穿龙薯蓣皂苷对再生障碍性贫血小鼠PPARγ,C/EBPα及脂肪分泌因子表达的影响
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摘要
目的:观察穿龙薯蓣皂苷对再生障碍性贫血小鼠的治疗作用及其对过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPARγ),CCATT增强子结合蛋白α(CCAAT-enhancer binding proteinα,C/EBPα),脂联素(Adiponectin),瘦素(Leptin)的影响,探究其在骨髓间充质干细胞脂肪化过程中发挥效应的可能机制。方法:BALB/c小鼠随机分为正常组及模型组,模型组小鼠经60Coy照射联合尾静脉输注DBA/2小鼠淋巴细胞混悬液法造模。模型评估成功后,将成模小鼠随机分为6组,分别为模型组,穿龙薯蓣皂苷低、中、高剂量组(37. 44,74. 88,149. 76 mg·kg-1·d-1),环孢素组(23. 5 mg·kg-1·d-1),雷公藤多苷组(9. 36 mg·kg-1·d-1),相应药物灌胃14 d。干预完成后,检测各组小鼠外周血象并制作骨髓细胞涂片评价骨髓增生情况。采用贴壁法分离培养各组小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)并进行成脂诱导;采用实时荧光定量聚合酶链式反应(Real-time PCR),蛋白免疫印迹法(Western blot)检测各组小鼠BMMSCs中PPARγ,C/EBPα,Adiponectin,Leptin mRNA和蛋白表达。结果:与正常组比较,模型组小鼠外周血白细胞(white blood cell,WBC),血红蛋白(hemoglobin,HGB),血小板(blood platelet,PLT)明显降低(P <0. 05);与模型组比较,各治疗组小鼠血象均有不同程度回升,以穿龙薯蓣中剂量组最优(P <0. 05)。与正常组比较,模型组小鼠BMMSCs中PPARγ,C/EBPα,Adiponectin,Leptin mRNA和蛋白表达明显升高(P <0. 05);与模型组比较,各治疗组小鼠上述指标均被不同程度抑制,其中穿龙薯蓣皂苷中剂量组和环孢素组PPARγ,C/EBPα,Adiponectin,Leptin mRNA和蛋白表达明显下调,优于雷公藤多苷组(P <0. 05)。结论:穿龙薯蓣皂苷能够促进再障小鼠外周血象恢复,改善骨髓造血。穿龙薯蓣皂苷可抑制AA小鼠BMMSCs PPARγ,C/EBPα的表达,减少脂肪细胞分泌的Adiponectin,Leptin,进而有效地抑制BMMSCs向脂肪细胞的过度分化。
Objective: To observe the effect of diosgenin on aplastic anemia(AA) mice and peroxisome proliferator activated receptor γ(PPARγ),CCAAT-enhancer binding protein α(C/EBPα),Adiponectin,Leptin,in order to discuss the potential mechanism of bone marrow mesenchymal stem cells in the process of adipemia.Method: BALB/c mice were randomly divided into control group and model group. The model group was established by60 Coy irradiation combined with tail vein infusion with lymphatic suspension cells of DBA/2 mice.After successful evaluation of the model,the mice were randomly divided into 6 groups: model group,low,medium and high-dose diosgenin groups(37. 44,74. 88,149. 76 mg·kg-1·d-1),cyclosporine group(23. 5 mg·kg-1·d-1),and tripterygium glycoside group(9. 36 mg·kg-1·d-1),and given corresponding drugs by gavage for 14 days. After the intervention,the peripheral blood of mice in each group was detected,and bone marrow smears were collected to evaluate the proliferation of bone marrow. Bone marrow mesenchymal stem cells(BMMSCs) were isolated and cultured by adherent method and induced by adipogenesis. The mRNA and protein expressions of PPARγ,C/EBPα,Adiponectin,Leptin in BMMSCs were detected by quantitative real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) and Western blot. Result: The white blood cell(WBC),hemoglobin(HGB)and blood platelet(PLT) in peripheral blood of model group were significantly lower than those of normal group(P < 0. 05). The hemogram of each treatment group was higher than that of model group to varying degrees. The middle-dose Dioscorea nipponica group had the most obvious hemogram,with statistically significant differences compared with the model group(P < 0. 05). Compared with the control group,the mRNA and protein expressions of PPARγ,C/EBPα,Adiponectin and Leptin in BMMSCs of the model group increased significantly(P < 0. 05).Compared with the model group,the above indexes in the treatment groups were all decreased,the mRNA and protein expressions of PPARγ,C/EBPα,Adiponectin and Leptin in the middle-dose group diosgenin decreased obviously,which was better than those of Tripterygium glycoside group(P < 0. 05). Conclusion: Diosgenin can promote the recovery of peripheral blood in aplastic anemia mice and improve the hematopoiesis of bone marrow.Diosgenin can reduce the expressions of PPARγ and C/EBPα,the formation of adipocytes and the secretion of Adiponectin and Leptin in adipocytes,and effectively inhibit the process of adipose tissue derived from bone BMMSCs.
