褪黑素缓解糖皮质激素抑制MC3T3-E1细胞成骨分化与矿化的作用机制  

Melatonin rescues glucocorticoid-induced inhibition of osteoblast differentiation in MC3T3-E1 cells

作　　者：赵锐 朱悦[2] 陶琳[2] 单军[1] ZHAO Rui;ZHU Yue;TAO Lin;SHAN Jun(Minimally Invasive Orthopedic Surgery Ward,Shenyang Orthopedic Hospital,Shenyang 110044,Liaoning,China;Department of Orthopaedics,the First Hospital of China Medical University,Shenyang 110001,Liaoning,China)

机构地区：[1]沈阳市骨科医院微创骨科,沈阳110044 [2]中国医科大学附属第一医院骨外科,沈阳110001

出　　处：《医学研究生学报》2021年第1期8-13,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81472044);辽宁省自然科学基金(2019-BS-294)。

摘　　要：目的褪黑素能否挽救糖皮质激素所导致的成骨分化障碍尚不明确。文中旨在探讨褪黑素对糖皮质激素所致成骨分化障碍的抑制作用。方法应用地塞米松和褪黑素处理前成骨细胞系MC3T3-E1。将MC3T3-E1细胞分为对照组(基础培养液)、骨诱导组(骨分化细胞培养基)、地塞米松组(骨分化细胞培养基+100μmol/L地塞米松)、褪黑素组(骨分化细胞培养基+100μmol/L地塞米松+1μmol/L褪黑素);信号通路组(骨分化细胞培养基+100μmol/L地塞米松+1μmol/L褪黑素+1μmol/L LDN193189)与泛素化酶组(骨分化细胞培养基+100μmol/L地塞米松+1μmol/L褪黑素+5μmol/L MG132)。通过碱性磷酸酶(ALP)活性检测及染色、茜素红S染色及定量分析、qRT-PCR、Western blot评估细胞成骨分化和矿化。进一步分析BMP/Smad信号通路抑制剂(LDN193189)及泛素化酶抑制剂MG132对Runx2的影响。结果MC3T3-E1细胞中ALP、OCN、COLL-1、Runx2 mRNA的表达水平比较,褪黑素组较地塞米松组明显升高(P<0.05),地塞米松组、对照组较骨诱导组明显降低(P<0.05)。与对照组、地塞米松组比较,骨诱导组可以显著提升ALP酶活性、茜素红染色定量表达、Runx2蛋白表达(P<0.05);与地塞米松组比较,褪黑素组显著增加了ALP酶活性、茜素红染色定量表达、Runx2蛋白含量(P<0.05)。与褪黑素组比较,信号通路组显著降低了Runx2蛋白含量(P<0.05),泛素化酶组Runx2蛋白含量表达亦显著降低(P<0.05),同时伴随着Smurf2表达显著增高(P<0.05)。结论褪黑素通过BMP/Smad信号通路,缓解了地塞米松导致的MC3T3-E1细胞成骨分化的抑制,同时伴随着由Smurf2介导的泛素化途径参与。Objective High-dose glucocorticoid(GC)administration causes osteoporosis.Melatonin participates in the regulation of osteoblast proliferation and differentiation,especially low concentrations of melatonin,which enhance osteoblast osteogenesis.This study aims to evaluate the role of melatonin in glucocorticoid-induced inhibition of osteoblast differentiation.Methods In this study,an examination of the expression of osteoblast differentiation markers(ALP,OCN,COLL-1,Runx2)as well as ALP staining and enzymatic activity assay,Alizarin red S staining and quantitation,qRT-PCR and Western blotting were performed to determine the effects of dexamethasone(Dex)and melatonin on the osteoblast differentiation of MC3 T3-E1 cells.The molecular mechanisms were explored by using BMP/Smad signalling inhibitors(LDN193189)and proteasome inhibitor MG132.Results We found that 100μmol/L Dex significantly reduced osteoblast differentiation and mineralization in MC3 T3-E1 cells and that 1μmol/L melatonin attenuated these inhibitory effects.We found that LDN193189 abolished melatonin-induced differentiation and mineralization.Meanwhile,MG132 significantly reduced the expression of Runx2,along with increased the expression of Smurf2 in melatonin-treated and Dex-induced MC3 T3-E1 cells.Conclusion These findings suggest that melatonin rescues Dex-induced inhibition of osteoblast differentiation and mineralization in MC3 T3-E1 cells via the BMP/Smad signalling pathway,along with the involvement of the ubiquitination mediated by Smurf2.

关 键 词：糖皮质激素 褪黑素 成骨分化 BMP/Smad信号通路 SMURF2 

分 类 号：R68[医药卫生—骨科学]