丁酸钠对脂多糖诱导的奶牛乳腺上皮细胞系炎性损伤的修复作用

Repair Effects of Sodium Butyrate on Inflammatory Injury of Lipopolysaccharide-Induced Bovine Mammary Epithelial Cell Line

本研究拟通过分析丁酸钠对脂多糖(LPS)诱导的奶牛乳腺上皮细胞系(MAC-T细胞)炎性损伤的修复作用,进一步从体外角度阐述丁酸钠对奶牛乳腺健康的调控机制。在MAC-T细胞中添加不同浓度(0、1、10、100、1 000、10 000 ng/mL)的LPS,检测细胞活力,以确定LPS的适宜浓度,建立细胞氧化损伤模型;并进一步在MAC-T细胞中添加不同浓度(0、2、4、8、16、32μmol/L)的丁酸钠,检测细胞凋亡率,以确定丁酸钠的适宜浓度。最终选用1 000 ng/mL LPS和16μmol/L丁酸钠用于本试验。试验分为3组,分别为对照组、LPS处理组和LPS+丁酸钠处理组,分别对其细胞形态、氧化应激指标及凋亡蛋白mRNA表达水平进行检测。结果表明:1)对照组MAC-T细胞呈扁平的无规则形态,贴壁状态良好;而LPS处理组MAC-T细胞核固缩、破裂,并出现大面积死亡脱落现象;LPS+丁酸钠处理组MAC-T细胞边缘清楚,胞内颗粒较少,死亡脱落现象明显减少。2)与对照组相比,LPS处理组MAC-T细胞中超氧化物歧化酶(SOD)活性和总抗氧化力(T-AOC)显著降低(P<0.05),丙二醛(MDA)含量显著升高(P<0.05);与LPS处理组相比,LPS+丁酸钠处理组MAC-T细胞中SOD活性和T-AOC含量显著升高(P<0.05),MDA含量显著降低(P<0.05)。3)与对照组相比,LPS处理组MAC-T细胞中半胱天冬蛋白酶-3(Caspase-3)、半胱天冬蛋白酶-9(Caspase-9)、B细胞淋巴瘤/白血病-2相关X蛋白(Bax) mRNA表达水平显著或极显著升高(P <0.05或P <0.01),B细胞淋巴瘤/白血病-2(Bcl-2)mRNA表达水平极显著降低(P<0.01);与LPS处理组相比,LPS+丁酸钠处理组MAC-T细胞中Caspase-3、Caspase-9 mRNA表达水平显著下降(P<0.05),Bcl-2 mRNA表达水平显著升高(P<0.05)。由此可见,丁酸钠对LPS造成的MAC-T细胞氧化损伤起到了一定的修复作用,减少了细胞凋亡的发生。

This study intended to analyze the repair effects of sodium butyrate on inflammatory injury of lipopolysaccharide(LPS)-induced bovine mammary epithelial cell line(MAC-T),and to expound the regulatory mechanism of sodium butyrate on mammary gland health of dairy cows in vitro. The oxidative damage model was established by adding different concentrations(0,1,10,100,1 000 and 10 000 ng/mL)of LPS into MAC-T cells and determining the cell viability,to determine the appropriate concentration of LPS. In order to determine the appropriate concentration of sodium butyrate,the apoptosis rate of cells was detected by adding different concentrations(0,2,4,8,16 and 32μmol/L)of sodium butyrate into MAC-T cells. Finally,1 000 ng/mL LPS and 16μmol/L sodium butyrate were selected for this experiment. The experiment was divided into three groups:control group,LPS treatment group and LPS+sodium butyrate treatment group,and the cells morphology,oxidative stress indexes and of apoptosis protein mRNA expression levels were detected.The results showed as follows:1)the cells in the control group showed a flat,irregular shape and were well attached;the cells in the LPS treatment group had pyknosis and rupture of the nuclei,and a large area of death and shedding occurred;the cell edges in the LPS+sodium butyrate treatment group were clear,the intracellular particles were less,and the death and exfoliation were reduced obviously. 2)Compared with the control group,the superoxide dismutase(SOD)activity and total antioxidant stress capacity(T-AOC)in MAC-T cells of LPS treatment group were significantly decreased(P<0.05),and the malondialdehyde(MDA)content was significantly increased(P<0.05);compared with the LPS treatment group,the SOD activity and TAOC in MAC-T cells of LPS+sodium butyrate treatment group were significantly increased(P<0.05),and the MDA content was significantly decreased(P<0.05). 3)Compared with the control group,the mRNA expression levels of cysteinyl aspartate specific proteinase-3(Caspase-3),cysteinyl aspartate specific proteinase-9(Caspase-9)and B-cell lymphoma/leukaemia-2-associated X protein(Bax)in MAC-T cells of LPS+sodium butyrate treatment group were significantly increased(P<0.05 or P<0.01),and the B-cell lymphoma/leukaemia-2(Bcl-2)mRNA expression level was significantly decreased(P<0.05);compared with the LPS treatment group,the mRNA expression levels of Caspase-3 and Caspase-9 in MAC-T cells of LPS+sodium butyrate treatment group were significantly decreased(P<0.05),and the Bcl-2 mRNA expression level was significantly increased(P<0.05). In conclusion,sodium butyrate plays a certain role in repairing the oxidative damage of MAC-T cells caused by LPS and reduces the occurrence of cell apoptosis.[Chinese Journal of Animal Nutrition,2022,34(2):1276-1284]