谷氨酰胺对氧化应激状态下胎衣不下奶牛内质网应激的影响

doi:10.3864/j.issn.0578-1752.2025.07.015

摘要/Abstract

摘要：

【目的】明确谷氨酰胺是否可以通过PI3K途径减弱胎衣不下奶牛母体胎盘因氧化应激而引发的内质网应激作用，探讨谷氨酰胺的保护机制。【方法】依据产后12 h内胎衣能否正常排出作为胎衣不下组（retained fetal membranes， RFM）奶牛和健康组（NRFM）奶牛的判定标准，选择胎衣不下组奶牛与健康组奶牛各3头。在明确胎衣不下组与健康组奶牛血清中谷氨酰胺（glutamine，Gln）的含量的基础上，采用qRT-PCR和 Western blot方法检测两组奶牛母体胎盘组织中内质网应激（GRP78、p-PERK、PERK、p-IRE1α、IRE1α、ATF6）和细胞凋亡关键因子（Caspase-3、Caspase-8、Caspase-9、P53、Bcl-2、Bax）的表达变化情况。在体外试验中，使用400 μmol·L^-1的H₂O₂对奶牛子宫内膜上皮细胞（uterine caruncleepithelial cells，UCE）进行刺激，构建了体外奶牛子宫内膜上皮细胞氧化应激细胞模型。在该氧化应激细胞模型的基础上分别进行Gln预处理、PI3K抑制剂（LY294002）与谷氨酰胺共同预处理，采用免疫荧光技术检测细胞质中Ca²⁺的浓度变化，采用 qRT-PCR和 Western blot方法检测奶牛子宫内膜上皮细胞中内质网应激与凋亡相关指标的表达变化。【结果】当奶牛发生胎衣不下时，其血清Gln含量显著降低（P<0.01），其胎盘组织中内质网应激相关蛋白和基因GRP78（P<0.01）、p-PERK/PERK、p-IRE1α/IRE1α、ATF6（P<0.05）表达显著升高，凋亡相关蛋白与基因P53（P<0.01）、Caspase-3、Caspase-8、Caspase-9、Bax/Bcl-2（P<0.05）表达显著降低。在体外氧化应激模型中，H₂O₂ 刺激后UCEs中内质网应激相关蛋白和基因GRP78、p-PERK/PERK、p-IRE1α/IRE1α、ATF6（P<0.01）表达显著升高，与凋亡相关蛋白与基因P53、Caspase-3、Caspase-8、Caspase-9、Bax/Bcl-2（P<0.01）表达显著升高，细胞质中Ca²⁺浓度显著升高（P<0.01）。而与内质网应激组相比，Gln预处理后降低了细胞内质网应激与凋亡水平（P<0.01），细胞质中Ca²⁺浓度显著降低（P<0.01）。但当PI3K被抑制时，与仅使用谷氨酰胺预处理的Gln保护组相比，Gln无法降低奶牛子宫内膜上皮细胞中内质网应激与细胞凋亡相关蛋白与基因的表达，细胞质中Ca²⁺浓度显著升高（P<0.01）。【结论】当奶牛发生胎衣不下时，母体胎盘组织发生较强的内质网应激与细胞的异常凋亡，细胞的正常功能受损，是引发胎衣外排障碍的重要诱因。高水平的Gln可以通过PI3K/AKT途径调控内质网应激关键蛋白的表达，缓解因内质网应激造成的细胞功能障碍，从而减轻因氧化应激引发的内质网应激造成的胎衣外排障碍。

关键词: 胎衣不下, 氧化应激, 内质网应激, 谷氨酰胺, 奶牛子宫内膜上皮细胞

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

王薇, 罗春海, 贾红豆, 刘佳金, 李丹阳, 付世新. 谷氨酰胺对氧化应激状态下胎衣不下奶牛内质网应激的影响[J]. 中国农业科学, 2025, 58(7): 1451-1462.

WANG Wei, LUO ChunHai, JIA HongDou, LIU JiaJin, LI DanYang, FU ShiXin. Effect of Gln on Endoplasmic Reticulum Stress in Retained Fetal Membranes Cows Under Oxidative Stress via the PI3K/AKT Pathway[J]. Scientia Agricultura Sinica, 2025, 58(7): 1451-1462.

