Protocatechuic acid and quercetin attenuate ETEC-caused IPEC-1 cell inflammation and injury associated with inhibition of necroptosis and pyroptosis signaling pathways

Abstract

Background Necroptosis and pyroptosis are newly identified forms of programmed cell death, which play a vital role in development of many gastrointestinal disorders. Although plant polyphenols have been reported to protect intestinal health, it is still unclear whether there is a beneficial role of plant polyphenols in modulating necroptosis and pyroptosis in intestinal porcine epithelial cell line (IPEC-1) infected with enterotoxigenic Escherichia coli (ETEC) K88. This research was conducted to explore whether plant polyphenols including protocatechuic acid (PCA) and quercetin (Que), attenuated inflammation and injury of IPEC-1 caused by ETEC K88 through regulating necroptosis and pyroptosis signaling pathways. Methods IPEC-1 cells were treated with PCA (40 µM) or Que (10 µM) in the presence or absence of ETEC K88. Results PCA and Que decreased ETEC K88 adhesion and endotoxin level in cell supernatant. PCA and Que increased cell number and decreased lactate dehydrogenases (LDH) activity in cell supernatant. PCA and Que improved transepithelial electrical resistance (TEER) and reduced fluorescein isothiocyanate-labeled dextran (FD4) flux, and enhanced membrane protein abundance of occludin, claudin-1 and ZO-1, and rescued distribution of these tight junction proteins. PCA and Que also declined cell necrosis ratio. PCA and Que reduced mRNA abundance and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8, and down-regulated gene expression of toll-like receptors 4 (TLR4) and its downstream signals. PCA and Que down-regulated protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated-RIP1 (p-RIP1), p-RIP1/t-RIP1, t-RIP3, p-RIP3, mixed lineage kinase-like protein (MLKL), p-MLKL, motility related protein 1 (DRP1), phosphoglycerate mutase 5 (PGAM5) and high mobility protein 1 (HMGB1). Moreover, PCA and Que reduced protein abundance of nod-like receptor protein 3 (NLRP3), nod-like receptors family CARD domain-containing protein 4 (NLRC4), apoptosis-related macular protein (ASC), gasdermin D (GSDMD) and caspase-1. Conclusions In general, our data suggest that PCA and Que are capable of attenuating ETEC-caused intestinal inflammation and damage, which is maybe associated with inhibition of necroptosis and pyroptosis signaling pathways.