寻找高血糖与高尿酸血症协同加重内皮细胞损伤的共同作用靶点和分子机制，为糖尿病合并高尿酸血症患者心血管疾病的保护提供干预靶点。

用人脐静脉内皮细胞系(HUVEC-C)给予高糖(30 mmol/L，HG)、高尿酸(600 μmol/L，UA)和高糖(HG)＋高尿酸(UA)联合培养48 h。利用10 μmol/L阿司他丁阻断醛糖还原酶(AR)的活性。实时定量PCR检测内皮型一氧化氮合成酶(eNOS)和AR mRNA的表达；Western blot检测还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2 (NOX2)、NOX4、eNOS和AR蛋白的表达；共聚焦显微镜检测细胞内ROS的活性；一氧化氮(NO)试剂盒检测NO活性。

与单独HG组和UA组相比，HG＋UA共培养组明显降低eNOS mRNA水平和蛋白表达，减少NO产生，增加内皮细胞胞内ROS活性；HG＋UA共培养明显上调AR mRNA水平和蛋白表达。应用AR抑制剂能够明显增加HG＋UA组内皮细胞eNOS mRNA水平和蛋白表达，增加内皮细胞NO的分泌水平。AR抑制剂能够明显下调HG＋UA组内皮细胞NOX4的蛋白表达，对NOX2无影响；降低细胞内ROS的含量。

高血糖和高尿酸协同作用通过激活醛糖还原酶途径下调内皮细胞eNOS的表达，增加ROS活性，减少NO的产生，加重内皮细胞功能障碍；而抑制醛糖还原酶，能够通过抑制NOX4表达，阻断这种协同损伤作用。

To find the common target and molecular mechanism of the synergistic injury of the endothelial cell caused by high glucose and high uric acid for prevention from cardiovascular diseases in diabetes patients complicated with hyperuricemia.

The human umbilical vein endothelial cell line (HUVEC-C) was treated with high glucose (30 mmol/L, HG), high uric acid (600 μmol/L, UA) or HG plus UA for 48 hours. The activity of aldose reductase (AR) was blocked with alrestatin (10 μmol/L). Real-time quantitative PCR was used for detecting the mRNA expression of endothelial nitric oxide synthase (eNOS) and AR. Western blot was used to detect the expression of reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX)2, NOX4, eNOS and AR proteins. The intracellular activity of ROS was assayed with confocal microscopy. Nitric oxide (NO) concentration was measured with chemiluminescence assay kit.

Compared with the HG or UA, HG+ UA could significantly reduce not only the mRNA and protein levels of eNOS but also NO production, and increase the intracellular ROS generation in the endothelial cells. HG+ UA also remarkably increased AR mRNA level and protein expression. Furthermore, in the endothelial cells of the HG+ UA group, alrestatin significantly increased the mRNA and protein expression of eNOS as well as the secretion level of NO, while alrestatin obviously down-regulated both the protein expression of NOX4 and the intracellular content of ROS, but had no effects on NOX2.

In endothelial cells, via the aldose reductase pathway, the hyperglycemia and hyperuricemia synergistically decreased the expression of eNOS and the production of NO, but increased the ROS generation, aggravating the endothelial dysfunction, which could be blocked by inhibition of aldose reductase through down-regulation of NOX4.