Department of Endocrinology, Fujian Provincial Hospital, Fujian Medical University, Fuzhou, 350001, China.
Mol Cell Biochem. 2011 Jan;347(1-2):127-33. doi: 10.1007/s11010-010-0620-5. Epub 2010 Oct 19.
Our previous study found that blocking nuclear factor (NF)-κB signaling could protect human umbilical vein endothelial cells (HUVECs) from apoptosis and proliferation inhibition due to high glucose (HG). Intermittent HG makes glucose toxicity more significant. In this study, we aimed to investigate the effect of NF-κB pathway on HUVECs induced by intermittent HG (a daily alternating 5.5 or 30.5 mmol/l glucose). A recombinant adenovirus containing a RNAi cassette targeting the NF-κB/p65 gene was produced, and its silencing effect on p65 gene was detected by Western blot analysis in HUVECs cultured with intermittent HG. The subsequent effect on proliferation of HUVECs in the indicated conditions was measured by the AlamarBlue assay. The Bcl-2 expression was also detected by Western blot. The results showed that the expression of p65 protein could be inhibited efficiently by the RNAi adenovirus. Intermittent HG also induced the translocation of NF-κB in HUVECs. Inhibition of NF-κB with the RNAi adenovirus could prevent the effects. At the 6th day after HUVECs were exposed to intermittent HG, the proliferation of HUVECs with Ad-1566 was significantly higher than that of HUVECs with Ad-DEST (P < 0.01). Knockdown of NF-κB/p65 up-regulated the Bcl-2 expression of HUVECs under intermittent HG conditions (P < 0.01). These findings concluded that the NF-κB/p65-targeting RNAi adenovirus is an important tool, which can efficiently inhibit the expression of p65 gene in HUVECs. Intermittent HG reduces HUVECs proliferation with a concomitant increase in apoptosis. Knockdown of NF-κB/p65 partly protected HUVECs from proliferation inhibition and may reduce apoptosis.
我们之前的研究发现，阻断核因子 (NF)-κB 信号通路可以保护人脐静脉内皮细胞 (HUVECs) 免受高糖 (HG) 诱导的细胞凋亡和增殖抑制。间歇性 HG 会使葡萄糖毒性更加显著。在这项研究中，我们旨在研究 NF-κB 通路对间歇性 HG（每天交替 5.5 或 30.5mmol/l 葡萄糖）诱导的 HUVECs 的影响。我们制备了一个含有靶向 NF-κB/p65 基因的 RNAi 盒的重组腺病毒，并通过 Western blot 分析检测其在间歇性 HG 培养的 HUVECs 中对 p65 基因的沉默作用。随后，通过 AlamarBlue 测定法测量了在指定条件下对 HUVECs 增殖的影响。还通过 Western blot 检测了 Bcl-2 的表达。结果表明，RNAi 腺病毒可以有效地抑制 p65 蛋白的表达。间歇性 HG 也诱导了 HUVECs 中 NF-κB 的易位。用 RNAi 腺病毒抑制 NF-κB 可以阻止这种作用。在 HUVECs 暴露于间歇性 HG 后的第 6 天，用 Ad-1566 处理的 HUVECs 的增殖明显高于用 Ad-DEST 处理的 HUVECs（P<0.01）。在间歇性 HG 条件下，NF-κB/p65 的敲低上调了 HUVECs 的 Bcl-2 表达（P<0.01）。这些发现表明，靶向 NF-κB/p65 的 RNAi 腺病毒是一种重要的工具，可以有效地抑制 HUVECs 中 p65 基因的表达。间歇性 HG 降低了 HUVECs 的增殖，同时增加了细胞凋亡。NF-κB/p65 的敲低部分保护了 HUVECs 免受增殖抑制，并可能减少凋亡。
