自噬相关基因5下调抑制血管紧张素Ⅱ介导的巨噬细胞凋亡

Atg5 Deficiency Inhibited MacrophageApoptosisin Response to Angiotensin Ⅱ

目的探讨自噬相关基因5(autophagy-related gene5,Atg5)在血管紧张素Ⅱ(angiotensionⅡ,AngⅡ)诱导高血压心脏损伤中对巨噬细胞凋亡的影响。方法 40只雄性C57BL/6小鼠及10只Atg5+/-分为对照组,AngⅡ灌泵1d组,AngⅡ灌泵3d组,AngⅡ灌泵7d组和Atg5+/-灌泵组,每组10只。给予乙酸溶液(对照组)及AngⅡ(1500ng/kg*min)微量泵灌注。TUNEL染色观察心脏中细胞凋亡,免疫荧光共染明确凋亡细胞类型;qPCR检测Atg5+/-小鼠心脏中Atg5表达;TUNEL染色观察心脏中细胞凋亡,QuantiGenePlex(QGP)检测凋亡因子表达;体外AngⅡ刺激WT和Atg5+/-骨髓巨噬细胞,免疫荧光共染,检测巨噬细胞凋亡。结果 WT小鼠心脏于AngⅡ灌注1-7d后,TUNEL染色阳性细胞数显著增加,荧光共染显示凋亡细胞为巨噬细胞;Atg5+/-小鼠心脏中Atg5表达量较WT小鼠心脏降低53%;AngⅡ灌注7d后,与WT鼠相比,Atg5+/-小鼠心脏TUNEL染色阳性细胞数降低,促凋亡因子Caspase3、Caspase8、Caspase12表达降低。体外AngⅡ刺激下,Atg5+/-骨髓巨噬细胞较WT骨髓巨噬细胞凋亡减少。结论在AngⅡ致高血压心脏损伤中,Atg5下调可导致心脏中巨噬细胞凋亡下降。

Objective To determinate the role of autophagy-related gene 5 （Atg5） in macrophagesapoptosis in hypertension-induced cardiac damage. Methods 40 wildtypemiceand 20 Atg^5＋/- mice were divided randomly into control group and angiotensin II infusion group（at a rate of 1500ng/min＊kg）. TUNEL staining and immunostaining were used to detect apoptosis and the specific cell type. The mRNA level was determinated by realtime PCR and QuantiGenePlex.Macrophages used in in vitro study were isolated from the bone marrow. Results TUNEL staining showed an increase of apoptotic cells in heart during angiotensin II infusion, while imnunostaining showed a colocalization with F4/80, a marker of macrophage.Real-time PCRshowed a decreased mRNA expression of Atg5 in baseline level. And there was less macrophage apoptosis in Atg^5＋/-mice after angiotensin II infusion.QGP analysis showed a lower level of proapoptotic factor, such as Caspase3, Caspase8, Caspase12. Bone marrow derived macrophages apoptosis fromAtg^5＋/- mice exhibited less apoptosis after angiotensin II treatment compared with that of wildtype mice. Conclusions Our results demonstrate that Atg5 deficiency decreased angiotensin II infusion inducedmacrophages apoptosis.