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

发布时间：2018-03-26 17:22

本文选题：糖尿病性肾病　切入点：肾小球纤维化　出处：《中国药理学与毒理学杂志》2017年06期

【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(P0.05),而BMP7和SIRT1的荧光强度显著降低(P0.01)。(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(P0.01),BMP7表达下降(P0.01,P0.05)。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(P0.01);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(P0.01);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(P0.01),BMP7表达增加(P0.01);加FK866后,Nampt表达降低(P0.01),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。
[Abstract]:Objective to investigate the relationship between Namptase and bone morphogenetic protein 7 (BMP7) expression in diabetic glomerular cells and the mechanism of nicotinamide mononucleotide (NMN) in relieving glomerular inflammatory fibrosis in diabetic rats. Animal experiment: C57 / BL6 spontaneous diabetes mice and C57/BL6 wild-type mice, All of them were fed with common diet. When the blood glucose of spontaneous diabetic mice was 34.2 卤1.9)mmol Ln-1) and obvious renal tissue injury occurred, the renal tissues were taken for pathological section. Immunoconfocal assay was used to detect the expression of Nampt, NF- 魏 B p65, NF- 魏 B p65, silencing regulatory protein 1 (SIRT1) and BMP7 in rat glomerular Mesangial HBZYT-1) cells: glucose 200 mmol / L ~ (-1)). The expression of Nampt and BMP7 was detected by immunoblotting after 24 h treatment with different concentrations of NMN(50100 and 200 渭 mol L ~ (-1), and 96 h with glucose (200 mmol / L ~ (-1)). The expression of NF- 魏 B p65 and 伪 -smooth muscle actin (伪 -SMA-1) was detected by immunofluorescence assay at 96 h after treatment with glucose (200 mmol / L ~ (-1)), and the expression of NF- 魏 B p65 and 伪 -smooth muscle actin (伪 -SMA-1) was detected by immunofluorescence assay. NMN 100 渭 mol L ~ (1) and FK866 10 渭 mol L ~ (-1), a specific inhibitor of Nampt, were used to detect the expression of Nampttsil-BMP7 and NF- 魏 B p65 in HBZY _ (1) cells 24 h after treatment with Nampt specific inhibitor FK866 10 渭 mol L ~ (-1). Results: glomerular atrophy was observed in spontaneously diabetic mice. The fluorescence intensity of Nampt and NF- 魏 B p65 in glomerular cells was significantly higher than that in wild-type mice, while the fluorescence intensity of BMP7 and SIRT1 decreased significantly. After cultured with glucose 200 mmol / L for 48 and 72 h, the expression of Nampt was increased, and the expression of BMP7 was decreased. The expression of NMN 50100 and 200 渭 mol / L ~ (-1) was increased 24 h after the addition of NMN 50100 and 200 渭 mol / L ~ (-1). The results of immunofluorescence showed that, compared with the control group, the expression of BMP7 in each group was significantly higher than that in the control group (P _ (0.01) and P _ (0.01)), and the results of immunofluorescence showed that, compared with the control group, the expression of BMP7 in each group was significantly higher than that in the control group (P _ (0.01) and ~ (-1)). The fluorescence intensity of NF- 魏 B p65 and 伪 -SMA was increased after the treatment of glucose 200 mmol / L ~ (-1)). Compared with glucose 200 mmol L ~ (-1) group, the expression of nmpt and NF- 魏 B p65 decreased, and the expression of P0.01BMP7 increased; after adding FK866, the expression of P0.01- 魏 B p65 decreased, and the expression of P0.01- 魏 B p65 decreased, although the expression of P0.01- 魏 B p65 decreased, although the expression of P0.01- 魏 B p65 decreased, although the expression of P0.01- 魏 B p65 decreased, the expression of P0.01- 魏 B p65 decreased, and the expression of P0.01- 魏 B p65 decreased after treatment with Glucose-200 mmol / L ~ (-1). Conclusion under the condition of severe diabetes mellitus, inhibiting the overexpression of endogenous Nampt can up-regulate the expression of BMP7 and alleviate the inflammatory fibrosis of glomerular cells. NMN may influence the expression of BMP7 by interfering with Nampt.
【作者单位】：桂林医学院公共卫生学院;桂林医学院生物技术学院;桂林医学院药学院;
【基金】：国家自然科学基金(31060161);国家自然科学基金(81460164) 广西壮族自治区自然科学基金(2015GXNSF)~~
【分类号】：R587.2;R692.9

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