促红细胞生成素通过激活ERK和p38 MAPK抑制小鼠骨髓间充质干细胞成脂分化
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摘要
目的:探讨促红细胞生成素(EPO)是否抑制小鼠骨髓间充质干细胞(MSCs)分化为脂肪细胞及其机制。方法:分离提取小鼠骨髓MSCs,用3-异丁基-1-甲基黄嘌呤、胰岛素和地塞米松联合诱导成脂分化,实验组加入不同浓度EPO干预,诱导分化第20天油红O染色观察并进行定量分析细胞分化程度;MTT法观察不同浓度EPO对MSCs增殖能力的影响。实时荧光定量PCR检测过氧化物酶体增殖物激活受体γ(PPARγ)、CCAAT/增强子结合蛋白α(C/EBPα)、脂肪酸结合蛋白4(FABP4)和脂联素mRNA表达水平;Western blotting检测PPARγ及细胞外信号调节激酶(ERK)和p38丝裂原活化蛋白激酶(p38 MAPK)磷酸化水平。结果:诱导分化20 d,EPO能显著抑制MSCs的成脂分化,降低油红O染色后的吸光度值,诱导分化过程中各浓度的EPO不影响MSCs增殖活性。EPO能够下调成脂过程中PPARγ、C/EBPα、FABP4和脂联素mRNA表达,增加分化过程中ERK、p38 MAPK及PPARγ蛋白磷酸化。结论:EPO可能通过增加p38 MAPK和ERK蛋白磷酸化,下调PPARγ、C/EBPα、FABP4和脂联素mRNA表达,从而抑制MSCs成脂分化。
AIM: To explore the role of erythropoietin( EPO) in the differentiation of mouse bone marrow-derived mesenchymal stem cells( MSCs) into adipocytes. METHODS: The mouse MSCs were cultured using routine methods. The cells were induced to differentiate by the cocktail medium containing 3-isobutyl-1-methylxanthine,insulin and dexamethasone,and the cells in the experiment group were treated with EPO. On the 20th day of induced differentiation,the cells were detected by oil red O staining. The mRNA expression of peroxisome proliferator-activated receptor γ( PPARγ),CCAAT/enhancer-binding protein α( C/EBPα),fatty acid binding protien 4( FABP4) and adiponectin were determined by real-time fluorescence quantitative PCR. The phosphorylation levels of PPARγ,extracellular signal-regulated kinase( ERK) and p38 mitogen-activated protein kinase( p38 MAPK) were measured by Western blotting. MTT assay was adopted to detect the proliferation. RESULTS: During adipogenic induction,EPO decreased lipid accumulation,and inhibited the adipogenic differentiation of MSCs without cytotoxicity. The mRNA expression of PPARγ,C / EBPα,FABP4and adiponectin was significantly inhibited in induced cells. Moreover,EPO enhanced the activity of both p38 MAPK and ERK,and increased PPARγ phosphorylation. CONCLUSION: EPO significantly inhibits differentiation of mouse bone marrow-derived MSCs into adipocytes in vitro via reducing the mRNA expression of PPARγ,C / EBPα,FABP4 and adiponectin,which may be mediated by the p38 MAPK and ERK signaling pathways.
AIM: To explore the role of erythropoietin( EPO) in the differentiation of mouse bone marrow-derived mesenchymal stem cells( MSCs) into adipocytes. METHODS: The mouse MSCs were cultured using routine methods. The cells were induced to differentiate by the cocktail medium containing 3-isobutyl-1-methylxanthine,insulin and dexamethasone,and the cells in the experiment group were treated with EPO. On the 20th day of induced differentiation,the cells were detected by oil red O staining. The mRNA expression of peroxisome proliferator-activated receptor γ( PPARγ),CCAAT/enhancer-binding protein α( C/EBPα),fatty acid binding protien 4( FABP4) and adiponectin were determined by real-time fluorescence quantitative PCR. The phosphorylation levels of PPARγ,extracellular signal-regulated kinase( ERK) and p38 mitogen-activated protein kinase( p38 MAPK) were measured by Western blotting. MTT assay was adopted to detect the proliferation. RESULTS: During adipogenic induction,EPO decreased lipid accumulation,and inhibited the adipogenic differentiation of MSCs without cytotoxicity. The mRNA expression of PPARγ,C / EBPα,FABP4and adiponectin was significantly inhibited in induced cells. Moreover,EPO enhanced the activity of both p38 MAPK and ERK,and increased PPARγ phosphorylation. CONCLUSION: EPO significantly inhibits differentiation of mouse bone marrow-derived MSCs into adipocytes in vitro via reducing the mRNA expression of PPARγ,C / EBPα,FABP4 and adiponectin,which may be mediated by the p38 MAPK and ERK signaling pathways.
