目的：探讨高浓度葡萄糖对视网膜色素上皮细胞(ARPE-19)/血管内皮细胞(HUVEC)活性及对沉默信号调节蛋白6(SIRT6)表达水平的影响。

方法：将ARPE-19和HUVEC细胞在含不同浓度葡萄糖(5.5、25、50、75、100 mmol/L)培养基中培养48 h后光学显微镜下观察细胞形态； CCK-8法检测各组细胞活性； 流式细胞术检测各组细胞的凋亡水平； Western Blot检测细胞Bcl-2和SIRT6表达水平。将ARPE-19/HUVEC细胞经50 mmol/L葡萄糖处理后，培养48 h，采用免疫荧光染色检测SIRT6蛋白的亚细胞定位及表达水平； 另取相同处理的细胞，培养不同时间(0、6、12、24、36、48、72 h)，Western Blot检测细胞SIRT6蛋白随时间的变化趋势。构建SIRT6过表达的ARPE-19/HUVEC稳转细胞系，Western Blot检测SIRT6过表达对高糖(50 mmol/L葡萄糖)干预的ARPE-19/HUVEC中葡萄糖转运蛋白表达水平的影响。

结果：与5.5 mmol/L葡萄糖组相比，随着葡萄糖浓度的增加，ARPE-19/HUVEC细胞的数量减少，活性显著降低(均P<0.01)，且SIRT6蛋白表达水平显著降低(P<0.05)，该抑制水平呈浓度依赖性和时间依赖性(均P<0.05)。同时，高糖诱导细胞凋亡，降低Bcl-2蛋白的表达(P<0.05)。免疫荧光染色显示，高糖处理48 h后SIRT6蛋白主要定位于细胞核，并伴有核固缩和核碎裂现象，经高糖(50 mmol/L葡萄糖)培养不同时间后发现长时间高糖干预能降低ARPE-19/HUVEC细胞中SIRT6蛋白表达。经免疫荧光和Western Blot验证SIRT6过表达ARPE-19/HUVEC稳转细胞系成功构建。Western Blot结果分析显示，与HG+LV-con组相比，HG+LV-SIRT6组ARPE-19细胞中GLUT1与GLUT4的表达均无明显差异，GLUT3表达升高(P<0.05)； 与HG+LV-con组相比，HG+LV-SIRT6组HUVEC细胞中GLUT1的表达无明显差异，GLUT3、GLUT4表达升高(P<0.05)。

结论：高浓度葡萄糖可以降低ARPE-19/HUVEC细胞的增殖活性，诱导细胞凋亡，并下调SIRT6蛋白的表达，SIRT6可能通过促进葡萄糖转运蛋白的表达从而缓解高糖诱导视网膜细胞的损伤过程。

METHODS:ARPE-19 and HUVEC cells were cultured for 48 h in media containing various glucose concentrations(5.5, 25, 50, 75, 100 mmol/L)and cellular morphology was observed under an optical microscope.Cell viability was assessed using the CCK-8 assay. Flow cytometry was utilized to evaluate apoptosis levels, and Western blot analysis was conducted to measure the expression of Bcl-2 and SIRT6. ARPE-19/HUVEC cells were treated with 50 mmol/L glucose for 48 h, after which immunofluorescence staining was performed to determine the subcellular localization and expression level of SIRT6 protein. In parallel, cells receiving the same treatment were harvested at different time points(0, 6, 12, 24, 36, 48, and 72 h), and the temporal changes in SIRT6 protein expression were assessed by Western blot analysis. SIRT6 overexpressing stable ARPE-19/HUVEC cell lines were constructed. Western blot was performed to detect the effects of SIRT6 overexpression on the expression levels of glucose transporters in ARPE-19/HUVEC cells treated with high glucose(50 mmol/L glucose).

RESULTS:Compared with the 5.5 mmol/L glucose group, cell number was decreased and cell viability was significantly reduced in ARPE19/HUVEC cells with increasing glucose concentrations(all P<0.01). Meanwhile, the protein expression level of SIRT6 was markedly downregulated(P<0.05). Such inhibitory effects exhibited both concentration-and time-dependent patterns(all P<0.05). Furthermore, high glucose induced cell apoptosis and downregulated the protein expression of Bcl-2(P<0.05). Immunofluorescence staining revealed that SIRT6 protein was predominantly localized in the nucleus after 48 h of high-glucose treatment, accompanied by pyknosis and nuclear fragmentation. After incubation with highg-lucose medium(50 mmol/L glucose)for different durations, long-term high-glucose intervention was found to reduce SIRT6 protein expression in ARPE19/HUVEC cells. Successful construction of the SIRT6-overexpressing stable ARPE-19/HUVEC cell lines was confirmed via immunofluorescence and Western blotting. Western blot analysis revealed that, compared with the HG+LV-con group, the HG+LV-SIRT6 group exhibited no significant differences in GLUT1 or GLUT4 expression in ARPE-19 cells, whereas GLUT3 expression was elevated(P<0.05). Compared with the HG+LV-con group, the HG+LV-SIRT6 group showed no significant change in GLUT1 expression, while GLUT3 and GLUT4 levels were increased(both P<0.05).

CONCLUSION:High-concentration glucose-reduce the proliferative activity of ARPE-19/HUVEC cells, induce apoptosis, and downregulate SIRT6 expression. SIRT6 may alleviate high glucose-induced retinal cell damage by promoting the expression of glucose transporters.