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Title: [Effect and related mechanisms of RTA-408 on rat vascular smooth muscle cell calcification induced by advanced glycation end products].
Author: Xu Z, Suo CJ, Ruan YS, Tan RY, Zhang W, Niu TL.
Journal: Zhonghua Xin Xue Guan Bing Za Zhi; 2018 Jun 24; 46(6):475-479. PubMed ID: 29925185.
Abstract:
Objective: To investigate the effect and related mechanisms of RTA-408 on rat vascular smooth muscle cells (VSMCs) calcification induced by advanced glycation end products(AGE). Methods: VSMCs were isolated from the aorta of Sprague Dawley rats and cultured in vitro. The fifth generation of VSMCs were randomly divided into 4 groups with random number table including control group(cells were incubated with normal medium for 2 days, then incubated with bovine serum albumin for 5 days),AGE group (cells were incubated with normal medium for 2 days, then incubated with 200 mg/L AGE for 5 days), experimental group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with 200 mg/L AGE for 5 days),and RTA group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with bovine serum albumin for 5 days). Cytosolic calciumin VSMC was measured using arsenazo Ⅲ assay. Von Kossa staining was utilized to detect the calcium deposition.The contents of malondialdehyde(MDA) and superoxide dismutase(SOD) in VSMCs were tested by appropriate kits.The protein expressions of osteopontin (OPN), alkaline phosphatase (ALP), nuclear factor E2 related factor 2(Nrf2), and NAD(P)H: quinone oxidoreductase 1(NQO1) were examined using Western blot. Results: (1) Cytosolic calciumconcentration was significantly higher in AGE group than in control group((2.43±0.15) mmol/L vs. (1.23±0.09) mmol/L, P<0.01), which was significantly reduced in experimental group((1.62±0.18) mmol/L,P<0.01 vs. AGE group). (2) Calcium deposition in VSMCs was significantly upregulated in AGE group than in control group(3.64±0.50 vs. 1.00±0.12, P<0.01), and was downregulated in experimental group (1.56±0.37, P<0.01 vs. AGE group). (3) The MDA contents were higher((3.79±0.27) nmol/mg prot vs.(1.99±0.15) nmol/mg prot, P<0.01), while the SOD activities were lower((308.45±14.28) U/mg prot vs. (428.58±11.00) U/mg prot, P<0.01) in AGE group than in control group. The MDA contents were lower((2.37±0.19) nmol/mg prot vs. (3.79±0.27) nmol/mg prot, P<0.01),while the SOD activities were higher((391.03±22.92) U/mg prot vs. (308.45±14.28) U/mg prot, P<0.05)in experimental group compared with AGE group. (4) The relative expressions of OPN and ALP were higher in AGE group than in control group(3.06±0.21 vs. 1.00±0.07, and 2.89±0.29 vs. 1.00±0.10,both P<0.01), both (OPN(1.15±0.12) and ALP(1.45±0.15)) were downregulated in experimental group (both P<0.01 vs. AGE group). (5) The relative protein expressions of Nrf2 and NQO1 in experimental group were higher than AGE group(2.37±0.17 vs. 1.17±0.09, and 3.91±0.18 vs. 1.05±0.08, both P<0.01). Conclusion: Activation of nrf2/NQO1 signaling pathway by RTA-408 can reduce the AGE-induced VSMC calcification through attenuating oxidative injury. 目的： 探讨RTA-408对晚期糖基化终末产物（AGE）诱导的大鼠血管平滑肌细胞（VSMC）钙化的作用及其机制。 方法： 体外分离培养Sprague Dawley大鼠主动脉VSMC，将传代培养至第5代的细胞按照随机数字表法分为对照组（培养2 d后，更换为含牛血清蛋白的培养基继续培养5 d）、AGE组（培养2d后，更换为含200 mg/L AGE的培养基继续培养5 d）、实验组（使用含100 nmol/L RTA-408的培养基培养2 d后，更换为含200 mg/L AGE的培养基继续培养5 d）和RTA组（使用含100 nmol/L RTA-408的培养基培养2 d后，更换为含牛血清蛋白的培养基继续培养5 d）。收集各组VSMC，采用偶氮胂Ⅲ法检测细胞内钙离子含量，采用Von Kossa染色法检测细胞内相对钙沉积量，采用试剂盒检测细胞内丙二醛（MDA）含量及超氧化物歧化酶（SOD）活性，采用Western blot检测细胞内骨桥蛋白（OPN）、碱性磷酸酶（ALP）、核因子E2相关因子2（Nrf2）和醌氧化还原酶1（NQO1）蛋白含量。 结果： （1）AGE组细胞中钙离子含量高于对照组[（2.43±0.15）mmol/L比（1.23±0.09）mmol/L，P<0.01]，而实验组细胞中钙离子含量[（1.62±0.18）mmol/L]低于AGE组（P<0.01）。（2）AGE组细胞内相对钙沉积量高于对照组（3.64±0.50比1.00±0.12,P<0.01），而实验组细胞内相对钙沉积量（1.56±0.37）低于AGE组（P<0.01）。（3）与对照组比较，AGE组MDA含量较高[（3.79±0.27）nmol/mg蛋白比（1.99±0.15）nmol/mg蛋白，P<0.01]，而SOD活性较低[（308.45±14.28）U/mg蛋白比（428.58±11.00）U/mg蛋白，P<0.01]；实验组MDA含量[（2.37±0.19）nmol/mg蛋白]低于AGE组（P<0.01），而SOD活性[（391.03±22.92）U/mg蛋白]高于AGE组（P<0.05）。（4）AGE组OPN和ALP蛋白相对表达量均高于对照组[分别为3.06±0.21比1.00±0.07和2.89±0.29比1.00±0.10，P均<0.01]；实验组OPN（1.15±0.12）和ALP（1.45±0.15）的蛋白相对表达量均低于AGE组（P均<0.01）。（5）实验组Nrf2及NQO1蛋白相对表达量均高于AGE组（分别为2.37±0.17比1.17±0.09和3.91±0.18比1.05±0.08，P均<0.01）。 结论： RTA-408对AGE引起的大鼠主动脉VSMC钙化有保护作用，其机制可能与激活Nrf2/NQO1信号通路从而提高细胞抗氧化应激能力有关。.

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