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参考文献 31

[1]	HOOSHMANDABBASI R, ZERBE H, BAUERSACHS S, DE SOUSA N M, BOOS A, KLISCH K. Pregnancy-associated glycoproteins in cows with retained fetal membranes. Theriogenology, 2018, 105: 158-163. doi: S0093-691X(17)30462-4 pmid: 28982025
[2]	于程, 汤兆学. 奶牛胎衣不下的病因、临床症状和防治措施. 现代畜牧科技, 2018(3): 63.
	YU C, TANG Z X. Etiology, clinical symptoms and prevention measures of retained placenta in dairy cows. Modern Animal Husbandry Science & Technology, 2018(3): 63. (in Chinese)
[3]	GOHARY K, LEBLANC S J. Cost of retained fetal membranes for dairy herds in the United States. Journal of the American Veterinary Medical Association, 252(12): 1485-1489.
[4]	邢菲菲, 罗胜缤, 温小庆, 曹一鸣, 李美娟, 罗春海, 付世新. NEFA经MMPs途径对奶牛子宫内膜上皮细胞Ⅳ型胶原蛋白表达的影响. 中国兽医学报, 2022, 42(11): 2250-2255.
	XING F F, LUO S B, WEN X Q, CAO Y M, LI M J, LUO C H, FU S X. Effects of NEFA on expression of type Ⅳ collagen in endometrial epithelial cells of dairy cows via MMPs pathway. Chinese Journal of Veterinary Science, 2022, 42(11): 2250-2255. (in Chinese)
[5]	刘炳琦, 罗春海, 姚伟佳, 王薇, 刘佳金, 李丹阳, 付世新. miRNA-424-5p靶向VEGFA对胎衣不下奶牛胎盘胶原蛋白降解的影响. 中国农业科学, 2024, 57(15): 3083-3092. doi: 10.3864/j.issn.0578-1752.2024.15.013.
	LIU B Q, LUO C H, YAO W J, WANG W, LIU J J, LI D Y, FU S X. Mechanism of miRNA-424-5p regulate the decomposition of collagen on retained fetal membranes of dairy cows through VEGFA pathway. Scientia Agricultura Sinica, 2024, 57(15): 3083-3092. doi: 10.3864/j.issn.0578-1752.2024.15.013. (in Chinese)
[6]	张国华, 吕思文, 金振华, 王丽坤, 张莹, 鹿凌岩, 李烬, 张备. 奶牛胎衣不下的病因、临床症状和防治措施. 现代畜牧科技, 2021, 40(10): 84-85.
	ZHANG G H, LÜ S W, JIN Z H, WANG L K, ZHANG Y, LU L Y, LI J, ZHANG B. The etiology,clinical symptoms,and prevention and treatment measures of retained placenta in dairy cows. Modern Animal Husbandry Science & Technology, 2021, 40(10): 84-85. (in Chinese)
[7]	KANKOFER M. Antioxidative defence mechanisms against reactive oxygen species in bovine retained and not-retained placenta: Activity of glutathione peroxidase, glutathione transferase, catalase and superoxide dismutase. Placenta, 2001, 22(5): 466-472. pmid: 11373157
[8]	HAVAJNEH F M F. Antioxidants in dairy cattle health and disease. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Veterinary Medicine, 2014: 104-109.
[9]	赵璧忱. RFM奶牛血清代谢组学分析及Gln对奶牛RFM发生调控机制的研究[D]. 大庆: 黑龙江八一农垦大学, 2020.
	ZHAO B C. Serum metabolomic analysis of RFM dairy cows and study on the regulatory mechanism of Gln on RFM in dairy cows[D]. Daqing: Heilongjiang Bayi Agricultural University, 2020. (in Chinese)
[10]	庄敏, 谢钱龙, 檀灵芳, 庄捷. 橙皮素通过ROS介导内质网应激诱导吉非替尼耐药NCI-H1975细胞发生凋亡. 中国临床药理学与治疗学, 2024, 29(11): 1220-1231.
	ZHUANG M, XIE Q L, TAN L F, ZHUANG J. Hesperetin induces apoptosis in gefitinib-resistant NCI-H1975 cells through ROS mediated endoplasmic reticulum stress. Chinese Journal of Clinical Pharmacology and Therapeutics, 2024, 29(11): 1220-1231. (in Chinese)
