脱氢抗坏血酸对高糖诱导肾小管上皮细胞产生氧自由基的影响

目的 探讨脱氢抗坏血酸(DHA)对高糖诱导肾小管上皮细胞产生氧自由基的影响｡方法 (1)传代培养肾小管上皮细胞;(2)以Fe3+还原法检测细胞内抗坏血酸(AA)浓度,观察肾小管上皮细胞摄取AA和DHA的情况及葡萄糖和葡萄糖转运蛋白(GLUT)抑制剂细胞松弛素B(cytochalasin B)对其影响;(3)激光扫描共聚焦显微镜检测细胞内氧自由基,观察高糖诱导肾小管上皮细胞氧自由基产生的情况及不同浓度DHA对其影响｡结果 (1)AA不能由细胞外进入肾小管上皮细胞,而DHA可以进入,并且随着细胞外葡萄糖浓度的增加,其进入速度减慢｡细胞松弛素B则完全抑制了DHA进入到肾小管上皮细胞, 而固定葡萄糖浓度(25 mmol/L)时,随着DHA浓度的增加,DHA进入细胞内的速度逐渐增快｡(2)高糖快速诱导了肾小管上皮细胞氧自由基产生增多,DHA抑制了高糖的这种作用,并且该抑制作用在浓度小于､等于4 mmol/L的范围内呈浓度依赖性｡结论 (1)肾小管上皮细胞是依赖DHA利用VitC的细胞型,DHA进入该细胞依赖GLUT介导,高糖可抑制其进入细胞;(2)DHA可有效抑制高糖诱导的肾小管上皮细胞氧自由基产生增多,并在一定范围内呈浓度依赖性｡

Effect of dehydroascorbate on reactive oxygen species in tubular epithelial cell induced by high glucose

Objective To investigate the effect of dehydroascorbate on reactive oxygen species in tubular epithelial cell induced by high glucose. Methods Tubular epithelial cell was cultured in RPMI-1640 medium containing 10% newborn calf serum. Uptake assays for AA and DHA in tubular epithelial cell was done by using the method provided by Robert Root-Bernsteinz. The intracellular formation of ROS was detected with the fluorescent probe CM-H2DCFDA by using confocal microscopy. Results At a DHA concentration of 1 mmol/L, inereasing concentrations of glucose competitively inhibited DHA from entering into the cells such that the accumulation of DHA was smaller than half maximal at about 22 mmol/L glucose. Cytoehalasin B, a kind of hexose transporter inhibitor, inhibited DHA from entering into the cells. At a glucose eoneentration of 25 mmol/L, DHA entry into cells was strengthened with increased DHA level, and DHA inhibited intracellular ROS formation in a dose-dependent manner when DHA level was less than 4 mmol/L. However, the inhibitory effect was not observed at 8 mmol/L of DHA. Conclusions Tubular epithelial cells are DHA dependent.VitC exclusion from tubular epithelial cells through competition of glucose and DHA for common transport mechanism will deprive the cells of the central antioxidant and this could lead to ROS accumulation.