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  

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

作　　者：王薇 罗春海[1] 贾红豆 刘佳金 李丹阳 付世新[1] WANG Wei;LUO ChunHai;JIA HongDou;LIU JiaJin;LI DanYang;FU ShiXin(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163000,Heilongjiang)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163000

出　　处：《中国农业科学》2025年第7期1451-1462,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32473102);国家“十四五”重点研发计划(2023YFD1801100);黑龙江八一农垦大学研究生创新科研项目(YJSCX2023-Y34)。

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、-1 P53、Bcl-2、Bax)的表达变化情况。在体外试验中,使用400μmol·L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2+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Ca浓度显著降低(P<0.01)。但当PI3K被抑�【Objective】This study aimed to determine whether glutamine could alleviate endoplasmic reticulum stress in the 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 400μmol·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 Ca^(2+)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 concentrat

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 

分 类 号：S[农业科学]