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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.