摘要
目的探讨牛膝活性成分β-蜕皮甾酮对地塞米松诱导MLO-Y4骨样细胞凋亡的抑制作用及其可能的作用机制。方法 10μmol/L地塞米松(dexamethasone,Dex)作用于MLO-Y4骨样细胞,以PI3K/Akt抑制剂LY294002为对照,β-蜕皮甾酮(β-Ecdysone,β-Ecd)干预,分为6组:①正常组;②10μmol/L Dex模型组;③10μmol/L Dex+10μmol/Lβ-Ecd组;④10μmol/L Dex+10μmol/Lβ-Ecd+50μmol/L LY294002组;⑤10μmol/L Dex+0.1μmol/Lβ-Ecd组;⑥10μmol/L Dex+0.1μmol/Lβ-Ecd+50μmol/L LY294002组。作用48 h后,Annexin V-FITC/PI法检测细胞早期凋亡率,Western-Blot检测Akt1、Phospho-Akt(Thr308)、Connexin43(CX43)、Caspase9、Bad、Phospho-Bad蛋白表达。结果与正常组比较,Dex组细胞凋亡率升高,Caspase9、Bad蛋白表达升高,Akt1、CX43蛋白表达降低;与造模组比较,β-Ecd减弱Caspase9蛋白表达,使Akt1、CX43蛋白表达增强;LY294002使Akt1、CX43蛋白表达明显减弱,而β-Ecd并不能使LY294002降低的Akt1蛋白表达升高。结论 0.1μmol/L、10μmol/Lβ-蜕皮甾酮可能通过Akt信号途径干预地塞米松诱导的骨细胞凋亡。
Objective To investigate the effects of β-ecdysone on glucocorticoid-induced apoptosis by MLO-Y4 osteocyte-like cells and the possible mechanism. Methods MLO-Y4 cells were treated with 10 μM dexamethasone(Dex) and divided into 6 groups:(1) Control group;(2) 10 μmol/L Dex group;(3) 10 μmol/L Dex + 10 μmol/L β-Ecd group;(4) 10 μmol/L Dex +10 μmol/L β-Ecd + 50 μmol/L LY294002 group;(5) 10 μmol/L Dex +0.1 μmol/L β-Ecd group; and(6) 10 μmol/L Dex +0.1 μmol/L β-Ecd + 50 μmol/L LY294002 group. After 48 hours, the cell apoptosis was measured by Annexin V-FITC/PI assay. The protein expressions of Caspase9, Bad, Phospho-Bad, Akt1, Phospho-Akt(Thr308) and Connexin43(CX43) were examined with Western blotting. Results Comparing to those in control group, apoptosis rate, protein expressions of Caspase9 and Bad increased, and Akt1 and CX43 decreased in Dex group. Comparing to those in model groups, β-ecdysone inhibited Caspase9 expression, and increased the expressions of Akt1 and CX43. LY294002 significantly reduced the protein expressions of Akt1 and CX43. β-Ecdysone could not rescue the decreased Akt1 expression by LY294002. Conclusion Ecdysone of 0.1 μmol/L and 10 μmol/L may prevent the osteocyte apoptosis induced by glucocorticoid through Akt signal pathway.
引文
[1]Trijau S,De LG,Pradel V,et al.Osteoporosis prevention among chronic glucocorticoid users:results from a public health insurance database[J].RMD Open,2016,2(2):e000249.
[2]Komori T.Glucocorticoid signaling and bone biology[J].Horm Metab Res,2016,48(11):755-763.
[3]Buckley L,Guyatt G,Fink HA,et al.2017 American College of Rheumatology Guideline for the prevention and treatment of glucocorticoid-induced osteoporosis[J].Arthritis Rheumatol,2017,69(8):1095.
[4]Dai W,Zhang H,Zhong ZA,et al.β-Ecdysone augments peak bone mass in mice of both sexes[J].Clin Orthop Relat Res,2015,473(8):2495-2504.
[5]Duan P,Bonewald LF.The role of the wnt/β-catenin signaling pathway in formation and maintenance of bone and teeth[J].Int JBiochem Cell Biol,2016,77:23-29.
[6]Lems W F,Saag K.Bisphosphonates and glucocorticoid-induced osteoporosis:cons[J].Endocrine,2015,49(3):628-634.
[7]丁香莹,梁敏.凋亡在糖皮质激素性骨质疏松症中的研究进展[J].中国骨质疏松杂志,2017,23(12):1660-1663.
[8]任辉,魏秋实,江晓兵,等.糖皮质激素性骨质疏松的研究新进展[J].中国骨质疏松杂志,2014,20(9):1138-1142.
[9]Sato AY,Tu X,Mcandrews KA,et al.Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum(ER)stress in vitro and in vivo in female mice[J].Bone,2015,73(6):60-68.
[10]Davis HM,Pacheco-Costa R,Atkinson EG,et al.Disruption of the Cx43/mi R21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging[J].Aging Cell,2017,16(3):551-563.
[11]Kapur P,Wuttke W,Jarry H,et al.Beneficial effectsofβ-Ecdysone on the joint,epiphyseal cartilage tissue and trabecular bone in ovariectomized rats[J].Phytomedicine,2010,17(5):350-355.
[12]Kar R,Riquelme MA,Werner S,et al.Connexin 43 channels protect osteocytes against oxidative stress-induced cell death[J].J Bone Miner Res,2013,28(7):1611-1621.
[13]Gao J,Cheng TS,An Q,et al.Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43in osteocytes[J].Oncotarget,2016,7(19):26966-26978.
[14]Xia LJ,Wu YL,Zhang FC.Combination of cecropin XJ and LY294002 induces synergistic cytotoxicity,and apoptosis in human gastric cancer cells via inhibition of the PI3K/Akt signaling pathway[J].Oncology Letters,2017,14(6):7522-7528.
[15]Xu T,Pang Q,Wang Y,et al.Betulinic acid induces apoptosis by regulating PI3K/Akt signaling and mitochondrial pathways in human cervical cancer cells[J].Int J Molecul Med,2017,40(6):1669-1678.
[16]Zhou Q,Ye M,Lu Y,et al.Curcumin improves the tumoricidal effect of mitomycin C by suppressing ABCG2 expression in stem cell-like breast cancer cells[J].PLoS One,2015,10(8):e0136694.
