自发性糖尿病小鼠肾小球细胞Nampt和骨形态发生蛋白7的表达及烟酰胺单核苷酸在高糖条件下对大鼠肾小球系膜HBZY-1细胞纤维化的影响
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摘要
目的探讨糖尿病肾小球细胞烟酰胺磷酸核糖转移酶(Nampt)与骨形态发生蛋白7(BMP7)表达的关系及烟酰胺单核苷酸(NMN)缓解糖尿病肾小球细胞炎症纤维化的作用机制。方法 (1)动物实验:C57/BL6自发性糖尿病小鼠和C57/BL6野生型小鼠,均采取普通饲料喂养,当自发性糖尿病小鼠血糖>(34.2±1.9)mmol·L~(-1)并出现明显肾组织损伤时,取肾组织进行病理切片,免疫共聚焦法检测肾小球细胞Nampt、核转录因子κB p65(NF-κB p65)、沉默调节蛋白1(SIRT1)和BMP7的表达。(2)细胞实验:葡萄糖200 mmol·L~(-1)培养大鼠肾小球系膜HBZY~(-1)细胞,在不同时间(24,48和72 h)以及不同浓度NMN(50,100和200μmol·L~(-1))处理24 h时后,免疫印迹法检测Nampt和BMP7的表达;葡萄糖200 mmol·L~(-1)处理HBZY~(-1)细胞96 h,免疫荧光法检测NF-κB p65和α-平滑肌肌动蛋白(α-SMA)的表达;应用NMN 100μmol·L~(-1)和Nampt特异抑制剂FK866 10μmol·L~(-1)作用HBZY~(-1)细胞24 h后,免疫印迹法检测HBZY~(-1)细胞Nampt,BMP7和NF-κB p65表达。结果 (1)动物实验:自发性糖尿病小鼠肾小球明显萎缩,肾小球细胞Nampt和NF-κB p65的荧光强度比野生型小鼠明显升高(P<0.05),而BMP7和SIRT1的荧光强度显著降低(P<0.01)。(2)细胞实验:Western蛋白印迹检测显示,葡萄糖200 mmol·L~(-1)培养48和72 h,HBZY~(-1)细胞Nampt表达增加(P<0.01),BMP7表达下降(P<0.01,P<0.05)。葡萄糖200 mmol·L~(-1)条件下加NMN 50,100和200μmol·L~(-1)作用24 h,各组BMP7表达均增加(P<0.01);免疫荧光结果显示,与细胞对照组比较,葡萄糖200 mmol·L~(-1)处理HBZY~(-1)细胞,NF-κB p65和α-SMA的荧光强度升高(P<0.01);NMN干预后,与葡萄糖200 mmol·L~(-1)处理组比,Nampt和NF-κB p65表达降低(P<0.01),BMP7表达增加(P<0.01);加FK866后,Nampt表达降低(P<0.01),NF-κB p65表达下降,BMP7表达虽然有上升趋势,但其表达增高没有NMN组明显。结论严重糖尿病状态下,通过抑制内源性Nampt过表达能够上调BMP7,从而缓解肾小球细胞炎症纤维化作用,NMN可能通过干预Nampt影响细胞BMP7表达。
OBJECTIVE To investigate the mechanism of nicotinamide mononucleotide(NMN) on inflammation and fibrosis between endogenous nicotinamide phosphoribosyltransferase(NAMPT) and bone morphogenetic protein 7(BMP-7) in diabetic glomerular cells. METHODS(1) In vivo, spontaneous diabetic C57/BL6 mice and wild C57/BL6 mice were divided into two groups. When blood glucose was above(34.2±1.9) mmol·L~(-1), renal histology of diabetic mice became obvious. The protein expressions of Nampt and nuclear transcription factors-kappa B p65(NF-κB p65), silent mating type information regulation 2 homolog 1(SIRT1) and BMP7 were analyzed by lengths of immunofluorescence.(2) In vitro, rats′glomerular cells HBZY~(-1) were incubated with glucose 200 mmol·L~(-1)for different lengths of time(0,24,48 and 72 h) and at different concentrations of NMN(0,50,100 and 200 μmol·L~(-1)). The protein levels of Nampt and BMP7 were detected by Western blotting and the protein expressions of NF-κB p65 and α-SMA were measured by immunofluorescence assay. The protein levels of Nampt, BMP7 and NF-κB p65 were detected by Western blotting after HBZY~(-1) cells were treated with NMN 100 μmol·L~(-1)and FK86610 μmol·L~(-1)for 24 h. RESULTS(1) In vivo, the glomeruli of diabetic C57/BL6 mice showed obvious atrophy. Fluorescence intensity of Nampt was increased(P<0.05), but that of BMP7 and SIRT1 in renal glomeruli cells was decreased compared with the wild type(P<0.01).