Gln减轻LPS诱导奶牛睾丸支持细胞炎症损伤的作用  

Gln Attenuates LPS-induced Inflammatory Injury of Bos taurus Testis Sustentacular Cells

作　　者：丛霞[1] 曲明媚[1] 王龙光[1] 霍爱华[1] 迟世凯 曹荣峰[1] 田文儒[1] 李华涛[1] CONG Xia QU Ming- Mei WANG Long- Guang HUO Ai- Hua CHI Shi- Kai CAO Rong- Feng TIAN Wen-Ru LI Hua-Tao(College of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China)

机构地区：[1]青岛农业大学动物科技学院,青岛266109

出　　处：《农业生物技术学报》2016年第12期1855-1862,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31572590和No.31502138);山东省自然科学基金(No.BS2015NY001);山东省高校计划项目(No.J15LF03)

摘　　要：谷氨酰胺(glutamine,Gln)可以调节多种抗炎因子的表达,但是其能否减轻脂多糖(lipopolysaccharide,LPS)诱导的炎症损伤不得而知。本研究将传至第3代的奶牛(Bos taurus)睾丸支持细胞(sustentacular cell,SCs)分别与Gln(0.5,1,2,4和8 mmol/L)共培养12 h,更换培养液培养4 h后用试剂盒检测各组细胞存活率,当Gln浓度为2 mmol/L时细胞存活率为90.81%。将培养的睾丸支持细胞随机分为4个组:对照(空白)组、LPS(0.1μg/m L LPS共培养12 h)组、Gln(2 mmol/L的Gln共培养12 h)组和LPS+Gln(2 mmol/L的Gln共培养12 h后用0.1μg/m L LPS处理4 h)组,用q RT-PCR和Western blot分别检测热体克蛋白72(heat shock protein 72,HSP72),白介素-1受体相关激酶-M(interkeukin receptor-associated kinase-M,IRAK-M),Toll作用蛋白(toll-interacting protein,Tollip)和锌指蛋白(zinc finger protein,A20)的表达,用酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)法检测白介素-1β(interleukin-1β,IL-1β),白介素-6(IL-6),白介素-8(IL-8),白介素-10(IL-10),白介素-13(IL-13)和白介素-17(IL-17)的表达。结果显示,Gln能诱导SCs HSP72、IRAK-M、Tollip和A20的表达,且HSP72 m RNA和其蛋白的时效表达量分别在2和4 h最高。与空白对照组相比,LPS组细胞的HSP72、IRAK-M、Tollip和A20均显著升高(P<0.05),与LPS组相比,LPS+Gln组细胞的上述指标均显著降低(P<0.05)。同样,与空白组相比,LPS组细胞IL-1β、IL-6、IL-8、IL-10、IL-13和IL-17的表达量均显著升高(P<0.05),与LPS组相比LPS+Gln组细胞的上述相应指标的表达量均显著降低(P<0.05);上述结果说明,LPS能够诱导炎性因子、HSP72、IRAK-M、Tollip和A20炎症负反馈调节因子的表达。而Gln能够抑制LPS诱导的炎性因子、促进抑炎因子的表达,从而减少炎性损伤。研究结果为进一步筛选抗睾丸炎症的分子药物提供新的理论依据。Glutamine（Gln） can regulate expressions of several inflammatory cytokines while it is not clear if it can attenuate inflammatory injury induced by lipopolysaccharide（LPS）. The sustentacular cells（SCs） of Bos taurus on their third generation were cultured with various concentrations（0.5, 1, 2, 4 and 8 mmol/L） of Gln for 12 h and the survival rates of the cells were measured using CCK- 8 kit 4 h after changing medium. The cell survival rates were 90.81% treated with 2 mmol/L of Gln. The readily cultured SCs were randomly divided into 4 groups including control group（blank）, LPS group（cocultured with 0.1 μg/m L LPS for 12 h）,Gln group（cocultured with 2 mmol/L Gln for 12 h） and LPS＋Gln group（treated with 0.1 μg/m L LPS for 4 h after cocultured with 2 mmol/L Gln for 12 h）. Heat shock protein72（HSP72）, Interkeukin receptor-associated kinase- M（IRAK- M）, Toll- interacting protein（Tollip） and Zinc finger protein（A20）m RNA and protein expressions were detected by using q RT- PCR and Western blot, respectively. Interleukin- 1β（IL- 1β）,Interleukin- 6（IL- 6）, Interleukin- 8（IL- 8）, Interleukin- 10（IL- 10）, Interleukin- 13（IL- 13） and Interleukin- 17（IL-17） protein expression were measured by using Enzyme linked immunosorbent assay（ELISA）. The results showed that Gln could induce SCs HSP72, IRAK-M, Tollip and A20 expression. The HSP72, IRAK-M, Tollip and A20 levels were significantly（P〈0.05） increased compared to blank control group while the above indicators were all significantly（P〈0.05） decreased in LPS＋Gln group compared with LPS group. Similarly,IL-1β, IL- 6, IL- 8, IL- 10, IL- 13 and IL- 17 expressions were significantly（P〈0.05） increased in LPS group compared to the blank control group while they were all significantly（P〈0.05） decreased in LPS＋Gln group compared with LPS group. The above results demonstrated that LPS can induce expressions of inflammatory cytokines and negative feedback regulatory factors incl

关 键 词：牛睾丸支持细胞 脂多糖 谷氨酰胺 炎性因子 

分 类 号：S823.91[农业科学—畜牧学]