摘要
围产期奶牛能量负平衡(negative energy balance, NEB)可引起高非酯化脂肪酸(non-esterified fatty acids, NEFA)血症。大量NEFA被乳腺组织吸收,可诱发细胞凋亡。白藜芦醇(resveratrol, RES)是一种非黄酮类多酚有机化合物,具有抗炎抗凋亡等作用。目前,尚不清楚RES对高NEFA处理的乳腺上皮细胞(MAC-T)的周期阻滞和凋亡的影响。因此本研究主要探讨NEFA对MAC-T细胞周期阻滞和凋亡的影响以及RES是否能改善高浓度NEFA对MAC-T的病理作用。添加不同浓度的NEFA处理MAC-T,利用CCK-8细胞活力检测试剂盒检测细胞活力,通过Western blot检测细胞凋亡相关分子Caspase-3、Bcl-2、Bax和细胞周期调节因子Cyclin D1的蛋白表达水平,采用qRT-PCR法检测细胞凋亡相关因子Caspase-3、Bcl-2、Bax的mRNA转录水平,利用流式细胞仪检测细胞周期和细胞凋亡。结果显示,NEFA剂量依赖性地诱导MAC-T细胞凋亡,降低细胞活力,阻滞细胞周期。高NEFA显著增加Bax、Caspase-3的mRNA转录水平和Bax/Bcl-2、cleaved-Caspase-3/Caspase-3蛋白表达,降低细胞周期调节因子Cyclin D1的蛋白表达及Bcl-2 mRNA转录水平。此外,单独添加最高浓度80μmol/L的RES对MAC-T细胞活力无影响,且不会引起细胞凋亡。用高浓度NEFA处理细胞并分别添加10、20、40μmol/L RES,RES的添加可以缓解NEFA导致的Cyclin D1蛋白水平下降,降低Bax/Bcl-2和Cleaved-Caspase-3/caspase-3蛋白水平。同时,RES的添加还可以降低G2/M和S期细胞百分比和细胞凋亡。结果表明,NEFA可以引起细胞周期阻滞和细胞凋亡,RES能缓解NEFA诱导的MAC-T细胞周期阻滞和细胞凋亡。
Negative energy balance(NEB)during the perinatal period can induce high concentration of blood non-esterified fatty acids(NEFA).The NEFA were abundantly taken up by the bovine mammary epithelial cells,which can induce apoptosis.Resveratrol(RES),a non-flavonoid polyphenolic compound,has various of pharmacological effects,including anti-inflammation,anti-apoptosis,and others.The effects of RES on bovine mammary alveolar cell-T(MAC-T)cycle arrest and apoptosis induced by high NEFA are currently unknown.Therefore,this study aims to investigate the effect of NEFA on cell-cycle arrest and apoptosis of MAC-T,and whether RES can ameliorate the pathological effects exerted by high NEFA on MAC-T.The MAC-T cells were cultured with different concentrations of NEFA.Cell viability was assessed using CCK-8.Western blot was used to determine the expression levels of apoptosis-related proteins including Caspase-3,Bax,Bcl-2 and Cyclin D1.qRT-PCR was used to explore the mRNA levels of Caspase-3,Bax,Bcl-2.Flow cytometry was used to detect cell cycle and apoptosis.The results showed that NEFA dose-dependently induced apoptosis,decreased cell viability,and arrested the cell cycle of MAC-T.The mRNA levels of Bax,Caspase-3and protein ratios of Bax/Bcl-2,cleaved-Caspase-3/Caspase-3 were increased,while the protein expression of Cyclin D1and mRNA expression level of Bcl-2were downregulated significantly in response to high NEFA treatment.Treatment with 80μmol/L RES had little effect on cell viability of MAC-T cells and did not induce apoptosis.Importantly,10,20,and40μmol/L RES could alleviate the downregulation of Cyclin D1caused by high NEFA,reduce the protein ratios of Bax/Bcl-2,cleaved-Caspase-3/Caspase-3.Treatment with RES reduced the G2/M,S-phase cell population,as well as cell apoptosis.In summary,NEFA can cause cell cycle arrest and increase apoptosis,while RES can significantly prevent or reverse NEFA-induced MAC-T cell cycle arrest and apoptosis.
作者
曹丽光
刘思宇
栾奕
高文睿
刘恺
李国金
杨子凤
董忆飞
李心慰
高文文
刘国文
CAO Liguang;LIU Siyu;LUAN Yi;GAO Wenrui;LIU Kai;LI Guojin;YANG Zifeng;DONG Yifei;LI Xinwei;GAO Wenwen;LIU Guowen(College of Veterinary Medicine,Jilin University,Changchun 130062,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2024年第5期1032-1040,共9页
Chinese Journal of Veterinary Science
基金
国家自然科学基金青年基金资助项目(32302941)。
