p53激动剂nutlin-3对高糖培养的肾系膜细胞增殖的影响
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摘要
目的:观察p53激动剂nutlin-3对高糖培养的肾系膜细胞增殖、凋亡和细胞外基质表达的影响。方法:制备糖尿病肾病(DN)小鼠动物模型,对小鼠肾组织行PAS染色,观察病理形态学变化,免疫组化染色检测肾组织中p53的表达。对肾小球系膜细胞株SV40进行体外培养,根据实验需要将细胞分为甘露醇(Motl)组、正常糖浓度对照(NG)组、高糖(HG)组,高糖+nutlin-3(HG+Nut)组及nutlin-3对照(Nut)组,CCK-8法和台盼蓝染色活细胞计数检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Western blot法检测Ⅳ型胶原蛋白(Col-Ⅳ)、p53、p-p53、Bcl-2和Bax的蛋白水平。结果:DN小鼠肾组织中系膜细胞增生,细胞外基质增多,p53水平明显升高。高糖环境可以增强系膜细胞的活力,40μmol/L的nutlin-3对高糖培养下的系膜细胞有抑制作用。与HG组比较,HG+Nut组的p53、Bax和p-p53的蛋白水平显著升高(P<0.05),Bcl-2和Col-IV的蛋白水平降低(P<0.05),Bax/Bcl-2的比值大于1。与HG组比较,nutlin-3可促进高糖培养下的系膜细胞凋亡,凋亡率明显增高(P<0.05)。结论:高糖环境增强肾系膜细胞的增殖能力。p53激动剂可抑制高糖培养的肾系膜细胞的活力和Col-IV的表达,促进其凋亡。
AIM: To observe the effect of p53 agonist nutlin-3 on the proliferation, apoptosis and expression of extracellular matrix in the glomerular mesangial cells(MCs) cultured in high glucose, and to explore its possible mechanism. METHODS: After successful modeling of diabetic nephropathy(DN), PAS staining was performed on the kidney tissues to observe pathological changes. In addition, the p53 expression in the kidney tissue was detected by immunohistochemical staining. The mesangial cell SV40 was cultured in vitro and divided into mannitol(Motl) group, normal glucose(NG) group, high glucose(HG) group, high glucose plus nutlin-3(HG+Nut) group and nutlin-3 control(Nut) group. The cell viability was detected by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The apoptosis was analyzed by flow cytometry. The protein levels of collagen type IV(Col-IV), p53, p-p53, Bcl-2 and Bax were determined by Western blot. RESULTS: The significant increases in the proliferation of mesangial cells and the levels of p53 in renal tissue of diabetic nephropathy mice were observed. The results of CCK-8 assay showed that high glucose promoted the viability of mesangial cells, and nutlin-3 at 40 μmol/L inhibited the viability of mesangial cells in high glucose environment. Western blot analysis showed that the protein levels of p53, Bax and p-p53 were significantly increased(P<0.05), and the protein levels of Bcl-2 and Col-IV was decreased in HG+Nut group compared with HG group(P<0.05). Furthermore, the ratio of Bax/Bcl-2 was greater than 1. The results of flow cytometry showed that compared with HG group, nutlin-3 promoted the apoptosis of mesangial cells in high glucose environment, the apoptotic rate was significantly increased(P<0.05).CONCLUSION: High glucose promotes the proliferation of mesangial cells. p53 agonist inhibits the viability and promotes apoptosis of mesangial cells under high glucose.
AIM: To observe the effect of p53 agonist nutlin-3 on the proliferation, apoptosis and expression of extracellular matrix in the glomerular mesangial cells(MCs) cultured in high glucose, and to explore its possible mechanism. METHODS: After successful modeling of diabetic nephropathy(DN), PAS staining was performed on the kidney tissues to observe pathological changes. In addition, the p53 expression in the kidney tissue was detected by immunohistochemical staining. The mesangial cell SV40 was cultured in vitro and divided into mannitol(Motl) group, normal glucose(NG) group, high glucose(HG) group, high glucose plus nutlin-3(HG+Nut) group and nutlin-3 control(Nut) group. The cell viability was detected by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The apoptosis was analyzed by flow cytometry. The protein levels of collagen type IV(Col-IV), p53, p-p53, Bcl-2 and Bax were determined by Western blot. RESULTS: The significant increases in the proliferation of mesangial cells and the levels of p53 in renal tissue of diabetic nephropathy mice were observed. The results of CCK-8 assay showed that high glucose promoted the viability of mesangial cells, and nutlin-3 at 40 μmol/L inhibited the viability of mesangial cells in high glucose environment. Western blot analysis showed that the protein levels of p53, Bax and p-p53 were significantly increased(P<0.05), and the protein levels of Bcl-2 and Col-IV was decreased in HG+Nut group compared with HG group(P<0.05). Furthermore, the ratio of Bax/Bcl-2 was greater than 1. The results of flow cytometry showed that compared with HG group, nutlin-3 promoted the apoptosis of mesangial cells in high glucose environment, the apoptotic rate was significantly increased(P<0.05).CONCLUSION: High glucose promotes the proliferation of mesangial cells. p53 agonist inhibits the viability and promotes apoptosis of mesangial cells under high glucose.
