中药复方鹿角胶丸通过PI3-K/AKT信号通路调节破骨细胞凋亡  

Chinese medicine compound antler pill promotes osteoclast apoptosis through PI3K/AKT pathway

作　　者：于冬冬[1] 赵丹阳[2] 姚啸生[1] YU Dongdong1, ZHAO Danyang2, YAO Xiaosheng1(1. Department of Orthopedics, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China 2. Department of Neurology, Shenyang First People＇s Hospital, Shenyang 110041, Chin)

机构地区：[1]辽宁中医药大学附属医院,辽宁沈阳110032 [2]沈阳市第一人民医院神经内科,辽宁沈阳110032

出　　处：《中国骨质疏松杂志》2018年第7期874-878,共5页Chinese Journal of Osteoporosis

基　　金：辽宁省教育厅科学技术研究项目(L201621);辽宁省自然科学基金重点项目(20170540597);沈阳市科技局科技人才应用技术研究计划(18-014-4-47)

摘　　要：目的探索中药复方鹿角胶丸促进破骨细胞凋亡机制的实验研究。方法 RAW264.7细胞培养并传代,RANKL+MCSF诱导RAW264.7细胞向破骨细胞分化;MTS检测不同浓度的鹿角胶丸对破骨细胞增殖活性影响;倒置相差显微镜观察鹿角胶丸对破骨细胞形态学的影响;流式细胞仪检测鹿角胶丸及加入PI3K/AKT通路抑制剂LY294002后对破骨细胞凋亡的影响;Western blot检测Bax、Bcl-2、Caspase-3,Cleaved-Caspase-3、p-AKT、T-AKT的蛋白表达情况,以及检测加入LY294002后对凋亡通路蛋白的影响。结果 MTS显示各个浓度的鹿角胶丸(LJJW高、中、低)均能抑制破骨细胞的增殖活性,其中以LJJW(中浓度)作用24 h后具有明显的增殖活性抑制作用。加入鹿角胶丸后,破骨细胞透光度下降,凋亡的细胞皱缩,漂浮于培养基中。流式细胞仪检测结果显示鹿角胶丸促进破骨细胞凋亡,预处理LY294002后,鹿角胶丸的促破骨细胞凋亡作用受到抑制。免疫印迹结果显示,鹿角胶丸促进线粒体Bax表达,抑制Bcl-2的表达,同时促进Caspase-3的裂解。鹿角胶丸促进AKT磷酸化,加入LY294002后AKT的磷酸化受到抑制,预处理LY294002后鹿角胶丸的促AKT磷酸化作用被抑制。结论中药复方鹿角胶丸通过PI3K/AKT通路促进破骨细胞凋亡。Objective To explore the mechanism of osteoclast apoptosis promoted by traditional Chinese medicine compound antler pill. Methods RAW264. 7 cells were cultured and passaged. RAW264. 7 cells were induced into osteoclasts by RANKL ＋M-CSF. The proliferation of osteoclasts under different concentrations of antler pill was detected with MTS. The osteoclast morphology was observed after the treatment of antler pill. The apoptosis of osteoclasts was detected using flow cytometry. Western blotting was used to detect the protein expression of Bax,Bcl-2,caspase-3,cleaved-caspase-3,p-AKT,and T-AKT. Results MTS result showed that antler pill inhibited osteoclast proliferation at all concentrations,and at the middle concentration of osteoclast proliferation was inhibited obviously after 24 h. After addition of antler pill,the transmittance of osteoclasts decreased,and apoptotic cells shrunk and floated in the culture medium. Flow cytometry result showed that antler pill promoted apoptosis of osteoclasts. Pretreatment with LY294002 blocked the effect of antler pill on osteoclast apoptosis. Western blotting result showed that antler pill promoted mitochondrial Bax expression,inhibited the expression of Bcl-2,and promoted the cleavage of caspase-3.Phosphorylation of AKT promoted by antler pill was inhibited by LY294002 pretreatment. Conclusion Chinese medicine compound antler pill promotes osteoclast apoptosis through PI3 K/AKT pathway.

关 键 词：中医中药 鹿角胶丸 破骨细胞 凋亡 

分 类 号：R459.9[医药卫生—治疗学]