引文
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[2]Grimm C,Wenzel A,Groszer M,et al.HIF-1-induced erythropoietin in the hypoxic retinaprotects against light-induced retinal degeneration[J].Nat Med,2002,8(7):718-724.
[3]Digicaylioglu M,Bichet S,Marti HH,et al.Localization of specific erythropoietin binding sites in defined areas of the mouse brain[J].Proc Natl Acad Sci U S A,1995,92(9):3717-3720.
[4]Anagnostou A,Liu Z,Steiner M,et al.Erythropoietin receptor mRNA expression in human endothelial cells[J].Proc Natl Acad Sci U S A,1994,91(9):3974-3978.
[5]Kagaya Y,Asaumi Y,Wang W,et al.Current perspectives on protective roles of erythropoietin in cardiovascular system:erythropoietin receptor as a novel therapeutic target[J].Tohoku J Exp Med,2012,227(2):83-91.
[6]张新金,马业新,文渊,等.促红细胞生成素通过PI3-K/Akt信号通路抑制血管紧张素Ⅱ诱导的新生大鼠心脏成纤维细胞增殖[J].中国病理生理杂志,2009,25(2):293-298.
[7]Sirén AL,Fratelli M,Brines M,et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress[J].Proc Natl Acad Sci U S A,2001,98(7):4044-4049.
[8]Garcia P,Speidel V,Scheuer C,et al.Low dose erythropoietin stimulates bone healing in mice[J].J Orthop Res,2011,29(2):165-172.
[9]Hojman P,Brolin C,Gissel H,et al.Erythropoietin overexpression protects against diet-induced obesity in mice through increased fat oxidation in muscles[J].PLoS One,2009,4(6):e5894.
[10]Teng R,Gavrilova O,Suzuki N,et al.Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production[J].Nat Commun,2011,2:520.
[11]McGee SJ,Havens AM,Shiozawa Y,et al.Effects of erythropoietin on the bone microenvironment[J].Growth Factors,2012,30(1):22-28.
[12]Naveiras O,Nardi V,Wenzel PL,et al.Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment[J].Nature,2009,460(7252):259-263.
[13]Zhu RJ,Wu MQ,Li ZJ,et al.Hematopoietic recovery following chemotherapy is improved by BADGE-induced inhibition of adipogenesis[J].Int J Hematol,2013,97(1):58-72.
[14]Gurevitch O,Prigozhina TB,Pugatsch T,et al.Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment[J].Transplantation,1999,68(9):1362-1368.
[15]Shiozawa Y,Jung Y,Ziegler AM,et al.Erythropoietin couples hematopoiesis with bone formation[J].PLoS One,2010,5(5):e10853.
[16]Cristancho AG,Lazar MA.Forming functional fat:a growing understanding of adipocyte differentiation[J].Nat Rev Mol Cell Biol,2011,12(11):722-734.
[17]Wu Z,Rosen ED,Brun R,et al.Cross-regulation of C/EBPαand PPARγcontrols the transcriptional pathway of adipogenesis and insulin sensitivity[J].Mol Cell,1999,3(2):151-158.
[18]Lefterova MI,Lazar MA.New developments in adipogenesis[J].Trends Endocrinol Metab,2009,20(3):107-114.
[19]Camp HS,Tafuri SR.Regulation of peroxisome proliferator-activated receptorγactivity by mitogen-activated protein kinase[J].J Biol Chem,1997,272(16):10811-10816.
[20]Hu E,Kim JB,Sarraf P,et al.Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARγ[J].Science,1996,274(5295):2100-2103.