Objective: To observe the effect of diosgenin on aplastic anemia(AA) mice and peroxisome proliferator activated receptor γ(PPARγ),CCAAT-enhancer binding protein α(C/EBPα),Adiponectin,Leptin,in order to discuss the potential mechanism of bone marrow mesenchymal stem cells in the process of adipemia.Method: BALB/c mice were randomly divided into control group and model group. The model group was established by60 Coy irradiation combined with tail vein infusion with lymphatic suspension cells of DBA/2 mice.After successful evaluation of the model,the mice were randomly divided into 6 groups: model group,low,medium and high-dose diosgenin groups(37. 44,74. 88,149. 76 mg·kg-1·d-1),cyclosporine group(23. 5 mg·kg-1·d-1),and tripterygium glycoside group(9. 36 mg·kg-1·d-1),and given corresponding drugs by gavage for 14 days. After the intervention,the peripheral blood of mice in each group was detected,and bone marrow smears were collected to evaluate the proliferation of bone marrow. Bone marrow mesenchymal stem cells(BMMSCs) were isolated and cultured by adherent method and induced by adipogenesis. The mRNA and protein expressions of PPARγ,C/EBPα,Adiponectin,Leptin in BMMSCs were detected by quantitative real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) and Western blot. Result: The white blood cell(WBC),hemoglobin(HGB)and blood platelet(PLT) in peripheral blood of model group were significantly lower than those of normal group(P < 0. 05). The hemogram of each treatment group was higher than that of model group to varying degrees. The middle-dose Dioscorea nipponica group had the most obvious hemogram,with statistically significant differences compared with the model group(P < 0. 05). Compared with the control group,the mRNA and protein expressions of PPARγ,C/EBPα,Adiponectin and Leptin in BMMSCs of the model group increased significantly(P < 0. 05).Compared with the model group,the above indexes in the treatment groups were all decreased,the mRNA and protein expressions of PPARγ,C/EBPα,Adiponectin and Leptin in the middle-dose group diosgenin decreased obviously,which was better than those of Tripterygium glycoside group(P < 0. 05). Conclusion: Diosgenin can promote the recovery of peripheral blood in aplastic anemia mice and improve the hematopoiesis of bone marrow.Diosgenin can reduce the expressions of PPARγ and C/EBPα,the formation of adipocytes and the secretion of Adiponectin and Leptin in adipocytes,and effectively inhibit the process of adipose tissue derived from bone BMMSCs.
引文
[1] Yamazaki H. Acquired aplastic anemia[J]. Rinsho Ketsueki,2016,57(2):91-97.
[2] LI J, YANG S, LU S, et al. Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia[J]. PLo S One,2012,7(11):e47764.
