This study aimed to determine whether glutamine could alleviate endoplasmic reticulum stress in the maternal placenta of cows with retained fetal membranes (RFM) caused by oxidative stress through the PI3K pathway, and to explore the protective mechanism of glutamine.

Three cows with RFM and three healthy cows (NRFM) were selected based on whether the fetal membranes could be normally expelled within 12 hours after parturition. The content of glutamine (Gln) in the serum of RFM and NRFM cows was determined. The expression changes of endoplasmic reticulum stress (GRP78, p-PERK, PERK, p-IRE1α, IRE1α, and ATF6) and key apoptosis factors (Caspase-3, Caspase-8, Caspase-9, P53, Bcl-2, and Bax) in the maternal placenta of the two groups of cows were detected by qRT-PCR and Western blot. In the in vitro experiment, bovine uterine caruncle epithelial cells (UCEs) were stimulated with 400 μmol·L^-1 H₂O₂ to establish an in vitro oxidative stress model of bovine UCEs. On this basis, Gln pretreatment, PI3K inhibitor (LY294002) and glutamine co-pretreatment were performed. The concentration changes of Ca²⁺ in the cytoplasm were detected by immunofluorescence, and the expression changes of endoplasmic reticulum stress and apoptosis-related indicators in bovine UCEs were detected by qRT-PCR and Western blot.

When cows had RFM, the serum Gln content was significantly decreased (P<0.01), and the expression of endoplasmic reticulum stress-related proteins and genes GRP78 (P<0.01), p-PERK/PERK, p-IRE1α/IRE1α, ATF6 (P<0.05) in the placenta tissue was significantly increased, while the expression of apoptosis-related proteins and genes P53 (P<0.01), Caspase-3, Caspase-8, Caspase-9, Bax/Bcl-2 (P<0.05) was significantly decreased. In the in vitro oxidative stress model, the expression of endoplasmic reticulum stress-related proteins and genes GRP78, p-PERK/PERK, p-IRE1α/IRE1α, ATF6 (P<0.01) and apoptosis-related proteins and genes P53, Caspase-3, Caspase-8, Caspase-9, Bax/Bcl-2 (P<0.01) in UCEs were significantly increased after H₂O₂ stimulation, and the concentration of Ca²⁺ in the cytoplasm was significantly increased (P<0.01). However, compared with the Gln protection group pretreated only with glutamine, Gln could not reduce the expression of endoplasmic reticulum stress and apoptosis-related proteins and genes in bovine UCEs when PI3K was inhibited, and the concentration of Ca²⁺ in the cytoplasm was significantly increased (P<0.01).

When cows had RFM, the maternal placenta tissue underwent strong endoplasmic reticulum stress and abnormal apoptosis, which led to the damage of normal cell function and was an important cause of the disorder of fetal membrane expulsion. High levels of Gln could regulate the expression of key proteins in endoplasmic reticulum stress through the PI3K/AKT pathway, alleviate the cellular dysfunction caused by endoplasmic reticulum stress, and thereby reduce the placental expulsion disorder caused by endoplasmic reticulum stress induced by oxidative stress.

To investigate the effect of miRNA-424-5p on collagen degradation in placental tissues of dairy cows, and then to clarify the regulatory role and mechanism of miRNA-424-5p on the occurrence of retained fetal membranes (RFM) in dairy cows.

The expression level of miRNA-424-5p in maternal placenta tissues of normal cows and cows with RFM were firstly detected, and the expression levels of VEGFA, MMP-2, MMP-9 and COL-IV in maternal placenta tissues of normal cows and cows with RFM were detected by qRT-PCR and Western blot. Prediction of target genes for miRNA-424-5p was performed using bioinformatics analysis, the targeting relationship of miRNA-424-5p with VEGFA was verified using the dual luciferase assay. miRNA-424-5p mimics and miRNA-424-5p inhibitor were transfected in dairy endometrial epithelial cells for overexpression and silencing of miRNA-424-5p. Immunofluorescence was used to observe the changes of VEGFA expression in dairy endometrial epithelial cells. The expression of VEGFA, MMP-2, MMP-9 and COL-IV were detected by qRT-PCR and Western blot.

Compared with the healthy group, miRNA-424-5p mRNA expression level in maternal placental tissues of RFM group was significantly increased (P<0.01), the expression of VEGFA, MMP-2, MMP-9 mRNA and protein was highly significant decreased (P<0.01). COL-IV mRNA and protein was highly significant increased (P<0.01). There were 152 potential target genes of miRNA-424-5p, and VEGFA was confirmed to be the target gene of miRNA-424-5p by the results of dual luciferase assay.After overexpression and silencing of miRNA-424-5p, the immunofluorescence results showed that the expression of VEGFA was lower in the miRNA424-5p mimics group compared with the control group, while the expression of VEGFA was higher in the miRNA424-5p inhibitor group. The qRT-PCR and Western blot results showed that overexpression of miRNA-424-5p, the expression of target gene VEGFA mRNA and protein were significantly decreased, and the expression of MMP-2 and MMP-9 mRNA and protein were highly significantly down-regulated (P<0.01), and COL-IV mRNA and protein expression levels were highly significantly increased (P<0.01).

miRNA-424-5p can through VEGFA pathway to regulate the degradation of MMPs and collagen, and can cause the obstacle of extracellular collagen degradation, miRNA-424-5p is an important factor of causing the occurrence of retained fetal membranes.