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L-Leucine Promotes STAT1 and ISGs Expression in TGEV-Infected IPEC-J2 Cells via mTOR Activation.

ABSTRACT: L-leucine (Leu), as one of the effective amino acids to activate the mTOR signaling pathway, can alleviate transmissible gastroenteritis virus (TGEV) infection. However, the underlying mechanism by which Leu alleviates the virus infection has not been fully characterized. In particular, how Leu impacts TGEV replication through mTOR signaling has yet to be elucidated. In the present study, we found that TGEV proliferated efficiently in intestinal porcine epithelial cells (IPEC-J2 cells) as evidenced by the increase in viral contents by flow cytometry, the inhibition of cell proliferation by CCK-8 assay as well as the reduction of PCNA level by western blot. Besides, western blot analysis showed that STAT1 expression was markedly reduced in TGEV-infected cells. The results of ELISA revealed the inhibition of ISGs (ISG56, MxA, and PKR) expressions by TGEV infection. TGEV-induced mTOR and its downstream p70 S6K and 4E-BP1, STAT1 and ISGs downregulation were blocked by an mTOR activator-MHY1485 but not by an mTOR inhibitor-RAPA. Concurrently, mTOR activation by MHY1485 reduced the contents of TGEV and vice versa. Furthermore, Leu reversed the inhibition of STAT1 and ISGs by activating mTOR and its downstream p70 S6K and 4E-BP1 in TEGV-infected cells. Our findings demonstrated that Leu promoted the expressions of STAT1 and ISGs via activating mTOR signaling in IPEC-J2 cells, aiming to prevent TGEV infection.

SUBMITTER: Du J

PROVIDER: S-EPMC8339710 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-? concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC.

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L-Leucine Promotes STAT1 and ISGs Expression in TGEV-Infected IPEC-J2 Cells via mTOR Activation.

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