[3] Clarke S L, Robinson C E, Gimble J M. CAAT/enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor gamma2 promoter[J]. Biochem Biophys Res Commun,1997,240(1):99-103.
[4]王晓燕,余卫,罗英.再生障碍性贫血患者血浆脂联素水平与骨髓造血功能相关性研究[J].新医学,2012,43(3):181-183.
[5]刘国文,刘玉华,刘晓丹,等. AA病人血浆和骨髓瘦素及其受体表达与意义[J].青岛大学医学院学报,2014,50(6):493-495.
[6] WANG Y,YANT,MA L,et al. Effects of the total saponins from Dioscorea nipponica on immunoregulation in aplastic anemia mice[J]. Am J Chin Med,2015,43(2):289-303.
[7]刘宝山,马霖,苗润静,等.穿龙薯蓣皂苷调节再生障碍性贫血免疫共刺激分子的研究[J].中华中医药杂志,2015,30(12):4441-4443.
[8]姚军.淋巴细胞与再生障碍性贫血关系的实验研究[J].中华血液学杂志,1991,12(5):229-231.
[9] Dominici M,Le Blanc K,Mueller I,et al. Minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement[J]. Cytotherapy,2006,8(4):315-317.
[10]韦润红,陈艳丽,刘现辉.补髓生血汤对慢性再生障碍性贫血患者骨髓基质细胞相关细胞因子的影响[J].中国实验方剂学杂志,2016,23(6):192-197.
[11] YU H,ZHENG L,YIN L,et al. Protective effects of the total saponins from Dioscorea nipponica Makino against carbon tetrachloride-induced liver injury in mice through suppression of apoptosis and inflammation[J].Int Immunopharmacol,2014,19(2):233-244.
[12] LIU Y,LI H,FAN Y,et al. Antioxidant and antitumor activities of the extracts from chinese yam(Dioscorea opposite thunb.)flesh and peel and the effective compound[J]. J Food Sci,2016,81(6):1553-1564.
[13] WANG T,Choi R C,LI J,et al. Trillin,a steroidal saponin isolated from the rhizomes of Dioscorea nipponica, exerts protective effects against hyperlipidemia and oxidative stress[J]. J Ethnopharmacol,2011,139(1):214-220.
[14] ZHANG C,PENG J,WU S,et al. Dioscin promotes osteoblastic proliferation and differentiation via Lrp5 and ER pathway in mouse and human osteoblast-like cell lines[J]. J Biomed Sci,2014,21(1):30.
[15]刘宝山,纪超伦,杨向东,等.穿龙薯蓣皂苷抑制再生障碍性贫血p-mTOR/p-S6的机制研究[J].中国中医基础医学杂志,2014,20(12):1637-1641.
[16] CHEN Q,SHOU P,ZHENG C,et al. Fate decision of mesenchymal stem cells:adipocytes or osteoblasts[J].Cell Death Differ,2016,23(7):1128-1139.
[17] ZHANG J, ZHU X. Defectiveness of bone marrow mesenchymal stem cells in acquired aplastic anemia[J]. Chin J Contemp Pediatr,2015,1(17):100-106.
[18] LI J,YANG S,LU S,et al. Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia[J]. PLo S One,2012,7(11):e47764.
[19] Sugimura R, LI L. Shifting in balance between osteogenesis and adipogenesis substantially influences hematopoiesis[J]. J Mol Cell Biol,2010,2(2):61-62.
[20] FerréP. The biology of peroxisome proliferator-activated receptors:relationship with lipid metabolism and insulin sensitivity[J]. Diabetes,2004,53(1):43-50.
[21] Bionaz M,Monaco E,Wheeler M B. Transcription adaptation during in vitro adipogenesis and osteogenesis of porcine mesenchymal stem cells:dynamics of pathways,biological processes, up-stream regulators,and gene networks[J]. PLo S One, 2015,10(9):e137644.
