Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (7): 1451-1462.doi: 10.3864/j.issn.0578-1752.2025.07.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Effect of Gln on Endoplasmic Reticulum Stress in Retained Fetal Membranes Cows Under Oxidative Stress via the PI3K/AKT Pathway

WANG Wei(), LUO ChunHai, JIA HongDou, LIU JiaJin, LI DanYang, FU ShiXin()   

  1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163000, Heilongjiang
  • Received:2024-12-02 Accepted:2025-02-14 Online:2025-04-08 Published:2025-04-08
  • Contact: FU ShiXin

Abstract:

【Objective】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. 【Method】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. 【Result】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). 【Conclusion】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.

Key words: retained fetal membranes, oxidative stress, endoplasmic reticulum stress, glutamine, uterine caruncle epithelial

Table 1

Primer sequence"

基因名称Name 引物序列Primer sequence (5'→3') 长度 Length (bp)
β-actin F:CCGCAACCAGTTCGCCAT 180
R:CCCACGTACGAGTCCTTCTG
GRP78 F: GTGCCCACCAAGAAGTCTCA 92
R: GTCAGGGGTCGTTCACCTTC
PERK F: CACAGGGACCTCAAGCCTTC 98
R: TCCTCGTCTTGGTCCATTGC
IRE1α F: GCCATGAGGAATAAGAAGCACC 136
R: TGGCATGGTAGGTGTGTGAG
ATF6 F: TACTTCCAGCAGCACCCAAG 150
R: GCACCACGGTCTGACCTTTA
PI3K F:GGUUAAAGAUCCAGAAGUACA 148
R:UACUUCUGGAUCUUUAACCAU
AKT F:AGGACCTGGAGCAGCGTGAG 116
R:CCTCGCTCTCCAGATTCC
ATF4 F:GCTTAAGCCATGGCGCTTTT 132
R:ATGTTGCGAGGTTTTGGTGC
EIF2α F:ACCACCCTGGAGAGAACAGA 124
R:GTGACCACTTTGGGCTCCAT
CHOP F:CCTCGCTCTCCAGATTCC 96
R:TCCTTCATGCGTTGCTTC
Caspase-3 F:GACAGTGGTGCTGAGGATGACATG 168
R:TTCGCCAGGAAAAGTAACCAGGTG
Caspase-8 F:CCAGAAAGGTGTAGCCGTTGAGAC 144
R:CCCAGCAGAAAGTCAGCCTCATC
Caspase-9 F:GATCAGGCCAGGCAGCTAAT 152
R:CGGCTTTGATGGGTCATCCT
P53 F:CCCTGTCGTCCTTTGTCCC 128
R:AAGGGAAGGGGAATACGTGC
Bcl-2 F:TGTGGATGACCGAGTACCTGAACC 106
R:GCCAGACTGAGCAGTGCCTTC
Bax F:GGCTGGACATTGGACTTCCTTCG 114
R:ATGGTGAGCGAGGCGGTGAG

Fig. 1

Results of determination of serum Gln concentrations in NRFM and RFM cows * indicates significant difference compared with N group, P<0.05. ** indicates extremely significant difference compared with group N, P<0.01. The same as below"

Fig. 2

Expression changes of key factors of ER stress in maternal placentas of RFM cows A, B: The expression of ER stress-related proteins in cow maternal placentae is detected by Western blotting; C: The expression of ER stress-related genes in cow maternal placentae is detected by qRT-PCR"

Fig. 3

Expression changes of key factors of apoptosis-related factors in maternal placentas of RFM cows A, B: The expression of apoptosis-related factors proteins in cow maternal placentae is detected by Western blotting; C: The expression of apoptosis-related factors genes in cow maternal placentae is detected by qRT-PCR"

Fig.4

Regulation of the PI3K/AKT pathway by Gln A, B: The expression of p-PI3K/PI3K and p-AKT/AKT proteins in UCEs is detected by Western blotting; C: The expression of PI3K and AKT genes is detected by qRT-PCR. ** indicates that the difference is extremely significant compared with the control group, P<0.01. ## indicates a very significant difference compared with the PI3K inhibition group, P<0.01"

Fig. 5

Effect of Gln on gene and protein expression in ER stress and apoptosis pathways after inhibition of PI3K/AKT pathway A, B: The expression of PI3K/AKT/PERK/eIF2α/ATF4/CHOP pathway proteins in UCEs was detected by Western blotting; C: The expression of PI3K/AKT/PERK/eIF2α/ATF4/CHOP pathway genes in UCEs was detected by qRT-PCR. * indicates significant difference, P<0.05. ** indicates that the difference is extremely significant, P<0.01. The same as below"

Fig. 6

The expression of ER stress-related factors in oxidative stress state UCEs pretreated with high concentrations of Gln A, B: The expression of ER stress-related proteins in UCEs is detected by Western blotting; C: The expression of ER stress-related genes in UCEs is detected by qRT-PCR. * indicates significant difference, P<0.05. ** indicates that the difference is extremely significant, P<0.01"

Fig. 7

Effect of Gln on the expression of apoptosis-related factors after inhibition of the PI3K/AKT pathway A, B: Western blotting was used to detect the expression of apoptosis-related proteins in UCEs; C: qRT-PCR was used to detect the expression of apoptosis-related genes in UCEs"

Fig. 8

Ca2+ efflux after UECs treated with high Gln concentration under oxidative stress A: Control group; B: ER stress group; C: Gln protection group; D: PI3K inhibition group; E: Fluorescence intensity analysis"

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