论文部分内容阅读
氧化应激是糖尿病肾病的重要发病机制之一。过氧亚硝基阴离子(peroxynitrite,ONOO–)是参与氧化应激损伤的重要成员,与糖尿病及其并发症密切相关。该文观察高糖环境下ONOO–对系膜细胞合成纤连蛋白(fibronectin,FN)的影响,并探讨其作用机制。实验中,人肾小球系膜细胞分为4组:正常对照组、高糖组、高糖+尿酸组及高糖+AG490组。培养12,24,48 h后收集细胞及其上清液、并提取细胞总蛋白。采用酶联免疫吸附实验(ELISA)检测细胞上清液中FN的含量,采用免疫细胞化学和Western blot检测NT总蛋白(ONOO–生成的生物标志物)、p-JAK2及p-STAT3蛋白的表达。结果显示,与同期正常组相比,高糖组NT总蛋白、p-JAK2及p-STAT3的表达及FN含量明显增高(P<0.05),并且随着时间的延长表达逐渐增多,以48 h组最为显著;高糖+尿酸组,NT、p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05);高糖+AG490组,p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05),但NT表达与高糖组差异无统计学意义(P>0.05)。由此可见,高糖环境下系膜细胞中存在ONOO–的过量表达,ONOO–通过JAK/STAT信号途径促进系膜细胞FN的合成。
Oxidative stress is one of the important pathogenesis of diabetic nephropathy. Peroxynitrite (ONOO-) is an important member involved in oxidative stress injury and is closely related to diabetes mellitus and its complications. This study investigated the effect of ONOO- on the synthesis of fibronectin (FN) in mesangial cells under high glucose conditions and explored its mechanism. In the experiment, human mesangial cells were divided into 4 groups: normal control group, high glucose group, high glucose + uric acid group and high glucose + AG490 group. After culturing for 12, 24 and 48 h, the cells and their supernatants were collected and the total cellular protein was extracted. The content of FN in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of NT-total protein (ONOO-generated biomarker), p-JAK2 and p-STAT3 protein were detected by immunocytochemistry and Western blot . The results showed that compared with the normal control group, the expression of NT-total protein, p-JAK2 and p-STAT3 and the content of FN in the high glucose group were significantly increased (P <0.05), and gradually increased with the prolongation of time. (P <0.05). The levels of p-JAK2, p-STAT3 and FN in high glucose + AG490 group were significantly higher than those in high glucose + uric acid group (P <0.05), but there was no significant difference between NT group and high glucose group (P> 0.05). Thus, ONOO-overexpression is present in mesangial cells under high glucose conditions, while ONOO- promotes the synthesis of FN in mesangial cells via the JAK / STAT signaling pathway.