组胺抑制自噬流介导SARA奶牛PMN黏附增强  

Histamine inducedinhibition of autophagic flux mediates enhanced adhesion of PMN in SARA dairy cows

作　　者：李云飞 李聪宜 杨宇宸 冯献程 杜希良 李心慰 李小兵 王哲 宋玉祥 刘国文 LI Yunfei;LI Congyi;YANG Yuchen;FENG Xiancheng;DU Xiliang;LI Xinwei;LI Xiaobing;WANG Zhe;SONG Yuxiang;LIU Guowen(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》2021年第5期992-998,1010,共8页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31672621)。

摘　　要：亚急性瘤胃酸中毒(subacute rumen acidosis, SARA)奶牛存在系统性炎性反应,其分子机制尚不清楚。本研究通过检测健康奶牛(n=15)和SARA奶牛(n=15)外周血免疫生化指标、外周血中性粒细胞(PMN)黏附能力和自噬流的变化,以及体外病理浓度组胺(7μmol/L)对健康奶牛分离培养的PMN黏附和自噬流的影响,探明SARA奶牛高组胺血症对PMN黏附的影响及其分子机制。结果显示,SARA奶牛与健康奶牛相比平均日产奶量下降14.3%;SARA奶牛血清中脂多糖(LPS)的含量从不可检测升高到0.22 EU/mL左右,而组胺浓度从平均1.12μmol/L增加到6.32μmol/L;血清急性期反应蛋白内毒素结合蛋白(LBP)、触珠蛋白(Hp)和血清淀粉样蛋白(SAA)的浓度均有显著性增加,促炎因子肿瘤坏死因子(TNF-α)、白介素6(IL-6)和白介素1β(IL-1β)的平均浓度也有明显升高;免疫印迹结果显示,自噬标记分子微管相关蛋白1轻链3-β(LC3-II)和自噬降解底物P62蛋白水平增多,表明自噬流受阻;黏附试验显示SARA奶牛PMN黏附能力增强,且SARA奶牛PMN表面主要黏附分子白细胞功能相关抗原-1(CD11a)表达增加了约0.5倍,而巨噬细胞功能相关抗原-1(CD11b)表达增加了约3倍,表明SARA奶牛PMN自噬流抑制会导致黏附增强;体外结果显示,组胺可以导致自噬流阻断以及PMN黏附能力的增强,说明SARA奶牛PMN黏附增强主要由高组胺血症引起。结果表明,SARA奶牛高组胺血症可以通过抑制自噬流引起中性粒细胞黏附增强,进而导致系统性炎性反应。Subacute rumen acidosis(SARA) cows have systemic inflammatory response, however, its underlining molecular mechanism is not clear.In order to investigate the effect of hyperhistaminemia on PMN adhesion and its underlining molecular mechanism, the hematological immune indicators, and the adhesion ability and autophagic flux state of neutrophil(PMN) in healthy cows(n=15) and SARA dairy cows(n=15) were compared;the effect of histamine(7 μmol/L) on the adhesion and autophagic flux of PMN isolated from healthy dairy cows was detected in vitro.Compared with healthy cows, the average daily milk production of SARA cows decreased by 14.3%.The content of serum lipopolysaccharide(LPS) in SARA cows increased from undetectable to about 0.22 EU/mL,and the content of histamine increased from an average of 1.12 to 6.32 μmol/L.The acute phase reaction proteins, including LPS binding protein(LBP),haptoglobin(Hp),and serum amyloid(SAA),and the pro-inflammatory factors including tumor necrosis factor(TNF-α),interleukin 6(IL-6),and interleukin 1β(IL-1β) also increased significantly in SARA cows.The results of Western blot showed that the protein levels of autophagy marker LC3-II and autophagy degradation substrate P62 were increased in PMN of SARA cows,indicating that autophagy flux was blocked.Adhesion detecting experiments showed adhesion ability of PMN was enhanced and expression of lymphocyte function associated antigen-1(CD11 a)increased about 0.5 times and macrophage function-related antigen-1(CD11 b)increased about 3 times,suggesting that the SARA induced autophagy flux inhibition led to enhanced PMN adhesion ability.In vitro results confirmed that pathological concentration of histamine caused the blocking of the PMN autophagy flux and enhanced PMN adhesion ability,suggesting that the increase of PMN adhesion in SARA cows was mainly caused by hyperhistaminemia.In conclusion,hyperhistaminemia in SARA dairy cows increases the PMN adhesion by inhibiting the autophagic flux,which in turn leads to systemic inflammatory reacti

关 键 词：奶牛 亚急性瘤胃酸中毒 组胺 炎性反应 中性粒细胞 自噬 黏附 

分 类 号：S852.3[农业科学—基础兽医学] S823.91[农业科学—兽医学]