IL-6 and proET-1, there appeared to be an additive effect with treatment utilizing both luteolin and SN50, as the reduction was greater with dual treatment. Significance compared to TNF-α-stimulated group: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; N=6/group.

Figure 5:. Luteolin treatment reduces oxidative stress in HUVECs.

Treatment with TNF-α leads to significant increases in reactive oxygen species (ROS) and superoxide. Pretreatment with luteolin significantly reduced production of ROS and superoxide. While treatment with luteolin alone did not have an effect on superoxide production, ROS levels decreased such that levels were significantly lower than control. Cells were viewed under 40x magnification and scale bars represent 50μm. Fold change was calculated by normalizing to the average of the control group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; N=10/group.