[22]徐海玲. PPARγ在免疫介导的再生障碍性贫血小鼠骨髓中的表达及意义[D].太原:山西医科大学,2014.
[23] Madsen M S, Siersbaek R, Boergesen M, et al.Peroxisome proliferator-activated receptor and C/EBP synergistically activate key metabolic adipocyte genes by assisted loading[J]. Mol Cell Biol 2014,34(6):939-954.
[24]涂珺,罗新新,李冰涛,等.小檗碱对脂肪胰岛素抵抗细胞PPARγ及脂肪分泌因子表达的影响[J].中国中药杂志,2016,41(11):1983-1989.
[25] Martella E, Bellotti C, Dozza B, et al. Secreted adiponectin as a marker to evaluate in vitro the adipogenic differentiation of human mesenchymal stromal cells[J]. Cytotherapy,2014,16(11):1476-1485.
[26] Tripathy N K,Singh S P,Nityanand S. Enhanced adipogenicity of bone marrow mesenchymal stem cells in aplastic anemia[J]. Stem Cells International,2014,2014:1-6.
[27] Fernández-Riejos P,Najib S,Santos-Alvarez J,et al.Role of leptin in the activation of immune cells[J].Mediators Inflamm,2010,2010:568348.
[28] Reis B S,Lee K,Fanok M H,et al. Leptin receptor signaling in T cells is required for Th17 differentiation[J]. J Immunol,2015,194(11):5253-5260.
[29] Laharrague P,Larrouy D,Fontanilles A M,et al. High expression of Leptin by human bone marrow adipocytes in primary culture[J]. FASEB J,1998,12(9):747-753.
[30] YUE R,ZHOU B O,Shimada I S,et al. Leptin receptor promotes adipogenesis and reduces osteogenesis by regulating mesenchymal stromal cells in adult bone marrow[J]. Cell Stem Cell,2016,18(6):782-796.
[2] LI J, YANG S, LU S, et al. Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia[J]. PLo S One,2012,7(11):e47764.
[3] Clarke S L, Robinson C E, Gimble J M. CAAT/enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor gamma2 promoter[J]. Biochem Biophys Res Commun,1997,240(1):99-103.
[4]王晓燕,余卫,罗英.再生障碍性贫血患者血浆脂联素水平与骨髓造血功能相关性研究[J].新医学,2012,43(3):181-183.
[5]刘国文,刘玉华,刘晓丹,等. AA病人血浆和骨髓瘦素及其受体表达与意义[J].青岛大学医学院学报,2014,50(6):493-495.
[6] WANG Y,YANT,MA L,et al. Effects of the total saponins from Dioscorea nipponica on immunoregulation in aplastic anemia mice[J]. Am J Chin Med,2015,43(2):289-303.
[7]刘宝山,马霖,苗润静,等.穿龙薯蓣皂苷调节再生障碍性贫血免疫共刺激分子的研究[J].中华中医药杂志,2015,30(12):4441-4443.
[8]姚军.淋巴细胞与再生障碍性贫血关系的实验研究[J].中华血液学杂志,1991,12(5):229-231.
[9] Dominici M,Le Blanc K,Mueller I,et al. Minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement[J]. Cytotherapy,2006,8(4):315-317.
[10]韦润红,陈艳丽,刘现辉.补髓生血汤对慢性再生障碍性贫血患者骨髓基质细胞相关细胞因子的影响[J].中国实验方剂学杂志,2016,23(6):192-197.
[11] YU H,ZHENG L,YIN L,et al. Protective effects of the total saponins from Dioscorea nipponica Makino against carbon tetrachloride-induced liver injury in mice through suppression of apoptosis and inflammation[J].Int Immunopharmacol,2014,19(2):233-244.
[12] LIU Y,LI H,FAN Y,et al. Antioxidant and antitumor activities of the extracts from chinese yam(Dioscorea opposite thunb.)flesh and peel and the effective compound[J]. J Food Sci,2016,81(6):1553-1564.
