Baicalin inhibited PANX-1/P2Y6 signaling pathway activation in porcine aortic vascular endothelial cells infected by Glaesserella parasuis

Abstract

Glaesserella parasuis can induce endothelial barrier damage in piglets, although the mechanism by which this pathogen triggers inflammatory damage remains unclear. Baicalin possesses anti-inflammatory and anti-oxidant activities. However, whether baicalin can relieve endothelial barrier damage caused by Glaesserella parasuis infection has not yet been studied. Hence, we evaluated the ability of baicalin to counteract the changes induced by Glaesserella parasuis in porcine aortic vascular endothelial cells. The results showed that Glaesserella parasuis could upregulate the expression of pannexin 1 channel protein and promote the release of adenosine triphosphate, adenosine diphosphate, adenosine 3'-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate in porcine aortic vascular endothelial cells. The expression level of purinergic receptor P2Y6 was upregulated in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. In addition, Glaesserella parasuis could activate phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells. Baicalin could inhibit pannexin 1 channel protein expression, reduce adenosine triphosphate, adenosine diphosphate, adenosine 3'-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate release, and attenuate the expression level of P2Y6 in porcine aortic vascular endothelial cells induced by Glaesserella parasuis. Baicalin could also reduce the activation of phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. Our study report that Glaesserella parasuis could promote pannexin 1 channel protein expression, induce nucleosides substance release, and P2Y6 expression in porcine aortic vascular endothelial cells and baicalin could inhibit the expression levels of pannexin 1, nucleosides substance, and P2Y6 in the porcine aortic vascular endothelial cells induced by Glaesserella parasuis, which might be served as some targets for treatment of inflammation disease caused by Glaesserella parasuis.

Keywords: Baicalin; Glaesserella parasuis; Liquid chromatography–tandem mass spectrometry; Myosin light chain kinase-myosin light chain; P2Y6; Pannexin 1; Phospholipase C-Protein kinase C.

Graphical abstract

Fig. 1

The effects of probenecid on PANX-1 expression and MRS2578 on P2Y6 expression in Glaesserella parasuis–infected porcine aortic vascular endothelial cells (PAVECs). (A) PANX-1 messenger RNA (mRNA) expression determined with reverse transcription–quantitative polymerase chain (RT-qPCR). (B) P2Y6 mRNA expression determined with RT-qPCR. Each experiment was repeated at least three times. GPS, G. parasuis; PANX-1, pannexin 1. ^##p < 0.01 versus control; *p < 0.05; **p < 0.01.

Fig. 2

The effects of baicalin on PANX-1, P2Y6, and MLCK expression in Glaesserella parasuis–infected porcine aortic vascular endothelial cells (PAVECs). (A) PANX-1, (B) P2Y6 and (C) MLCK messenger RNA (mRNA) expression determined with reverse transcription–quantitative polymerase chain. Each experiment was repeated at least three times. PANX-1, pannexin 1; BA, baicalin; GPS, G. parasuis; MLCK, myosin light chain kinase. ^##p < 0.01 versus control; *p < 0.05; **p < 0.01.

Fig. 3

The effects of baicalin on PANX-1 expression in Glaesserella parasuis–infected porcine aortic vascular endothelial cells (PAVECs). PAVECs were pretreated with MRS2578 (10 μM), probenecid (100 μM), MRS2578 plus probenecid, or baicalin (50 μM) for 1 h, and then co-cultured with G. parasuis for 12 h. Meanwhile, an equal amount of medium or baicalin was added to isolated cells for the control groups. PANX-1 messenger RNA (mRNA) expression was determined with reverse transcription–quantitative polymerase chain. Each experiment was repeated at least three times. PANX-1, pannexin 1; BA, baicalin; GPS, G. parasuis. ^##p < 0.01 versus control; **p < 0.01.

Fig. 4

The effects of baicalin on P2Y6 expression in Glaesserella parasuis–infected porcine aortic vascular endothelial cells (PAVECs). (A) P2Y6 messenger RNA (mRNA) expression determined with reverse transcription–quantitative polymerase chain. (B) P2Y6 protein expression determined with Western blot. Each experiment was repeated at least three times. BA, baicalin; GPS, G. parasuis. ^##p < 0.01 versus control; **p < 0.01.

Fig. 5

The effects of baicalin on PLC β3 expression in Glaesserella parasuis–infected porcine aortic vascular endothelial cells (PAVECs). (A) PLC messenger RNA (mRNA) expression determined with reverse transcription–quantitative polymerase chain. (B) PLC β3 protein expression determine with Western blot. (C) Phosphorylated PLC β3 (p-PLC β3) expression determine with Western blot. (D) Ratio of p-PLC β3/PLC β3. Each experiment was repeated at least three times. BA, baicalin; GPS, G. parasuis; PLC β3, phospholipase C β3. ^##p < 0.01 versus control; *p < 0.05; **p < 0.01.

Fig. 6

The effects of baicalin on PKC expression in Glaesserella parasuis–infected porcine aortic vascular endothelial cells (PAVECs). (A) PKCα messenger RNA (mRNA) expression determined with reverse transcription–quantitative polymerase chain. (B) PKCα protein expression determined with Western blot. (C) Phosphorylated PKCα (p-PKCα) expression determined with Western blot. Each experiment was repeated at least three times. (D) Ratio of p-PKCα/PKCα. BA, baicalin; GPS: G. parasuis; PKCα: protein kinase C α. ^##p < 0.01 versus control; *p < 0.05; **p < 0.01.

Fig. 7

The effects of baicalin on the MLCK-MLC signaling pathway in G. parasuis–infected porcine aortic vascular endothelial cells (PAVECs). (A) MLCK messenger RNA (mRNA) expression determined with reverse transcription–quantitative polymerase chain. (B) MLCK protein expression determined with Western blot. (C) MLC and (D) phosphorylated MLC (p-MLC) expression were determined with Western blot. (E) Ratio of p-MLC/MLC. Each experiment was repeated at least three times. BA, baicalin; GPS, G. parasuis; MLC, myosin light chain; MLCK, myosin light chain kinase. ^##p < 0.01 versus control; *p < 0.05; **p < 0.01.