SIRT3在尿酸诱导的肾小球系膜细胞转分化中的表达变化及意义

Changes and significance of SIRT3 expression in cellular transdifferentiation induced by uric acid

摘要:

目的：探讨沉默信息调节因子 3（silent information regulator 3，SIRT3）在高浓度尿酸引起的小鼠肾小球系膜细胞53（mesangial cells 53，MC53）转分化过程中的表达变化和意义。方法：将不同浓度的尿酸（0.05、0.10、0.30 mmol/L）与小鼠MC53共孵育48 h后，以2’，7’－二氯荧光素二乙酸酯为荧光探针，用荧光显微镜和流式细胞仪检测各组活性氧（reactive oxygen species，ROS）水平，用real-time PCR和Western blot检测各组SIRT3以及转分化标志物α平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、转化生长因子β1（transforming growth factor-β1，TGF-β1）、纤连蛋白（fibronectin，FN）mRNA和蛋白的表达；通过SIRT3 siRNA的转染检测它对氧化应激及细胞转分化的影响。结果：与对照组相比，尿酸能诱导肾小球系膜细胞ROS水平呈浓度依赖性上调（F=98.018，P=0.000），SIRT3mRNA及蛋白表达量呈浓度依赖性下降（mRNA：F=81.622，P=0.000；蛋白：F=38.551，P=0.000），其α-SMA、TGF-β1、FN mRNA和蛋白的表达量均呈浓度依赖性上调（mRNA：F=162.222、22.111、26.960，P=0.000、0.000、0.000；蛋白：F=31.431、39.381、44.642，P=0.000、0.000、0.000）；转染SIRT3 siRNA之后再进行高尿酸孵育，ROS水平及α-SMA、TGF-β1、FN蛋白的表达与单独尿酸孵育组相比均明显上升（ROS：F=24.883，P=0.001；蛋白：F=8.741、78.778、45.403，P=0.017、0.000、0.000）。结论：高浓度尿酸能诱导MC53细胞转分化，SIRT3可能与高浓度尿酸引起的细胞转分化有着密切关系。

Abstract:

Objective：To explore the role of the silent information regulator 3（SIRT3） in the transdifferentiation process of mouse’s glomerular mesangial cells（MC53） caused by high levels of uric acid. Methods：The MC53 cells were cultured in media containing different concentrations of uric acid（0.05，0.1，0.3 mmol/L） for 48 h. The intracellular reactive oxygen species（ROS） level was deter－mined with fluorescence microscope and flow cytometry. The mRNA and protein expression levels of SIRT3，α-smooth muscle actin （α-SMA），transforming growth factor-β1（TGF-β1），fibronectin（FN） were evaluated through real-time PCR and Western blot. The effect of SIRT3 on oxidative stress and cell transdifferentiation was assessed by transfection of siSIRT3 into MC53 cells. Results：UA increased ROS production in a concentration-dependent manner with a maximal stimulation at 0.3 mmol/L. UA stimulated the down regulation of SIRT3 and upregulation of the α-SMA，TGF-β1 and FN mRNA and proteins in a concentration manner. The ROS level and the expression of α-SMA，TGF-β1，FN proteins were elevated in cells transfected with siSIRT3 after incubation with uric acid. Conclusion：High concentration of uric acid can induce MC53 cells transdifferentiation，SIRT3 may play an important role in cell trans－differentiation.