精氨酸对热应激处理的奶牛原代小肠上皮细胞增殖和凋亡的影响

Effects of L-Arginine on Proliferation and Apoptosis of Primary Intestinal Epithelial Cells of Dairy Cows Treated with Heat Stress in vitro

本试验旨在研究精氨酸(L-Arginine,L-Arg)对热应激奶牛原代小肠上皮细胞增殖和凋亡的影响,探究精氨酸对热应激奶牛小肠上皮细胞损伤的修复作用。首先以体外培养的奶牛原代小肠上皮细胞为模型,分为对照组(Con组,37℃)、试验组[42℃培养6 h后,更换成无精氨酸培养基(HS组)或者不同浓度(2、4、6、8、10 mmol·L^(-1))精氨酸的培养基在37℃培养12 h],采用CCK-8检测细胞活力,筛选出精氨酸最佳修复浓度。然后采用生长状态良好的奶牛原代小肠上皮细胞,分为对照组(Con组,37℃培养)、热应激组(HS组,42℃培养6 h后37℃培养12 h)、6 mmol·L^(-1)精氨酸组(6 mmol·L^(-1)L-Arg组,42℃培养6 h后更换6 mmol·L^(-1)精氨酸培养基37℃培养12 h),DAPI染色观察细胞形态变化,微板法检测乳酸脱氢酶(LDH)漏出量,流式细胞仪检测细胞周期,实时荧光定量PCR和酶联免疫吸附试验(ELISA)分别检测半胱氨酸蛋白水解酶-3(Caspase-3)、半胱氨酸天冬氨酸蛋白水解酶-9(Caspase-9)、Bcl-2相关X蛋白(Bax)以及B淋巴细胞瘤-2(Bcl-2)的mRNA和蛋白的相对表达量来研究精氨酸对热应激奶牛小肠上皮细胞增殖及凋亡的影响。结果表明:在2~6 mmol·L^(-1)范围内,与HS组相比,添加L-Arg可提高热应激小肠上皮细胞的活性,其中浓度为6 mmol·L^(-1)时活力最高(P<0.05)。故选用6 mmol·L^(-1)作为后续试验中L-Arg组的试验浓度。与Con组相比,HS组细胞核形态不规则,染色质浓缩,LDH的漏出量显著升高(P<0.05),S期细胞比率显著减低(P<0.05);促凋亡基因Bax、Caspase-9、Caspase-3的mRNA和蛋白相对表达量显著升高(P<0.05),而抑凋亡基因Bcl-2的蛋白含量显著降低(P<0.05)。上述结果表明,热应激促进了奶牛小肠上皮细胞的凋亡。与HS组相比,添加6 mmol·L^(-1)L-Arg后的奶牛小肠上皮细胞染色质浓缩减少,LDH漏出量显著降低(P<0.05),S期细胞比率显著升高(P<0.05);促凋亡基因Bax、Caspase-3、Caspase-9的mRNA和蛋白相对表达量显著降低(P<0.05),而抑凋亡基因Bcl-2的mRNA和蛋白相对表达量显著升高(P<0.05)。由此可见,6 mmol·L^(-1)精氨酸能够促进体外培养的热应激奶牛小肠上皮细胞增殖,减少细胞的凋亡,缓解热应激对奶牛小肠上皮细胞的损伤。

The aim of this study was to investigate the effect of L-Arginine(L-Arg)on the proliferation and apoptosis of bovine intestinal epithelial cells injured by heat stress.The bovine primary intestinal epithelial cells cultured in vitro were divided into control group(Con group,37℃)and experimental groups(after being cultured at 42℃for 6 h,the medium without L-Arg(HS group)or the medium with different concentrations(2,4,6,8,10 mmol·L^(-1))of L-Arg were changed to culture at 37℃for 12 h).The cell viability was detected by CCK-8 and the best healing concentration of L-Arginine was screened.Then the third generation bovine intestinal epithelial cells with good condition were divided into control group(Con group,cultured at 37℃),heat stress group(HS group,cultured at 42℃for 6 h and then cultured at 37℃for 12 h)and 6 mmol·L^(-1)L-Arg group(42℃for 6 h and then replaced with 6 mmol·L^(-1)L-Arg medium at 37℃for 12 h).The morphological changes of cells were observed by DAPI staining,the leakage of lactate dehydrogenase(LDH)was detected by micro method,and the cell cycle was detected by flow cytometry.The relative expression levels of cysteine proteolytic enzyme-3(Caspase-3),cysteine aspartate proteolytic enzyme-9(Caspase-9),Bcl-2-associated X protein(Bax)and B lymphocyte tumor-2(Bcl-2)mRNA and protein were detected by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay(ELISA)to evaluate the effect of L-Arg on the proliferation and apoptosis of bovine intestinal epithelial cells injured by heat stress.The results showed that in the range of 2~6 mmol·L^(-1),compared with HS group,the activity of intestinal epithelial cells was increased by adding L-Arg under heat stress and the activity was the highest when the concentration of L-Arg was 6 mmol·L^(-1)(P<0.05),which could be used as the experimental concentration of L-Arg group in the follow-up experiment.Compared with Con group,the nuclear morphology of HS group was irregular,chromatin was condensed,the leakage of LDH was significantly increased(P<0.05),the ratio of cells at S stage was significantly decreased(P<0.05),the relative expression levels of Bax,Caspase-9,Caspase-3 mRNA and protein were significantly increased(P<0.05),while the protein content of Bcl-2 was significantly decreased in HS group(P<0.05).These results indicated that heat stress promoted the apoptosis of intestinal epithelial cells in dairy cows.Compared with HS group,the nuclear morphology was regular and the leakage of LDH in intestinal epithelial cells of dairy cows treated with 6 mmol·L^(-1)L-Arg were significantly decreased(P<0.05),the S stage cell ratio increased(P<0.05),and the relative expression levels of Bax,Caspase-3,Caspase-9 mRNA and protein were significantly decreased(P<0.05),while the relative expression levels of Bcl-2 mRNA and prfotein were significantly increased(P<0.05).It is concluded that L-Arg can promote the proliferation of small intestinal epithelial cells injured by heat stress,and reduce cell apoptosis thus alleviate the damage of heat stress on intestinal epithelial cells of dairy cows in vitro.