[11]	张雨欣, 周小杰, 于浩然, 董婉玉, 杨永春, 王晓杜, 周莹珊, 宋厚辉. 内质网应激与氧化应激. 农业生物技术学报, 2022, 30(10): 2009-2024.
	ZHANG Y X, ZHOU X J, YU H R, DONG W Y, YANG Y C, WANG X D, ZHOU Y S, SONG H H. Endoplasmic reticulum stress and oxidative stress. Journal of Agricultural Biotechnology, 2022, 30(10): 2009-2024. (in Chinese)
[12]	MING S X, TIAN J, MA K, PEI C B, LI L, WANG Z Y, FANG Z Y, LIU M, DONG H, LI W J, ZENG J W, PENG Y H, GAO X F. Oxalate-induced apoptosis through ERS-ROS-NF-κB signalling pathway in renal tubular epithelial cell. Molecular Medicine, 2022, 28(1): 88. doi: 10.1186/s10020-022-00494-5 pmid: 35922749
[13]	LIU J, ZONG C G, YU X, DING Y, CHANG B, WANG R Y, SANG L X. Alanyl-glutamine (Ala-gln) ameliorates dextran sulfate sodium (DSS)-induced acute colitis by regulating the gut microbiota, PI3K-Akt/NF-κB/STAT3 signaling, and associated pulmonary injury. ACS Infectious Diseases, 2023, 9(4): 979-992. doi: 10.1021/acsinfecdis.3c00014 pmid: 36917734
[14]	孙汇蕾, 仲庆振, 李明晔, MOLAWA M N. 热休克蛋白70上调表达对热应激猪小肠上皮细胞凋亡的影响. 动物营养学报, 2020, 32(4): 1867-1874. doi: 10.3969/j.issn.1006-267x.2020.04.046
	SUN H L, ZHONG Q Z, LI M Y, N M. Effects of up-regulated expression of heat shock protein 70 on apoptosis in intestinal epithelial cells of heat-stressed piglets. Chinese Journal of Animal Nutrition, 2020, 32(4): 1867-1874. (in Chinese)
[15]	马焦. Hsp70通过调控p38MAPK信号介导细胞内钙超载与凋亡参与原代大鼠心肌细胞氧糖剥夺: 复氧损伤[D]. 苏州: 苏州大学, 2019.
	MA J. Hsp70 participates in oxygen-glucose deprivation and reoxygenation injury in primary rat cardiomyocytes by mediating intracellular calcium overload and apoptosis through p38MAPK signaling pathway[D]. Suzhou: Soochow University, 2019. (in Chinese)
[16]	TODOROVA V, VANDERPOOL D, BLOSSOM S, NWOKEDI E, HENNINGS L, MRAK R, KLIMBERG V S. Oral glutamine protects against cyclophosphamide-induced cardiotoxicity in experimental rats through increase of cardiac glutathione. Nutrition, 2009, 25(7/8): 812-817.
[17]	葛亚楠, 陈建军, 吴腾飞, 郑振东. 尪五味子乙素通过PI3K/Akt信号通路调控内质网应激促进胃癌细胞凋亡. 解剖科学进展, 2022, 28(6): 755-758.
	GE Y N, CHEN J J, WU T F, ZHENG Z D. Schisandra B regulates endoplasmic reticulum stress and promotes gastric cancer cell apoptosis through PI3K/Akt signaling pathway. Progress of Anatomical Sciences, 2022, 28(6): 755-758. (in Chinese)
[18]	邵帅. 黄芪甲苷通过介导PI3K/AKT/Nrf2信号通路和抑制内质网应激对脂多糖诱导的MC3T3-E1细胞损伤的保护作用[D]. 锦州: 锦州医科大学, 2022.
	SHAO S. Protection of Astragaloside IV on LPS-induced MC3T3-E1 cells injury by mediating PI3K/AKT/Nrf2 signaling pathway and inhibiting endoplasmic reticulum stress[D]. Jinzhou: Jinzhou Medical University, 2022. (in Chinese)
[19]	ZHENG C Y, ZOU X, ZHAO B C, ZHANG M L, LIN H J, LUO C H, XU Z M, SHAO L Y, FU S X. miRNA-185 regulates retained fetal membranes of cattle by targeting STIM1. Theriogenology, 2019, 126: 166-171.
[20]	HOLEČEK M. Origin and roles of alanine and glutamine in gluconeogenesis in the liver, kidneys, and small intestine under physiological and pathological conditions. International Journal of Molecular Sciences, 2024, 25(13): 7037.
