尿酸活化ERK1/2信号通路刺激大鼠肾小球系膜细胞增殖

目的 探讨尿酸（UA）对大鼠肾小球系膜细胞（GMC）增殖的影响及其可能的机制。 方法 体外培养大鼠GMC，应用不同浓度的UA（50、100、300 μmol/L）刺激或应用细胞外信号调节激酶（ERK1/2）特异性抑制剂U0126（10 μmol/L）、NADPH氧化酶特异性抑制剂夹竹桃麻素（500 μmol/L）、线粒体复合体Ⅰ抑制剂鱼藤酮（10 μmol/L）预处理 30 min 后，再加入UA（300 μmol/L）。于实验终点收集细胞，应用3H-TdR掺入法、细胞计数及流式细胞术测定GMC增殖和细胞周期变化；应用实时定量PCR、Western印迹法检测细胞周期素cyclin D1和cyclin A2的表达及ERK1/2的磷酸化水平；应用荧光探针2，7-二氯二氢荧光素乙酰乙酸（DCFDA）检测细胞内活性氧（ROS）的变化。 结果 （1）与对照组相比，3H-TdR掺入法和细胞计数均显示，UA呈剂量依赖性促进GMC增殖，300 μmol/L UA刺激组其细胞数为对照组的1.5倍以上。（2）流式细胞术显示，UA呈剂量依赖性减少G1/G0期细胞数，增加S期细胞数，300 μmol/L UA刺激组其S期细胞数为对照组的2倍以上。（3）UA呈剂量依赖性促进系膜细胞周期蛋白cyclin D1和cyclin A2的表达。（4）UA呈剂量依赖性促进系膜细胞ERK1/2磷酸化且U0126能够抑制UA诱导的GMC增殖。细胞计数和3H-TdR掺入法分别显示，U0126的抑制率分别是UA 300 μmol/L刺激组的22%和31%（均P < 0.05）。（5）UA呈剂量依赖性促进ROS产生增加，夹竹桃麻素能够明显抑制UA诱导的ROS生成、ERK1/2的磷酸化和系膜细胞增殖（均P < 0.05），而鱼藤酮对其无明显影响。 结论 UA可促进GMC增殖，其可能的机制为UA诱导NADPH 氧化酶来源的 ROS 产生增加，从而激活ERK1/2信号通路，引起周期蛋白表达增加，促进GMC增殖。

Uric acid stimulates rat glomerular mesangial cells proliferation via activation of extracellular signal-regulated kinases

Objective To explore the effect of uric acid (UA) on the rat glomerular mesangial cells (GMCs) and its possible mechanism in vitro. Methods Cultured rat GMCs were treated with various concentrations of UA (50 μmol/L, 100 μmol/L, 300 μmol/L) in the presence or absence of U0126, apocynin, rotenone. The incorporation of 3H-thymidine (3H-TdR) and cell counting were used to measure GMCs proliferation. GMCs cell-cycle was analyzed by flow cytometry. CyclinD1 and cyclin A2 expression were determined by real-time PCR and Western blotting analysis. ERK1/2 phosphorylation was detected by Western blotting. Reactive oxygen species (ROS) was measured by 2, 7-dichlorofluorescein diacetate (DCFDA) fluorescence. Results (1) Uric acid increased GMCs proliferation in dose-dependent manner compared with the control groups, as assessed by 3H-TdR incorporation and cell counting. GMCs proliferation induced by 300 μmol/L uric acid was increased by more than 1.5-fold assessed by both of the methods. (2) Uric acid decreased cell number in G1/G0 phase and increased cell number in S phase in dosedependent manner, as assessed by flow cytometry. (3) Uric acid iuduced cyclin D1 and cyclin A2 expression in dose-dependent manner. (4)Uric acid increased pospho-ERK1/2 in dose-dependent manner and ERK1/2 specific inhibitor U0126 could suppress uric acid-induced cell proliferation.The inhibition percentage of U0126 was 22% and 31% assassed by cell counting and 3H-TdR incorporation, respectively. (5) Uric acid increased ROS production in dose-dependent manner.NADPH oxidase inhibitor apocynin could also significantly inhibit uric acid-induced ROS production, ERK phosphorylation and cell proliferation. In contrast, rotenone had no effect on them.Conclusion Uric acid can stimulate rat GMCs proliferation, partially by the activation of ERK pathway via NADPH oxidase-derived ROS generation.