高糖诱导内皮-脂肪细胞转分化的机制探讨
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摘要
目的探讨高糖刺激内皮细胞(endothelial cells,ECs)是否可通过内皮-间充质细胞转分化(endothelial-to-mesenchymal transition,EndMT),继而转分化为脂肪细胞(即内皮-脂肪细胞转分化),从而介导糖尿病(diabetes mellitus,DM)骨质疏松形成。方法 (1)体内实验:建立DM大鼠模型,随机分为正常对照(control,CTL)组和DM组。双能X线和micro-CT检测骨代谢特征。骨组织HE染色检测骨髓脂肪组织含量。骨组织荧光和Western印迹检测骨髓EndMT相关标记物(内皮标记物CD31和间充质标记物FSP1);(2)体外实验:培养原代人主动脉内皮细胞,分为对照(normal glucose,NG)组和高糖(high glucose,HG)组。免疫荧光和Western印迹检测高糖对ECs EndMT相关标记物变化。继而使用脂肪培养基对高糖刺激后ECs进行1周诱导分化,Western印迹检测脂肪相关标记物(PPAR-γ)的水平,油红O染色检测脂滴形成情况。结果 DM组大鼠较CTL组体重明显下降,血糖、血肌酐、尿素氮、24 h尿蛋白显著升高(P<0.05);骨密度扫描和micro-CT结果示,与CTL组相比,DM大鼠股骨和腰椎骨密度显著下降,松质骨变少(骨体积和骨小梁数目减少,骨小梁离散度增加)(P<0.05)。皮质骨无明显改变(P>0.05);骨组织Western印迹结果示DM大鼠CD31表达减少,FSP1表达增加(P<0.05);免疫荧光结果示DM组骨髓CD31和FSP1表达重叠;体外高糖干预ECs,Western印迹和免疫荧光结果显示HG组细胞CD31表达减少,FSP1表达增加(P<0.05)。进一步诱导脂肪细胞分化一周,HG组PPAR-γ表达水平显著升高(P<0.05),油红O染色阳性。结论高糖诱导ECs发生内皮-脂肪细胞转分化,从而参与了DM骨质疏松的发生。
Objective To investigate whether high-dose of glucose induces endothelial-to-adipocyte transition via endothelial-to-mesenchymal transition(EndMT) in endothelial cells(ECs), and mediates osteoporosis in diabetes mellitus(DM). Methods(1) In vivo experiment: a DM rat model was established. Rats were randomly divided into control(CTL) group and DM group. Dual energy X-ray Absorptiometry and micro-CT were performed to detect the skeletal changes. HE staining of the bone was used to detect the bone marrow fat. Bone tissue immunofluorescence and Western blotting were used to determine the EndMT-related markers(ECs marker CD31 and mesenchymal marker FSP1).(2) In vitro experiment: primary human aortic ECs were cultured. The cells were divided into normal glucose(NG) group and high glucose(HG) group. Immunofluorescence and Western blotting were used to determine the changes of EndMT-related markers. And then these cells were transformed into adipogenic culture medium for 1 week. Western blotting and oil red O staining were performed to detect the adipocytes. Results Compared to those in the CTL group, the body weight decreased in DM group, while blood glucose, serum creatinine, urea nitrogen, and 24-hour proteinuria increased(all P<0.05). Bone mineral density of the femur and lumbar vertebrae decreased in DM rats compared to that in CTL group. Bone volume/tissue volume and the number of trabecular number decreased, and the trabecular separation increased in DM group(P<0.05). There was no significant change in cortical bone(P>0.05). The expression of CD31 decreased and the expression of FSP1 increased in DM rats compared to those in CTL group(P<0.05). Bone tissue fluorescence showed overlapping expression of CD31 and FSP1 in bone marrow of DM group in vitro. ECs were stimulated by high-dose of glucose. Western blotting and fluorescence result showed a decrease in CD31 and an increase in FSP1(P<0.05). After further induction of adipocyte differentiation for one week, the expression level of PPAR-γ increased significantly(P<0.05) and oil red O staining was positive in HG group. Conclusion High-dose of glucose induces endothelial-to-adipocyte transition in ECs, and is involved in DM-related osteoporosis.
