精氨酸对热应激诱导奶牛中性粒细胞凋亡的影响  

Effects of L-Arginine on neutrophil apoptosis induced by heat stress in dairy cows

作　　者：王超 姜尚 李心慰 宋玉祥 冯海华 WANG Chao;JIANG Shang;LI Xinwei;SONG Yuxiang;FENG Haihua(College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2024年第5期1008-1013,1018,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32102737,32273059)。

摘　　要：旨在研究热应激对奶牛中性粒细胞(polymorphonuclear granulocyte, PMN)凋亡的影响,并探究精氨酸(L-arginine,L-Arg)是否对热应激诱导下的奶牛PMN具有保护作用。利用从健康奶牛分离培养的PMN构建体外试验模型,分为对照组(Con组,37℃)和热应激组(HS组,42℃培养0.0、0.5、1.0、1.5、2.0、2.5、3.0 h),通过CCK-8法检测细胞活性,确定最适热处理时间为2.5 h;将从健康奶牛分离得到的PMN与不同浓度(2、4、6、8、10 mmol/L)L-Arg预孵育30 min后,再置于42℃下培养2.5 h,利用CCK-8法检测细胞活性,最终筛选出L-Arg的最佳使用浓度为4 mmol/L。通过Western blot检测热休克蛋白70(HSP70)和凋亡关键蛋白c-CASP 3的蛋白丰度,流式细胞术检测PMN凋亡率。结果显示,相对于Con组,HS组PMN HSP70蛋白的丰度显著升高(P<0.01),c-CASP 3与CASP 3的蛋白丰度比显著升高(P<0.01),同时PMN凋亡率显著升高(P<0.01);而4 mmol/L的L-Arg预孵育可显著缓解热应激对奶牛PMN的c-CASP 3与CASP 3的蛋白丰度(P<0.01)和凋亡(P<0.05)的影响。这些结果表明,热应激能引起奶牛PMN凋亡,而L-Arg能缓解热应激对奶牛PMN的促凋亡作用。This study aims to investigate the effect of heat stress on the apoptosis of polymorphonuclear granulocyte(PMN)in dairy cows,and to investigate whether L-arginine(L-Arg)has a protective effect on PMN induced by heat stress.In vitro test models were constructed using isolated and cultured PMN from healthy dairy cows,which were divided into the control group(Con group,37℃)and the experimental group(HS group,cultured at 42℃for 0.0,0.5,1.0,1.5,2.0,2.5,3.0 h).Cell activity was detected by CCK-8 method,the optimal heat treatment time was determined to be 2.5 h.Then,the PMN isolated from healthy cows was pre-incubated with L-Arg at different concentrations(2,4,6,8,10 mmol/L)for 30 min,and then cultured at 42℃for 2.5 h.CCK-8 method was used to detect the cell activity,and the optimal concentration of L-Arg was found to be 4 mmol/L.Western blot was used to detect the activation of heat shock protein 70(HSP70)and c-CASP 3,a key protein of apoptosis,and flow cytometry was used to measure the apoptosis rate of PMN.The results showed that compared with the Con group,after PMN culture at 42℃for 2.5 h,in the HS group,the expression of HSP70 protein was significantly increased(P<0.01),and the protein abundance ratio of c-CASP 3 to CASP 3 was significantly increased(P<0.01).The apoptosis rate of PMN was significantly increased(P<0.01).After incubating PMN with 4 mmol/L L-Arg and then cultured at 42℃for 2.5 h,it was found that compared with the HS group,L-Arg could significantly reduce the protein abundance ratio of c-CASP 3 to CASP 3(P<0.01),and significantly reduce the apoptosis rate of PMN(P<0.05).These results suggest that heat stress can induce the apoptosis of PMN in dairy cows,and L-Arg can alleviate the proapoptotic effect of heat stress on PMN in dairy cows.

关 键 词：精氨酸 奶牛 热应激 中性粒细胞 凋亡 

分 类 号：S816.72[农业科学—饲料科学]