[13] WANG T,Choi R C,LI J,et al. Trillin,a steroidal saponin isolated from the rhizomes of Dioscorea nipponica, exerts protective effects against hyperlipidemia and oxidative stress[J]. J Ethnopharmacol,2011,139(1):214-220.
[14] ZHANG C,PENG J,WU S,et al. Dioscin promotes osteoblastic proliferation and differentiation via Lrp5 and ER pathway in mouse and human osteoblast-like cell lines[J]. J Biomed Sci,2014,21(1):30.
[15]刘宝山,纪超伦,杨向东,等.穿龙薯蓣皂苷抑制再生障碍性贫血p-mTOR/p-S6的机制研究[J].中国中医基础医学杂志,2014,20(12):1637-1641.
[16] CHEN Q,SHOU P,ZHENG C,et al. Fate decision of mesenchymal stem cells:adipocytes or osteoblasts[J].Cell Death Differ,2016,23(7):1128-1139.
[17] ZHANG J, ZHU X. Defectiveness of bone marrow mesenchymal stem cells in acquired aplastic anemia[J]. Chin J Contemp Pediatr,2015,1(17):100-106.
[18] LI J,YANG S,LU S,et al. Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia[J]. PLo S One,2012,7(11):e47764.
[19] Sugimura R, LI L. Shifting in balance between osteogenesis and adipogenesis substantially influences hematopoiesis[J]. J Mol Cell Biol,2010,2(2):61-62.
[20] FerréP. The biology of peroxisome proliferator-activated receptors:relationship with lipid metabolism and insulin sensitivity[J]. Diabetes,2004,53(1):43-50.
[21] Bionaz M,Monaco E,Wheeler M B. Transcription adaptation during in vitro adipogenesis and osteogenesis of porcine mesenchymal stem cells:dynamics of pathways,biological processes, up-stream regulators,and gene networks[J]. PLo S One, 2015,10(9):e137644.
[22]徐海玲. PPARγ在免疫介导的再生障碍性贫血小鼠骨髓中的表达及意义[D].太原:山西医科大学,2014.
[23] Madsen M S, Siersbaek R, Boergesen M, et al.Peroxisome proliferator-activated receptor and C/EBP synergistically activate key metabolic adipocyte genes by assisted loading[J]. Mol Cell Biol 2014,34(6):939-954.
[24]涂珺,罗新新,李冰涛,等.小檗碱对脂肪胰岛素抵抗细胞PPARγ及脂肪分泌因子表达的影响[J].中国中药杂志,2016,41(11):1983-1989.
[25] Martella E, Bellotti C, Dozza B, et al. Secreted adiponectin as a marker to evaluate in vitro the adipogenic differentiation of human mesenchymal stromal cells[J]. Cytotherapy,2014,16(11):1476-1485.
[26] Tripathy N K,Singh S P,Nityanand S. Enhanced adipogenicity of bone marrow mesenchymal stem cells in aplastic anemia[J]. Stem Cells International,2014,2014:1-6.
[27] Fernández-Riejos P,Najib S,Santos-Alvarez J,et al.Role of leptin in the activation of immune cells[J].Mediators Inflamm,2010,2010:568348.
[28] Reis B S,Lee K,Fanok M H,et al. Leptin receptor signaling in T cells is required for Th17 differentiation[J]. J Immunol,2015,194(11):5253-5260.
[29] Laharrague P,Larrouy D,Fontanilles A M,et al. High expression of Leptin by human bone marrow adipocytes in primary culture[J]. FASEB J,1998,12(9):747-753.
[30] YUE R,ZHOU B O,Shimada I S,et al. Leptin receptor promotes adipogenesis and reduces osteogenesis by regulating mesenchymal stromal cells in adult bone marrow[J]. Cell Stem Cell,2016,18(6):782-796.
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