干预糖尿病患者血管病变:二氯醋酸二异丙胺对内皮细胞的功能效应

Intervention of angiopathy in patients with diabetes mellitus: Effect of diisopropylamine dichloroacetate on the function of endothelial cells

目的:了解丙酮酸脱氢酶激活剂二氯醋酸二异丙胺对内皮细胞功能指标的影响。方法:将传代培养的人脐静脉内皮细胞接种至6孔板,培养48～72h至90%融合。将每孔分别分组:对照组:葡萄糖浓度为5.5mmol/L;高糖组:葡萄糖浓度为30mmol/L;二氯醋酸二异丙胺+高糖组:浓度为1×10-5,1×10-4,1×10-3mol/L的二氯醋酸二异丙胺,加高糖处理48h。用2.5g/L胰酶消化收集细胞;固定后充分混匀制成1×109L-1细胞悬液;加RNA酶A37℃水浴45min;加入碘化丙啶50g/L混匀,4℃避光放置60min,尼龙网滤过,上流式细胞仪分析细胞DNA含量,计数10000个细胞,以亚G1峰细胞为凋亡细胞,计算细胞凋亡率。以体外原代培养的人脐静脉内皮细胞为靶细胞观察二氯醋酸二异丙胺对高糖诱导的血管内皮细胞功能指标一氧化氮和可溶性细胞间黏附分子1及细胞凋亡的影响;用改进的DPN诱导分析法检测二氯醋酸二异丙胺对人脐静脉内皮细胞中丙酮酸脱氢酶活性的影响。结果:①高糖组细胞一氧化氮浓度[(590.77±56.00)μmol/L]明显低于对照组[(799.47±51.77)μmol/L,P<0.01],可溶性细胞间黏附分子1水平明显高于对照组(P<0.001)。二氯醋酸二异丙胺+高糖组细胞一氧化氮浓度明显高于高糖组(P<0.05～0.01),可溶性细胞间黏附分子1水平明显低于高糖组(P<0.01)。②不同浓度的二氯醋酸二异丙胺可呈浓度依赖性激活丙酮酸脱氢酶活性,高浓度组与中、低浓度二氯醋酸二异丙胺组相比,差异有显著性意义(P<0.05)。结论:二氯醋酸二异丙胺可以通过对丙酮酸脱氢酶的激活来纠正内皮细胞功能紊乱。

AIM： To investigate the influence of pyruvate dehydrogenase （PDH） activator diisopropylamine dichloroacetate （DIPA） on the indexes of endothelial cell function. METHODS： The subcuhured human umbilical vein endothelial cells （HUVECs） were plated into 6-well plates and cultured for 48 to 72 hours until 90% HUVECs were confiuenced. HUVECs in each well were divided into 3 groups： control group （5.5 mmol/L glucose）; high glucose group （30 mmol/L glucose）; DIPA＋ high glucose group： （1×10^-5, 1×10^-4, 1×10^-3 mol/L DIPA added high glucose cultured for 48 hours. Venous endothelial cells were isolated by 2.5 g/L trypsin digestion. Cell suspension was fixed with 700 mL/L ice-cold ethanol at 4 ℃, and then were fully mixed into cell suspension of 1×10^9 L^-1, then they were incubated with RNase at 37 ℃ for 45 minutes and propidium iodide （PI） 50 g/L at 4 ℃ in avoidance of light for 60 minutes. The DNA content was analyzed by flow cytometer after the cells were filtered with nylon mash. 10 000 cells were counted and the cells at sub G1 peak were taken as the apoptosis cells to calculate the apoptosis rate. The effects of DIPA on the endothelial dysfunction indexes of the levels of nitric oxide （NO） and soluble intercellular adhesion molecule-1 （slCAM-1） induced by high glucose and apoptosis were studied taking the primarily cultured HUVECs in vitro as the target cells. The influence of DIPA on the activity of pyruvat dehydrogenase （PDH） in HUVECs was assayed with the improved method of DPN induced chemistry. RESULTS： ①High glucose group had obviously lower NO level [（590.77±56.00） μmol/L] than the control group [（799.47±51.77） μmol/L] （P 〈 0.01）, but had markedly higher s1CAM-1 level （P 〈 0.01）. The NO level in the DIPA＋high glucose group was obviously higher than that in the high glucose group （P 〈 0.05 to 0.01）, while the sICAM-1 level was obviously lower than that in the high glucose group （P 〈 0.01）. ② DIPA of different concentrations could activate the activity of PDH in a concentrationdependent manner. High concentration DIPA group had significant difference from those in the middle and low concentration DIPA groups （P 〈 0.05）. CONCLUSION： DIPA can correct the endothelial cell dysfunctions induced by high glucose through activation of PDH, which provides new evidence for the earlier prevention and the secondary rehabilitation of angiopathy of diabetes mellitus.