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目的观察不同水平尿酸对体外培养的肾小管上皮细胞还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)及活性氧簇(ROS)的影响,探讨尿酸损害肾小管上皮细胞的可能机制。方法分离肾小管上皮细胞,将其分为对照组与尿酸干预组(尿酸干预组又分0.1、0.2、0.4、0.8 mmol.L-14个亚组),每组重复6孔;紫外分光光度法测定肾小管上皮细胞内NADPH蛋白水平;光泽精化学发光法测定肾小管上皮细胞内超氧阴离子(O—2)生成量;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)与琼脂糖凝胶电泳法检测肾小管上皮细胞凋亡。结果 24 h后,0.1 mmol.L-1尿酸干预组NADPH酶蛋白水平与O 2—生成量较对照组仅轻微增高,0.2、0.4、0.8 mmol.L-1尿酸干预组NADPH酶蛋白水平与O2—生成量均较对照组显著增高(Pa<0.01)。对照组细胞均未形成明显的DNA凋亡梯带,0.1、0.2、0.4、0.8 mmol.L-1尿酸干预组均可见明显的DNA凋亡梯带,0.4、0.8 mmol.L-1尿酸干预组DNA凋亡梯带较0.1、0.2 mmol.L-1尿酸干预组更为明显。TUNEL结果显示:对照组,0.1、0.2、0.4、0.8 mmol.L-1尿酸干预组的凋亡率分别为(17.5±1.0)‰、(72.4±12.4)‰、(136.8±13.4)‰、(328.7±32.6)‰、(427.2±51.5)‰,0.1、0.2、0.4、0.8 mmol.L-1尿酸干预组细胞凋亡率均明显高于对照组(Pa<0.01),各尿酸干预组之间细胞凋亡率随着尿酸干预浓度的增高而增高;相关分析发现肾小管上皮细胞凋亡率与肾小管上皮细胞内NADPH酶蛋白水平及O2—生成量呈显著正相关(r=0.765、0.792,Pa<0.01)。结论肾小管上皮细胞在持续高尿酸的作用下,可激活细胞内NADPH酶蛋白表达,释放O—2,启动氧化应激级联反应,使肾小管上皮细胞持续受损而导致肾小管上皮细胞凋亡增加。
Objective To observe the effects of different levels of uric acid on the reduction of nicotinamide adenine dinucleotide phosphate (NADPH) and reactive oxygen species (ROS) in renal tubular epithelial cells cultured in vitro, and to explore the possible mechanism of uric acid on renal tubular epithelial cells. Methods The renal tubular epithelial cells were isolated and divided into control group and uric acid intervention group (0.1, 0.2, 0.4, 0.8 mmol.L-14 subgroups in uric acid intervention group) with 6 replicates in each group. Ultraviolet spectrophotometry The level of NADPH in renal tubular epithelial cells was measured by spectrophotometry. The production of superoxide anion (O-2) in renal tubular epithelial cells was determined by lucigenin chemiluminescence assay. The expression of TUNEL (terminal deoxynucleotidyl transferase mediated nick end labeling) Tubular epithelial cell apoptosis was detected by agarose gel electrophoresis. Results After 24 h, the level of NADPH enzyme and the production of O 2 in the intervention group were only slightly higher than those in the control group, while the levels of NADPH enzyme in the intervention group with 0.2, 0.4 and 0.8 mmol·L -1 uric acid were significantly lower than those in the control group - Generations were significantly higher than the control group (Pa <0.01). No apoptotic DNA ladder was found in the control cells. Significant apoptotic DNA ladder was observed in 0.1,0.2,0.4,0.8 mmol.L-1 uric acid intervention group, and 0.4,0.8 mmol.L-1 uric acid intervention group DNA apoptosis ladder than 0.1, 0.2 mmol.L-1 uric acid intervention group more obvious. The results of TUNEL showed that the apoptotic rates in the control group were (17.5 ± 1.0) ‰, (72.4 ± 12.4) ‰ and (136.8 ± 13.4) ‰, respectively, in the groups of 0.1, 0.2, 0.4 and 0.8 mmol.L- 328.7 ± 32.6) ‰, (427.2 ± 51.5) ‰, 0.1,0.2,0.4,0.8 mmol.L-1 uric acid intervention group were significantly higher than the control group (Pa <0.01), uric acid intervention group The apoptosis rate increased with the increase of uric acid intervention concentration. Correlation analysis showed that there was a significant positive correlation between the apoptosis rate of tubular epithelial cells and the levels of NADPH enzyme and O2- production in renal tubular epithelial cells (r = 0.765,0.792, Pa <0.01). CONCLUSION: Sustained high uric acid can activate NADPH enzyme protein expression and release O-2 in renal tubular epithelial cells, initiate the oxidative stress cascade, and cause persistent damage of renal tubular epithelial cells and lead to tubular epithelial cell apoptosis Death increased.