Baicalin Alleviates ADAM17/EGFR Axis-Induced Peritonitis in Weaned Piglets Infected by Glaesserella parasuis

Abstract

Glaesserella parasuis (GPS) is a Gram-negative, pathogenic bacterium that colonizes the upper respiratory tract of piglets and causes Glässer's disease with peritonitis under stress conditions. The mechanism underlying GPS-induced peritonitis in piglets remains unclear. Baicalin is one of the main active ingredients of Huangqin (Scutellaria baicalensis), which has a significant anti-inflammatory effect on inflammatory diseases. Therefore, this study aimed to elucidate the molecular mechanism by which baicalin alleviates GPS-induced peritonitis in piglets, specifically focusing on the role of the ADAM17/EGFR signaling axis. We investigated the effects of baicalin in vitro using porcine peritoneal mesothelial cells (PPMCs) and in vivo in GPS-infected piglets. Our results showed that baicalin reduced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in PPMCs and the peritoneum of piglets after GPS infection. Concurrently, baicalin significantly reduced the upregulation of disintegrin and metalloproteinase 17 (ADAM17), phosphorylated epidermal growth factor receptor (p-EGFR)/EGFR, and phosphorylated extracellular signal-regulated kinase (p-ERK)/ERK induced by GPS infection in PPMCs and the peritoneum of piglets. Crucially, in vitro mechanistic investigations revealed that baicalin can significantly reduce the upregulation of ADAM17, p-EGFR/EGFR, p-ERK/ERK, TNF-α, IL-1β, and IL-6 induced by ADAM17 overexpression in PPMCs. Furthermore, ADAM17 small interfering RNA can significantly reduce the upregulation of ADAM17, p-EGFR/EGFR, p-ERK/ERK, TNF-α, IL-1β, and IL-6 induced by GPS infection in PPMCs. These findings demonstrate that baicalin can inhibit the expression of inflammatory factors TNF-α, IL-1β, and IL-6 through the ADAM17/EGFR axis, and then alleviate the peritonitis caused by GPS in piglets. This provides a theoretical basis for developing novel non-antibiotic strategies, including phytochemical therapeutics and feed additives, for preventing and controlling GPS.

Keywords: Glässer’s disease; anti-inflammatory effects; antibiotic resistance; small interfering RNA; swine; traditional Chinese medicine.

Figure 1

The cytotoxicity of baicalin to porcine peritoneal mesothelial cells. The cells were treated with 6.25–800 μg/mL of baicalin for 12 h. BA stands for baicalin.

Figure 2

Baicalin inhibited the expression of inflammatory proteins in porcine peritoneal mesothelial cells infected with Glaesserella parasuis. Baicalin reduced the upregulation of TNF-α, IL-1β, and IL-6. GPS stands for Glaesserella parasuis; BA stands for baicalin; TNF-α stands for tumor necrosis factor alpha; IL-1β stands for interleukin-1 beta; IL-6 stands for interleukin-6; GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase. Significant differences are denoted as follows: ## represents p < 0.01 for the comparison between the control group and the GPS infection group; * and ** represent p < 0.05 and p < 0.01, respectively, for the comparisons between the GPS infection group and the baicalin groups.

Figure 3

Baicalin inhibited the expression of ADAM17/EGFR axis-related proteins in porcine peritoneal mesothelial cells infected with Glaesserella parasuis. Baicalin reduced the upregulation of ADAM17, p-EGFR/EGFR, and p-ERK/ERK. GPS stands for Glaesserella parasuis; BA stands for baicalin; ADAM17 stands for disintegrin and metalloproteinase 17; EGFR stands for epidermal growth factor receptor; p-EGFR stands for phosphorylated EGFR; ERK stands for extracellular signal-regulated kinase; p-ERK stands for phosphorylated ERK; GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase. Significant differences are denoted as follows: # and ## represent p < 0.05 and p < 0.01, respectively, for the comparisons between the control group and the GPS infection group; * and ** represent p < 0.05 and p < 0.01, respectively, for the comparisons between the GPS infection group and the baicalin groups.

Figure 4

Baicalin inhibited the expression of ADAM17/EGFR axis-related proteins and inflammatory proteins induced by ADAM17 overexpression in porcine peritoneal mesothelial cells. (A) Baicalin reduced the upregulation of ADAM17, p-EGFR/EGFR, and p-ERK/ERK. (B) Baicalin reduced the upregulation of TNF-α, IL-1β, and IL-6. OvADAM17 stands for overexpression of ADAM17; BA stands for baicalin; ADAM17 stands for disintegrin and metalloproteinase 17; EGFR stands for epidermal growth factor receptor; p-EGFR stands for phosphorylated EGFR; ERK stands for extracellular signal-regulated kinase; p-ERK stands for phosphorylated ERK; TNF-α stands for tumor necrosis factor alpha; IL-1β stands for interleukin-1 beta; IL-6 stands for interleukin-6; GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase. Significant differences are denoted as follows: ## represents p < 0.01 for the comparison between the control group and the OvADAM17 group; * and ** represent p < 0.05 and p < 0.01, respectively, for the comparisons between the OvADAM17 group and the baicalin groups.

Figure 5

ADAM17 small interfering RNA (siRNA) inhibited the expression of ADAM17/EGFR axis-related proteins and inflammatory proteins in porcine peritoneal mesothelial cells infected by Glaesserella parasuis. (A) ADAM17 siRNA reduced the upregulation of ADAM17, p-EGFR/EGFR, and p-ERK/ERK. (B) ADAM17 siRNA reduced the upregulation of TNF-α, IL-1β, and IL-6. NC stands for negative control siRNA; siRNA stands for ADAM17 siRNA; GPS stands for Glaesserella parasuis; ADAM17 stands for disintegrin and metalloproteinase 17; EGFR stands for epidermal growth factor receptor; p-EGFR stands for phosphorylated EGFR; ERK stands for extracellular signal-regulated kinase; p-ERK stands for phosphorylated ERK; TNF-α stands for tumor necrosis factor alpha; IL-1β stands for interleukin-1 beta; IL-6 stands for interleukin-6; GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase. Significant differences are denoted as follows: # and ## represent p < 0.05 and p < 0.01, respectively, for the comparisons between the negative control siRNA group and the negative control siRNA plus GPS group; * and ** represent p < 0.05 and p < 0.01, respectively, for the comparisons between the corresponding negative control siRNA and ADAM17 siRNA groups, both in the presence and absence of GPS infection.

Figure 6

Baicalin alleviated inflammatory protein expression via the ADAM17/EGFR axis in the peritoneum of piglets infected with Glaesserella parasuis. (A) Baicalin reduced the upregulation of ADAM17, p-EGFR/EGFR, and p-ERK/ERK. (B) Baicalin reduced the upregulation of TNF-α, IL-1β, and IL-6. GPS stands for Glaesserella parasuis; BA stands for baicalin; ADAM17 stands for disintegrin and metalloproteinase 17; EGFR stands for epidermal growth factor receptor; p-EGFR stands for phosphorylated EGFR; ERK stands for extracellular signal-regulated kinase; p-ERK stands for phosphorylated ERK; TNF-α stands for tumor necrosis factor alpha; IL-1β stands for interleukin-1 beta; IL-6 stands for interleukin-6; GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase. Significant differences are denoted as follows: ## represents p < 0.01 for the comparison between the control group and the GPS challenge group; * and ** represent p < 0.05 and p < 0.01, respectively, for the comparisons between the GPS challenge group and the baicalin treatment groups.