MAPK/JNK信号通路介导血管紧张素Ⅱ促进成骨细胞凋亡的研究

MAPK/JNK signaling pathway mediates AngiotensinⅡto promote osteoblast apoptosis

目的研究血管紧张素Ⅱ(AngⅡ)调控小鼠成骨细胞凋亡的分子机制。方法用10-6 mol/L AngⅡ刺激小鼠成骨细胞24 h,利用原位末端转移酶标记技术检测成骨细胞凋亡情况。并将成骨细胞分为4组,对照组(α-MEM培养基处理细胞1 h);AngⅡ组(10-6 mol/L AngⅡ处理细胞1 h);AngⅡ+SP600125组[使用c-Jun氨基末端激酶(JNK)通路抑制剂(SP600125)预处理细胞1 h,再用AngⅡ处理1h];AngⅡ+vehicle组(加入对应量的二甲基亚砜预处理1 h,再用AngⅡ处理细胞1 h)。利用免疫印迹法检测凋亡信号通路相关蛋白(Bax、Bcl-2、细胞色素C)及应激活化蛋白激酶(SAPK)/JNK信号通路的表达,同时使用试剂盒检测Caspase-3活性。结果10-6 mol/L AngⅡ处理后,成骨细胞凋亡数目增加2倍,同时细胞内SAPK/JNK通路活化,Bcl-2表达明显降低,Bax表达明显增加,细胞色素C从细胞核内向细胞质内转移增加,Caspase-3活性明显增加,而使用SP600125后可明显抑制上述改变。结论AngⅡ可促进小鼠成骨细胞凋亡,凋亡效应可由SAPK/JNK通路所介导。

Objective To analyze the effects of Angiotensin(Ang)Ⅱon osteoblast apoptosis and underlying molecular mechanism.Methods The mouse osteoblasts were cultured in vitro,and treated with or without 10-6 mol/L AngⅡfor 24 h,cell apoptosis was detected by TUNEL.Osteoblasts were divided into four groups,control group(α-MEM medium treatment for 1 h);AngⅡgroup(10-6 mol/L AngⅡtreatment for 1 h);AngⅡ+SP600125 group[c-Jun N-terminal kinase(JNK)inhibitor SP600125 pre-treatment for 1 h,then 10-6 mol/L AngⅡtreatment for 1 h];AngⅡ+vehicle group(corresponding amount of dimethyl sulfoxide pre-treatment for 1 h,then 10-6 mol/L AngⅡtreatment for 1 h).Bax,Bcl-2,cytochrome C expressions and stress-activated protein kinase(SAPK)/JNK activation were evaluated by Western blot,and Caspase-3 activity was measured.Results After being stimulated with 10-6 mol/L AngⅡ,number of apoptotic cells were increased by 2-time.Expression of Bcl-2 was decreased,while the expression of Bax and translocation of cytochrome C from mitochondria to cytosol were highly increased.And Caspase-3 activity was enormously elevated.However,these above changes could be alleviated by pre-treatment of SP600125.Conclusion AngⅡpromote apoptosis in osteoblast through SAPK/JNK signaling pathway,which could provide insights into potential links between hypertension and bone loss-related diseases.