BHBA对体外培养牛肝细胞氧化应激状态及抗氧化关键酶活性和mRNA表达的影响  

Effect of BHBA on oxidative stress states and the activity and mRNA level of antioxidant key enzyme in primary culture hepatocyte in vitro

作　　者：刘兆喜[1] 王建国[1] 李玉[1] 李心慰[1] 杨文涛[1] 陈灰[1] 史晓霞[1] 宋玉祥[1] 周长领 王哲[1] 刘国文[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]滁州温氏畜牧有限公司,安徽滁州235500

出　　处：《中国兽医学报》2013年第5期715-721,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31172372/C180802)

摘　　要：评价氧化应激状态有助于理解奶牛代谢紊乱性疾病(如酮病等)的发病机制。本研究以牛肝细胞体外培养模型为基础,通过细胞培养液中添加不同浓度的β-羟丁酸(BHBA),运用分光光度方法和实时荧光定量PCR(real-time PCR)技术,分别测定细胞裂解上清液中过氧化氢(H2O2)和丙二醛(MDA)的含量、总抗氧化能力(TCA)和过氧化氢酶(CAT)、总谷胱甘肽过氧化物酶(T-GSH-Px)和总超氧化物歧化酶(T-SOD)的活性,以及CAT、GSH-Px、Mn-SOD和Cu/Zn SOD mRNA表达水平的变化。结果显示,随着BHBA浓度的增加,肝细胞中H2O2和MDA的含量呈剂量依赖性增加,添加BHBA 24h后,各组H2O2和MDA的含量均显著高于0、12和48h。各时间点TCA含量随添加BHBA浓度的增加而降低。添加BHBA 12h后,低浓度组(0.6mmol/L BHBA)T-SOD、T-GSH-Px和CAT的活性及CAT、GSH-Px、Mn-SOD和Cu/Zn SOD mRNA表达均显著高于对照组,高浓度组(1.2和2.4mmol/L BHBA)T-SOD、T-GSH-Px和CAT的活性及CAT、GSH-Px、Mn-SOD和Cu/Zn SOD mRNA表达均低于对照组;添加BHBA 24、48h后,T-SOD、T-GSH-Px和CAT的活性及CAT、GSH-Px、Mn-SOD和Cu/Zn SOD mR-NA表达均随BHBA浓度的增呈剂量依赖性抑制。结果表明,添加低浓度的BHBA可以维持体外培养肝细胞中氧化抗氧化系统的动态平衡,但高浓度BHBA影响该动态平衡,导致肝细胞处于氧化应激状态,引起氧化应激损伤,进而影响肝细胞的正常生理功能。Evaluating oxidative stress states has contributed to comprehend the pathogenesis of metabolic disorders in dairy cows. In this study,based on the cultured hepatocyte model in vitro, different concentration of BHBA were supplemented to culture medium. We determined the content of H202 MDA and, TCA, and the activity of CAT, T-GSH-Px and T-SOD, and the mRNA levels of CAT, GSH-Px, Mn-SOD and Cu/Zn SOD in cell supernatant. The results showed that with the increase of BHBA concentration, the contents of H202 and MDA had dose dependently increased effect,were significantly higher at 24 h than 0 h,12 h and 48 h. TCA content decreases as BHBA concentration in different time point. After 12 h,the activity of T-SOD,T-GSH-Px and CAT in low concentration（0. 6 mmol/L BHBA） and the mRNA expression of CAT,GSH-Px, Mn-SOD and Cu/Zn SOD were higher than the control group, while the activity of T-SOD, T-GSH-Px and CAT in high concentration（1.2 and 2.4 mmol/L BHBA）and the mRNA expression of CAT,GSH-Px,Mn-SOD and Cu/Zn SOD were lower than the control group. After 24 and 48 h supplement BHBA, the activity of T-SOD, T-GSH-Px and CAT and the mRNA expression of CAT,GSH-Px,Mn-SOD and Cu/Zn SOD had dose dependently inhibitory effect with the increase of BHBA concentration. The results indicated that low concentration BHBA could maintain dy- namic balance of oxidant-antioxidant system in primary culture hepatocyte in vitro. However, high concentration BHBA influence dynamic balance, result in oxidative stress of hepatocyte, cause oxidative stress injury and affect normal physiology function of hepatoeyte.

关 键 词：Β-羟丁酸 肝细胞 氧化应激 酮病 

分 类 号：S856.5[农业科学—临床兽医学]