引文
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[9] 姚亚兰, 王金霞, 阚春明, 等. 单次大剂量对比多次小剂量STZ诱导昆明小鼠糖尿病肾病的研究[J]. 安徽医科大学学报, 2017, 52(12):1786-1790.
[10] Lei CT, Tang H, Ye C, et al. MDM2 contributes to high glucose-induced glomerular mesangial cell proliferation and extracellular matrix accumulation via Notch1[J]. Sci Rep, 2017, 7(1):10393.
[11] Momand J, Zambetti GP, Olson DC, et al. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation[J]. Cell, 1992, 69(7):1237-1245.
[12] Amaral JD, Xavier JM, Steer CJ. Targeting the p53 pathway of apoptosis[J]. Curr Pharm Design, 2010, 16(22):2493-2503.
[13] Ju M, Pae HO, Kim WS, et al. Role of reactive oxygen species in p53 activation during cisplatin-induced apoptosis of rat mesangial cells[J]. Eur Rev Med Pharmaco, 2014, 18(8):1135-1141.
[14] Shi H, Zhang A, He Y, et al. Effects of p53 on aldosterone-induced mesangial cell apoptosis in vivo and in vitro[J]. Mol Med Rep, 2016, 13(6):5102-5108.
[2] Kolset SO, Reinholt FP. Diabetic nephropathy and extracellular matrix[J]. J Histochem Cytochem, 2012, 60 (12):976-986.
[3] 王谦, 于玫, 龚慕辛, 等. 高浓度葡萄糖对大鼠肾小球系膜细胞增殖的影响[J]. 中国病理生理杂志, 2003, 19(12):1691-1692, 1708.
[4] Chiarelli F, Gaspari S. Role of growth factors in diabetic kidney disease[J]. Horm Metab Res, 2009, 41(8):585-593.
[5] Tervaert TW, Mooyaart AL, Amann K, et a1. Pathologic classification of diabetic nephropathy[J]. J Am Soc Nephrol, 2010, 21(4):556-563.
[6] Zhang GY, Wang DD, Cao Z, et al. Sitagliptin ameliorates high glucose-induced cell proliferation and expression of the extracellular matrix in glomerular mesangial cells[J]. Exp Ther Med, 2017, 14(4):3862-3867.
[7] 刘意, 赵双林. 白细胞介素22抗体抑制Snail1高表达对糖尿病肾病的影响[J]. 中国病理生理杂志, 2018, 34(5):939-944.
[8] Waasdorp M, Duitman J, Florquin S, et al. Vorapaxar treatment reduces mesangial expansion in streptozotocin-induced diabetic nephropathy in mice[J]. Oncotarget, 2018, 9(31):21655-21662.
[9] 姚亚兰, 王金霞, 阚春明, 等. 单次大剂量对比多次小剂量STZ诱导昆明小鼠糖尿病肾病的研究[J]. 安徽医科大学学报, 2017, 52(12):1786-1790.
[10] Lei CT, Tang H, Ye C, et al. MDM2 contributes to high glucose-induced glomerular mesangial cell proliferation and extracellular matrix accumulation via Notch1[J]. Sci Rep, 2017, 7(1):10393.
[11] Momand J, Zambetti GP, Olson DC, et al. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation[J]. Cell, 1992, 69(7):1237-1245.
[12] Amaral JD, Xavier JM, Steer CJ. Targeting the p53 pathway of apoptosis[J]. Curr Pharm Design, 2010, 16(22):2493-2503.
[13] Ju M, Pae HO, Kim WS, et al. Role of reactive oxygen species in p53 activation during cisplatin-induced apoptosis of rat mesangial cells[J]. Eur Rev Med Pharmaco, 2014, 18(8):1135-1141.
[14] Shi H, Zhang A, He Y, et al. Effects of p53 on aldosterone-induced mesangial cell apoptosis in vivo and in vitro[J]. Mol Med Rep, 2016, 13(6):5102-5108.
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