Protective Effects of Luteolin on Glaesserella parasuis-Induced Injury: An In Vitro Study with Porcine Vascular Endothelial Cells

Abstract

Background:Glaesserella parasuis (GPS) is a conditional pathogen that colonizes the upper respiratory tract in pigs and causes Glässer's disease, resulting in high morbidity and mortality in piglets. GPS infection increases the vascular endothelial permeability, but the mechanism has not been fully elucidated. Luteolin (Lut) is a naturally occurring flavonoid found in plants such as vegetables, herbs, and fruits, but its potential to treat the increased vascular endothelial permeability caused by GPS infection has not been evaluated. Results: This study revealed that GPS infection induces increased vascular endothelial permeability in porcine iliac artery endothelial cells (PIECs) by increasing the gene expressions of tumor necrosis factor (TNF), interleukin 6 (IL-6), IL-8, and IL-1β, and by regulating F-actin cytoskeleton reorganization. Mechanistically, GPS infection or Cluster of differentiation 44 (CD44) overexpression significantly increased the expressions of vascular-endothelial-permeability-related proteins (CD44; vascular endothelial growth factor (VEGFA); matrixmetalloProteinase-3 (MMP-3); MMP-9; and SRC proto-oncogene, non-receptor tyrosine kinase (c-Src)) and increased the vascular endothelial permeability; these changes were alleviated by a Lut treatment or CD44 silencing in the PIECs. Conclusions: This study comprehensively illustrates the potential targets and molecular mechanism of Lut in alleviating the GPS-induced increase in vascular endothelial permeability. The CD44 pathway and Lut may be an effective target and antibiotic alternative, respectively, to prevent the increased vascular endothelial permeability caused by GPS.

Keywords: CD44; Glaesserella parasuis; infection; luteolin; vascular endothelial permeability.

Figure 1

Lut-modulated GPS-induced increase in the expression of inflammatory genes in the PIECs. (A) The cytotoxicity of Lut on the PIECs was determined via a CCK8 kit. The PIECs were treated with Lut (1–100 μg/mL) for 12 h. (B) RT-qPCR results showing that Lut alleviated the GPS-induced expression of inflammatory genes in the PIECs. The PIECs were pretreated with Lut for 1 h and then cocultured with GPS for another 12 h. ## p < 0.01 for the control group vs. the GPS group; * p < 0.05 for the Lut group vs. the GPS group for (B) (or control group for (A)), ** p < 0.01 for the Lut group vs. the GPS group for (B) (or control group for (A)).

Figure 2

Lut reversed the F-actin cytoskeletal reorganization induced by GPS. The PIECs were pretreated with Lut for 1 h and then cocultured with GPS for another 12 h. The intensity of F-actin staining was quantified via ImageJ software (https://imagej.net/ij/download.html). ## p < 0.01 for the control group vs. the GPS group; ** p < 0.01 for the Lut group vs. the GPS group.

Figure 3

Lut counteracted the increase in vascular endothelial permeability-related proteins expression induced caused by GPS. Lut reduced the expression of vascular-endothelial-permeability-related proteins induced by GPS in the PIECs, including CD44, VEGFA, MMP-3, MMP-9, and c-Src induced by GPS treated in the PIECs. ImageJ software was used for the densitometry. # p < 0.05 and ## p < 0.01 for the control group vs. the GPS group; * p < 0.05 and ** p < 0.01 for the control group vs. the GPS group.

Figure 4

Lut alleviated the CD44 overexpression–induced increased expression of vascular endothelial permeability-related proteins in PIECs. Pretreatment with Lut alleviated the CD44-overexpression-induced upregulation of vascular-endothelial-permeability-related proteins, including CD44, VEGFA, MMP-3, MMP-9, and c-Src, in the PIECs. ImageJ software was used for densitometry. OvCD44 indicates CD44 overexpression. # p < 0.05 and ## p < 0.01 for the control group vs. the OvCD44 group; * p < 0.05 and ** p < 0.01 for the control group vs. the OvCD44 group.

Figure 5

Silencing CD44 reduced the GPS–induced increase in the expression of vascular endothelial permeability-related proteins in PIECs. Silencing CD44 decreased the GPS-induced upregulation of vascular-endothelial-permeability-related proteins, including CD44, VEGFA, MMP-3, MMP-9, and c-Src, in the PIECs. ImageJ software was used for the densitometry. siRNA indicates the silencing of CD44. NC indicates negative control siRNA. * p < 0.05; ** p < 0.01.