Objective To investigate whether high-dose of glucose induces endothelial-to-adipocyte transition via endothelial-to-mesenchymal transition(EndMT) in endothelial cells(ECs), and mediates osteoporosis in diabetes mellitus(DM). Methods(1) In vivo experiment: a DM rat model was established. Rats were randomly divided into control(CTL) group and DM group. Dual energy X-ray Absorptiometry and micro-CT were performed to detect the skeletal changes. HE staining of the bone was used to detect the bone marrow fat. Bone tissue immunofluorescence and Western blotting were used to determine the EndMT-related markers(ECs marker CD31 and mesenchymal marker FSP1).(2) In vitro experiment: primary human aortic ECs were cultured. The cells were divided into normal glucose(NG) group and high glucose(HG) group. Immunofluorescence and Western blotting were used to determine the changes of EndMT-related markers. And then these cells were transformed into adipogenic culture medium for 1 week. Western blotting and oil red O staining were performed to detect the adipocytes. Results Compared to those in the CTL group, the body weight decreased in DM group, while blood glucose, serum creatinine, urea nitrogen, and 24-hour proteinuria increased(all P<0.05). Bone mineral density of the femur and lumbar vertebrae decreased in DM rats compared to that in CTL group. Bone volume/tissue volume and the number of trabecular number decreased, and the trabecular separation increased in DM group(P<0.05). There was no significant change in cortical bone(P>0.05). The expression of CD31 decreased and the expression of FSP1 increased in DM rats compared to those in CTL group(P<0.05). Bone tissue fluorescence showed overlapping expression of CD31 and FSP1 in bone marrow of DM group in vitro. ECs were stimulated by high-dose of glucose. Western blotting and fluorescence result showed a decrease in CD31 and an increase in FSP1(P<0.05). After further induction of adipocyte differentiation for one week, the expression level of PPAR-γ increased significantly(P<0.05) and oil red O staining was positive in HG group. Conclusion High-dose of glucose induces endothelial-to-adipocyte transition in ECs, and is involved in DM-related osteoporosis.
引文
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[15] 颜春鲁,李盛华,郭爱军,等.藤黄健骨胶囊对去卵巢骨质疏松大鼠骨密度和骨代谢的影响[J].中国骨质疏松杂志,2015,21(8):914-917.
[16] Hardouin P,Rharass T,Lucas S.Bone marrow adipose tissue:To be or not to be a typical adipose tissue?[J].Front Endocrinol,2016,7:85.
[17] Horwitz EM.Building bone from blood vessels[J].Nat Med,2010,16(12):1373-1374.
[18] Lin SC,Lee YC,Yu G,et al.Endothelial-to-osteoblast conversion generates osteoblastic metastasis of prostate cancer[J].Dev Cell,2017,41(5):467-480.
[2] Kurra S,Fink DA,Siris ES.Osteoporosis-associated fracture and diabetes[J].Endocrinol Metab Clin North Am,2014,43(1):233-243.
[3] Vestergaard P.Discrepancies in bone mineral density and fracture risk in patients with type 1 and type 2 diabetes-a meta-analysis[J].Osteoporos Int,2007,18(4):427-444.
[4] Cortet B,Lucas S,Legroux-Gerot I,et al.Bone disorders associated with diabetes mellitus and its treatments[J].Joint Bone Spine,2018 Aug 8.doi:10.1016/j.jbspin.2018.08.002.
[5] Kim TY,Schafer AL.Diabetes and bone marrow adiposity[J].Curr Osteoporos Rep,2016,14(6):337-344.
[6] Napoli N,Chandran M,Pierroz DD,et al.Mechanisms of diabetes mellitus-induced bone fragility[J].Nat Rev Endocrinol,2017,13(4):208-219.
[7] 倪利华,刘必成,汤日宁.慢性肾脏病骨密度下降研究进展[J].中华医学杂志,2018,98(4):317-320.
[8] Medici D.Endothelial-mesenchymal transition in regenerative medicine[J].Stem Cells Int,2016,2016:6962801.
[9] Zhu DD,Tang RN,Lv LL,et al.Interleukin-1beta mediates high glucose induced phenotypic transition in human aortic endothelial cells[J].Cardiovasc Diabetol,2016,15:42.
[10] 汤日宁,朱冬冬,韩雨晨,等.厄贝沙坦对糖尿病大鼠心肌内皮细胞转分化的作用[J].中华肾脏病杂志,2015,31(5):351-358.
[11] 汤日宁,伍敏,刘宏,等.高糖诱导人主动脉内皮细胞-软骨转分化[J].中华肾脏病杂志,2013,29(3):183-188.
[12] Lecka-Czernik B.Diabetes,bone and glucose-lowering agents:basic biology[J].Diabetologia,2017,60(7):1163-1169.
[13] Kawai M,de Paula FJ,Rosen CJ.New insights into osteoporosis:the bone-fat connection[J].J Intern Med,2012,272(4):317-329.
[14] Lecka-Czernik B,Baroi S,Stechschulte LA,et al.Marrow fat-a new target to treat bone diseases[J]?.Curr Osteoporos Rep,2018,16(2):123-129.
[15] 颜春鲁,李盛华,郭爱军,等.藤黄健骨胶囊对去卵巢骨质疏松大鼠骨密度和骨代谢的影响[J].中国骨质疏松杂志,2015,21(8):914-917.
[16] Hardouin P,Rharass T,Lucas S.Bone marrow adipose tissue:To be or not to be a typical adipose tissue?[J].Front Endocrinol,2016,7:85.
[17] Horwitz EM.Building bone from blood vessels[J].Nat Med,2010,16(12):1373-1374.
[18] Lin SC,Lee YC,Yu G,et al.Endothelial-to-osteoblast conversion generates osteoblastic metastasis of prostate cancer[J].Dev Cell,2017,41(5):467-480.