阿仑膦酸钠在地塞米松诱导C2C12细胞自噬中的作用机制探讨  

Study on the mechanism of alendronate in dexamethasone induced autophagy in C2C12 cells

作　　者：田爱现 马剑雄[2] 马信龙[2] 李岩 TIAN Ai-xian;MA Jian-xiong;MA Xin-long;LI Yan(Department of Biomaterials and Histomorphology,Tianjin Key Laboratory of Orthopaedics Biomechanics and Medical Engineering,Tianjin 300050,China;Biomechanics Research Laboratory,Institute of Orthopaedics,Tianjin Hospital(Tianjin Hospital,Tianjin University),Tianjin Key Laboratory of Orthopaedics Biomechanics and Medical Engineering,Tianjin 300050,China)

机构地区：[1]天津市天津医院(天津大学天津医院)骨科研究所生物材料与组织形态学研究室,天津市骨科生物力学与医学工程重点实验室,300050 [2]天津市天津医院(天津大学天津医院)生物力学研究室,300050

出　　处：《天津医药》2021年第9期921-925,共5页Tianjin Medical Journal

基　　金：国家自然科学基金面上项目(81871777,11772226);天津市卫生健康科技项目(RC20204);天津市卫健委中医中西医结合科研课题(2017008)。

摘　　要：目的探讨阿仑膦酸钠(ALN)在地塞米松(Dexa)诱导C2C12细胞自噬中的作用及机制。方法以C2C12细胞为研究对象,设对照组(DMSO处理),Dexa组(100μmol/L Dexa处理),Dexa+ALN组(100μmol/L Dexa+1.0μmol/L ALN处理)。分别以0、0.1、0.5、1.0μmol/L ALN处理C2C12细胞48 h后,采用CCK-8法检测C2C12细胞增殖水平,筛选ALN合适剂量;HE染色法检测C2C12细胞分化;采用Image J统计肌小管直径和融合指数;Western blot检测C2C12细胞肌蛋白肌球蛋白重链(MHC)、肌肉特异性环指蛋白1(MuRF1)和自噬相关蛋白微管相关蛋白1轻链3(LC3)、Beclin-1的表达变化;免疫荧光标记检测C2C12细胞MHC表达水平。结果选取ALN合适剂量1.0μmol/L进行后续实验。HE染色和Image J结果显示ALN剂量为1.0μmol/L时,肌小管直径和融合指数显著增加(P<0.01)。Western blot结果显示,与对照组相比,Dexa组细胞MuRF1蛋白表达水平升高;而与Dexa组相比,Dexa+ALN组MuRF1蛋白表达水平显著下调,自噬相关蛋白LC3、Beclin-1的表达增多(P<0.01)。免疫荧光结果显示,与对照组相比,Dexa组MHC蛋白荧光表达强度(红光)明显降低;与Dexa组相比,Dexa+ALN组MHC蛋白荧光表达强度表达显著升高。结论1.0μmol/L ALN能有效促进C2C12细胞增殖分化,改善Dexa对C2C12细胞分化的抑制作用,抑制肌降解蛋白MuRF1表达,其机制可能与适度激活自噬信号通路有关。Objective To investigate the role and mechanism of alendronate(ALN)in dexamethasone(Dexa)induced autophagy in C2C12 cells.Methods C2C12 cells were divided into the control group(DMSO treatment),Dexa group(100μmol/L Dexa treatment)and Dexa+ALN group(100μmol/L Dexa+1.0μmol/L ALN treatment).C2C12 cells were treated with 0,0.1,0.5 and 1.0μmol/L ALN for 48 h,respectively.The proliferation level of C2C12 cells was detected by CCK-8 method,and the appropriate dose of ALN was screened.The differentiation of C2C12 cells was detected by hematoxylin-eosin(HE)staining.The diameter and fusion index of myotubule were measured by Image J.Western blot assay was used to detect the expression levels of myoglobin MHC,MuRF1 and autophagy related proteins LC3 and Beclin-1 in C2C12 cells.Immunofluorescence was used to detect the myosin heavy chain(MHC)expression of C2C12 cells.Results The appropriate dose of ALN was 1.0μmol/L for subsequent experiments.HE staining and Image J showed that the diameter and fusion index of myotubule were significantly increased when the dose of ALN was 1.0μmol/L(P<0.01).Western blot results showed that compared with the control group,MuRF1 protein increased in Dexa group,while the expression of MuRF1 protein was significantly down-regulated in Dexa+ALN group than that of Dexa group,and autophagy related proteins LC3 and Beclin-1 increased(P<0.01).Immunofluorescence results showed that compared with the control group,the fluorescence expression intensity(red light)of MHC protein was significantly decreased in Dexa group,and the fluorescence expression intensity of MHC protein was significantly increased in Dexa+ALN group than that of Dexa group.Conclusion The 1.0μmol/L ALN can effectively promote the proliferation and differentiation of C2C12 cells,improve the inhibitory effect of Dexa on the differentiation of C2C12 cells,and inhibit the expression of myodegradable protein MuRF1.The mechanism may be related to the moderate activation of autophagy signaling pathway.

关 键 词：二膦酸盐类 地塞米松 自噬 Beclin-1蛋白 阿仑膦酸钠 C2C12细胞 信号通路 

分 类 号：R349.54[医药卫生—基础医学]