# LPS–TLR4 signaling attenuates CHOP-mediated apoptosis under endoplasmic reticulum stress conditions during porcine embryonic development

## TL;DR

LPS activates TLR4 to reduce cell death in pig embryos under stress, improving their survival and development.

## Contribution

Discovers a new non-immune role for TLR4 in protecting embryos from ER stress via CHOP suppression.

## Key findings

- LPS improved blastocyst formation and cell numbers in ER-stressed embryos.
- LPS reduced CHOP and ATF4 levels, indicating reduced ER stress signaling.
- TLR4 knockdown reversed LPS's protective effects, confirming its essential role.

## Abstract

Persistent endoplasmic reticulum (ER) stress impairs early embryonic development by inducing apoptosis through C/EBP homologous protein (CHOP). Toll-like receptor 4 (TLR4), traditionally recognized for its role in innate immunity, has recently emerged as a modulator of intracellular stress responses. Lipopolysaccharide (LPS), a natural TLR4 agonist derived from Gram-negative bacteria, elicits both pro-inflammatory and cytoprotective effects depending on the cellular context and dosage. This study aimed to elucidate the role of TLR4 signaling in the regulation of CHOP-mediated apoptosis during porcine preimplantation development under ER stress.

Porcine embryos were treated with tunicamycin (TM, 5 nM) to induce ER stress and co-treated with LPS (10 μM) to activate TLR4 signaling. Developmental competence was assessed by blastocyst formation rates, total cell number, and markers of apoptosis and autophagy.

LPS treatment significantly improved blastocyst formation rates compared to TM groups (TM: 37.50 ± 4.77% vs. TM+LPS: 52.89 ± 4.86%). Consistent with this improvement, the total cell number per blastocyst was significantly restored by LPS co-treatment (Control: 55.63 ± 2.15 vs. TM: 38.61 ± 2.57; TM+LPS: 48.84 ± 0.83), confirming enhanced cell proliferation under ER stress conditions. LPS co-treatment markedly reduced CHOP protein expression and suppressed ATF4 expression, indicating alleviation of PERK-ATF4-CHOP signaling. Additionally, autophagy and apoptosis were attenuated, as evidenced by a significantly decreased LC3-II/LC3-I ratio and a reduced number of TUNEL-positive cells. Notably, TLR4 knockdown abolished these LPS-mediated protective effects, confirming the requirement of TLR4 in mitigating ER stress-induced damage.

These findings demonstrated that LPS-mediated TLR4 signaling suppressed CHOP-induced apoptosis and autophagy under persistent ER stress, thereby improving embryonic viability. This study provides novel mechanistic insights into the non-canonical role of TLR4 in early embryonic development and highlights its therapeutic potential for improving in vitro embryo culture systems.