(2) In vitro, HBZY~(-1) cells were cultured in glucose 200 mmol·L~(-1)for 48 and 72 h. The protein expression of NAMPT was increased, but that of BMP7 was decreased(P<0.05, P<0.01). Expressions of NF-κB p65 and α-SMA were increased(P<0.01) by immunofluorescence. The expression of BMP7 was increased after treatment with glucose200 mmol · L~(-1), followed by NMN 50, 100 and 200 μmol · L~(-1)for 24 h(P<0.01). The expressions of NAMPT and NF-κB p65 were decreased(P<0.01). The expressions of Nampt and NF-κB p65 in glucose 5.6 mmol·L~(-1)+FK866 and glucose 5.6 mmol·L~(-1)+ NMN groups were increased(P<0.01), but the expression of BMP7 did not change. CONCLUSION Upregulation of endogenous Nampt obviously intervenes in BMP7 expression in the process of glomerular inflammatory fibrosis in severe diabetes.NMN can affect the protein expression of BMP7 via a special Nampt signaling pathway.
OBJECTIVE To investigate the mechanism of nicotinamide mononucleotide(NMN) on inflammation and fibrosis between endogenous nicotinamide phosphoribosyltransferase(NAMPT) and bone morphogenetic protein 7(BMP-7) in diabetic glomerular cells. METHODS(1) In vivo, spontaneous diabetic C57/BL6 mice and wild C57/BL6 mice were divided into two groups. When blood glucose was above(34.2±1.9) mmol·L~(-1), renal histology of diabetic mice became obvious. The protein expressions of Nampt and nuclear transcription factors-kappa B p65(NF-κB p65), silent mating type information regulation 2 homolog 1(SIRT1) and BMP7 were analyzed by lengths of immunofluorescence.(2) In vitro, rats′glomerular cells HBZY~(-1) were incubated with glucose 200 mmol·L~(-1)for different lengths of time(0,24,48 and 72 h) and at different concentrations of NMN(0,50,100 and 200 μmol·L~(-1)). The protein levels of Nampt and BMP7 were detected by Western blotting and the protein expressions of NF-κB p65 and α-SMA were measured by immunofluorescence assay. The protein levels of Nampt, BMP7 and NF-κB p65 were detected by Western blotting after HBZY~(-1) cells were treated with NMN 100 μmol·L~(-1)and FK86610 μmol·L~(-1)for 24 h. RESULTS(1) In vivo, the glomeruli of diabetic C57/BL6 mice showed obvious atrophy. Fluorescence intensity of Nampt was increased(P<0.05), but that of BMP7 and SIRT1 in renal glomeruli cells was decreased compared with the wild type(P<0.01).(2) In vitro, HBZY~(-1) cells were cultured in glucose 200 mmol·L~(-1)for 48 and 72 h. The protein expression of NAMPT was increased, but that of BMP7 was decreased(P<0.05, P<0.01). Expressions of NF-κB p65 and α-SMA were increased(P<0.01) by immunofluorescence. The expression of BMP7 was increased after treatment with glucose200 mmol · L~(-1), followed by NMN 50, 100 and 200 μmol · L~(-1)for 24 h(P<0.01). The expressions of NAMPT and NF-κB p65 were decreased(P<0.01). The expressions of Nampt and NF-κB p65 in glucose 5.6 mmol·L~(-1)+FK866 and glucose 5.6 mmol·L~(-1)+ NMN groups were increased(P<0.01), but the expression of BMP7 did not change. CONCLUSION Upregulation of endogenous Nampt obviously intervenes in BMP7 expression in the process of glomerular inflammatory fibrosis in severe diabetes.NMN can affect the protein expression of BMP7 via a special Nampt signaling pathway.