[21]	COQUEIRO A Y, ROGERO M M, TIRAPEGUI J. Glutamine as an anti-fatigue amino acid in sports nutrition. Nutrients, 2019, 11(4): 863.
[22]	LIN M Y, LING P Y, HE Q W, CHEN D Z, ZHENG L S, TANG L S, JIANG S W. Low acrosin activity is associated with decreased Spam1/acrosin expression and GSH deficiency-caused premature acrosome release of human sperm cells. Cell and Tissue Research, 2023, 394(3): 529-545. doi: 10.1007/s00441-023-03826-x pmid: 37833433
[23]	LIU T, SUN L, ZHANG Y B, WANG Y L, ZHENG J. Imbalanced GSH/ROS and sequential cell death. Journal of Biochemical and Molecular Toxicology, 2022, 36(1): e22942.
[24]	张勇涛, 李霞, 李晓齐, 买丽亚木古丽·阿布都克尤木, 张贵程, 齐鲁. 内质网应激通路相关蛋白IRE1、PERK、ATF6在舌鳞癌中的表达及其功能的生物信息学分析. 生物化工, 2023, 9(5): 1-6, 18.
	ZHANG Y T, LI X, LI X Q, MAILIYAGULIMU·ABUDUKEYOUMU, ZHANG G C, QI L. Bioinformatics analysis of the expression and function of endoplasmic reticulum stress pathway-related proteins IRE1, PERK and ATF6 in squamous carcinoma of the tongue. Biological Chemical Engineering, 2023, 9(5): 1-6, 18. (in Chinese)
[25]	LIN R J, MA M J, HAN B, ZHENG Y, WANG Y P, ZHOU Y N. Esophageal cancer stem cells reduce hypoxia-induced apoptosis by inhibiting the GRP78-perk-eIF2α-ATF4-CHOP pathway in vitro. Journal of Gastrointestinal Oncology, 2023, 14(4): 1669-1693.
[26]	ZHANG B H, WAN S S, LIU H, QIU Q M, CHEN H, CHEN Z Y, WANG L, LIU X H. Naringenin alleviates renal ischemia reperfusion injury by suppressing ER stress-induced pyroptosis and apoptosis through activating Nrf2/HO-1 signaling pathway. Oxidative Medicine and Cellular Longevity, 2022, 2022: 5992436.
[27]	LI J Y, YANG K Y, DAI S S, HE S Y, LIU R R, GUO Q Y, LIU F. ERS mediated by GRP-78/PERK/CHOP signaling is involved in fluoride-induced ameloblast apoptosis. Biological Trace Element Research, 2024, 202(3): 1103-1114.
[28]	MICHALAK M. Calreticulin: Endoplasmic reticulum Ca²⁺ gatekeeper. Journal of Cellular and Molecular Medicine, 2024, 28(5): e17839.
[29]	姚伟佳, 罗春海, 刘佳金, 王薇, 李丹阳, 刘炳琦, 付世新. 过表达miRNA-424-5p靶向AKT3对奶牛子宫内膜上皮细胞凋亡的影响. 中国畜牧兽医, 2024, 51(8): 3635-3642. doi: 10.16431/j.cnki.1671-7236.2024.08.040
	YAO W J, LUO C H, LIU J J, WANG W, LI D Y, LIU B Q, FU S X. Effect of overexpression of miRNA-424-5p targeting AKT3 on apoptosis of endometrial epithelial cells in dairy cows. China Animal Husbandry & Veterinary Medicine, 2024, 51(8): 3635-3642. (in Chinese)
[30]	MIYAZAKI T, KANATSU-SHINOHARA M, OGONUKI N, MATOBA S, OGURA A, YABE-NISHIMURA C, ZHANG H L, POMMIER Y, TRUMPP A, SHINOHARA T. Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal divisionin vitro. Development, 2023, 150(20): dev201157.
[31]	周乐, 常晨城, 王宇, 满达, 石彩霞, 张文广. PI3K/Akt/mTOR 信号通路调控牛细胞生长作用的研究进展. 饲料研究, 2022, 45(9): 138-142.
	ZHOU L, CHANG C C, WANG Y, MAN D, SHI C X, ZHANG W G. Research progress of PI3K/Akt/mTOR signaling pathway in regulating the growth of bovine cells. Feed Research, 2022, 45(9): 138-142. (in Chinese)

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