橙皮苷调控Bcl-2/Bax-Caspase3信号通路阻抑H2O2诱导的奶牛乳腺上皮细胞凋亡  

Hesperidin inhibits H2O2-induced apoptosis of bovine mammary epithelial cells by regulating Bcl-2/Bax-Caspase3 signal pathway

作　　者：刘嘉烁 彭灿 汤少勋[2] 韩雪峰[2] 谭支良 LIU Jia-shuo;PENG Can;TANG Shao-xun;HAN Xue-feng;TAN Zhi-liang(School of Horticulture,Hunan Agricultural University,Changsha,Hunan 410128,China;Institute of SubtropicalAgroecology,Chinese Academy of Sciences/Key Laboratory of Subtropical Agroecological Processes/NationalEngineering Laboratory of Livestock and Poultry Breeding Pollution Control and Resource Technology/Southern Central Experimental Station of Animal Nutrition and Feed Science in Ministry ofAgriculture and Rural Affairs,Changsha,Hunan 410125,China)

机构地区：[1]湖南农业大学园艺学院,湖南长沙410128 [2]中国科学院亚热带农业生态研究所/亚热带农业生态过程重点实验室/畜禽养殖污染控制与资源化技术国家工程实验室/农业农村部中南动物营养与饲料科学观测实验站,湖南长沙410125

出　　处：《南方农业学报》2023年第10期3037-3046,共10页Journal of Southern Agriculture

基　　金：中国科学院战略性先导科技专项(XDA26040304)。

摘　　要：【目的】探析橙皮苷对奶牛乳腺上皮细胞凋亡的阻抑效果及其分子作用机制,为其在促进奶牛乳腺器官生长发育和维持泌乳功能领域的应用提供理论依据。【方法】以1000μmol/L H_(2)O_(2)处理奶牛乳腺上皮细胞8 h构建细胞凋亡模型,再用120μg/mL橙皮苷处理24 h,然后采用CCK-8法测定奶牛乳腺上皮细胞活性,通过激光共聚焦显微镜、流式细胞仪和DNA凝胶电泳检测细胞凋亡情况,利用实时荧光定量PCR检测奶牛乳腺上皮细胞的Bcl-2、Bax和Caspase3基因表达水平,并以Western blotting检测Bcl-2、Bax和Caspase3蛋白的表达变化。【结果】与空白对照组(CK)相比,H_(2)O_(2)组奶牛乳腺上皮细胞活性极显著下降(P<0.01,下同),发生凋亡的细胞比例极显著升高,且DNA片段呈梯状条带;橙皮苷组奶牛乳腺上皮细胞活性显著升高(P<0.05,下同),且细胞凋亡程度低,发生凋亡的细胞数目较少。与H_(2)O_(2)组相比,H_(2)O_(2)+橙皮苷组奶牛乳腺上皮细胞活性显著上升,细胞凋亡比例极显著降低,且DNA片段的梯状条带减少。在Bax、Bcl-2和Caspase3基因及其蛋白表达方面,H_(2)O_(2)组奶牛乳腺上皮细胞中的Bax和Caspase3基因及其蛋白表达水平显著或极显著高于CK组,而Bcl-2基因及其蛋白表达水平较低;橙皮苷组奶牛乳腺上皮细胞的Bax和Caspase3基因及其蛋白表达水平显著低或极显著于CK组,Bcl-2基因及其蛋白表达水平则均高于CK组;与H_(2)O_(2)组相比,H_(2)O_(2)+橙皮苷组奶牛乳腺上皮细胞的Bax和Caspase3基因及其蛋白表达极显著下调,而Bcl-2基因及其蛋白表达极显著上调。【结论】橙皮苷通过Bcl-2/Bax-Caspase3信号通路阻抑H_(2)O_(2)诱导的奶牛乳腺上皮细胞凋亡,有效提高细胞活性,故推测橙皮苷对促进乳腺器官发育和维持泌乳能力具有良好的正向效应。【Objective】The aim of this study was to investigate the inhibitory effects of hesperidin on apoptosis of bo-vine mammary epithelial cells and its molecular mechanism,so as to provide a theoretical basis for its application in pro-moting the growth and development of mammary gland and maintaining lactation function in cows.【Method】Apoptosis model of bovine mammary epithelial cells was generated by treating it with 1000μmol/L H_(2)O_(2)for 8 h,and then treated with 120μg/mL hesperidin for 24 h.Cell viability was measured by CCK-8 assay,and apoptosis was detected by confocal microscopy,flow cytometer and DNA gel electrophoresis.The expression levels of Bcl-2,Bax and Caspase3 genes were measured by real-time fluorescence quantitative PCR and the protein expression levels of Bcl-2,Bax and Caspase3 were measured by Western blotting.【Result】Compared with the blank control group(CK),the bovine mammary epithelial cell viability was decreased extremely significantly(P<0.01,the same below)and the apoptosis proportion was extremely sig-nificantly increased in H_(2)O_(2)group,with the DNA fragments appearing as a ladder pattern;the bovine mammary epithelial cell viability was improved significantly in hesperidin group(P<0.05,the same below),while apoptosis rate was de-creased and the number of apoptotic cells was lower in hesperidin group.Compared with H_(2)O_(2)group,the bovine mam-mary epithelial cell viability was significantly increased and apoptosis rate was extremely significantly decreased,and DNA ladder fragments were decreased in H_(2)O_(2)+hesperidin group.In terms of genes expression of Bax,Bcl-2,and Cas-pase3 and proteins expression,in H_(2)O_(2)group,Bax and Caspase3 genes expression and protein expression levels were sig-nificantly up-regulated,while Bcl-2 gene expression and its protein expression levels were down-regulated compared with CK group.In hesperidin group,Bax and Caspase3 gene expression and their protein expression levels were significantly or extremely significantly down-regulated com

关 键 词：奶牛 橙皮苷 乳腺上皮细胞 细胞凋亡 Bcl-2/Bax-Caspase3信号通路 

分 类 号：S823.91[农业科学—畜牧学]