引文
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[10]Li RX,Yiu WH,Tang SC.Role of bone morphogenetiprotein-7 in renal fibrosis[J].Front Physiol,2015,6:114.
[11]Lee SY,Kim SI,Choi ME.Therapeutic targets for treating fibrotic kidney diseases[J].Transl Res,2015,165(4):512-530.
[12]Yeh CH,Chang CK,Cheng MF,Lin HJ,Cheng JT.The antioxidative effect of bone morphogenetic protein-7 against high glucose-induced oxidative stress in mesangial cells[J].Biochem Biophys Res Commun,2009,382(2):292-297.
[13]Abreu JG,Ketpura NI,Reversade B,De Robertis EM.Connective-tissue growth factor(CTGF)modulates cell signalling by BMP and TGF-beta[J].Nat Cell Biol,2002,4(8):599-604.
[14]GalíM,Van Gool F,Rongvaux A,Andris F,Leo O.The nicotinamide phosphoribosyltransferase:a molecular link between metabolism,inflammation,and cancer[J].Cancer Res,2010,70(1):8-11.
[15]Hasmann M,Schemainda I.F K866,a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase,represents a novel mechanism for induction of tumor cell apoptosis[J].Cancer Res,2003,63(21):7436-7442.
[2]Kitada M,Kume S,Koya D.Role of sirtuins in kidney disease[J].Curr Opin Nephrol Hypertens,2014,23(1):75-79.
[3]Salminen A,Huuskonen J,Ojala J,Kauppinen A,Kaarniranta K,Suuronen T.Activation of innate immunity system during aging:NF-k B signaling is the molecular culprit of inflamm-aging[J].Ageing Res Rev,2008,7(2):83-105.
[4]Tsujimura T,Idei M,Yoshikawa M,Takase O,Hishikawa K.Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases[J].World J Stem Cells,2016,8(9):288-296.
[5]Yuan A,Lee Y,Choi U,Moeckel G,Karihaloo A.Chemokine receptor Cxcr4 contributes to kidney fibrosis via multiple effectors[J].Am J Physiol Renal Physiol,2015,308(5):F459-F472.
[6]Wang SL,Yang CQ,Qi XL,Yuan M,Chang YZ,Yang L,et al.Inhibitory effect of bone morphogenetic protein-7 on hepatic fibrosis in rats[J].Int J Clin Exp Pathol,2013,6(5):897-903.
[7]Anil Kumar P,Welsh GI,Saleem MA,Menon RK.Molecular and celular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus[J].Front Endocrinol(Lausanne),2014,5:151.
[8]Coward R,Fornoni A.Insulin signaling:implications for podocyte biology in diabetic kidney disease[J].Curr Opin Nephrol Hypertens,2015,24(1):104-110.
[9]St?y J,Edghill EL,Flanagan SE,Ye H,Paz VP,Pluzhnikov A,et al.Insulin gene mutations as a cause of permanent neonatal diabetes[J].Proc Natl Acad Sci USA,2007,104(38):15040-15044.
[10]Li RX,Yiu WH,Tang SC.Role of bone morphogenetiprotein-7 in renal fibrosis[J].Front Physiol,2015,6:114.
[11]Lee SY,Kim SI,Choi ME.Therapeutic targets for treating fibrotic kidney diseases[J].Transl Res,2015,165(4):512-530.
[12]Yeh CH,Chang CK,Cheng MF,Lin HJ,Cheng JT.The antioxidative effect of bone morphogenetic protein-7 against high glucose-induced oxidative stress in mesangial cells[J].Biochem Biophys Res Commun,2009,382(2):292-297.
[13]Abreu JG,Ketpura NI,Reversade B,De Robertis EM.Connective-tissue growth factor(CTGF)modulates cell signalling by BMP and TGF-beta[J].Nat Cell Biol,2002,4(8):599-604.
[14]GalíM,Van Gool F,Rongvaux A,Andris F,Leo O.The nicotinamide phosphoribosyltransferase:a molecular link between metabolism,inflammation,and cancer[J].Cancer Res,2010,70(1):8-11.
[15]Hasmann M,Schemainda I.F K866,a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase,represents a novel mechanism for induction of tumor cell apoptosis[J].Cancer Res,2003,63(21):7436-7442.