SIRT1对高糖高脂培养胰岛微血管内皮细胞炎性反应激活的影响

Roles of SIRT1 in the inflammatory activation of high-glucose and lipid cultured islet microvascular endothelial cells

目的分析高糖、高脂环境下胰岛微血管内皮细胞（IMVC）分泌E-选择素、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β的变化，研究沉默信息调节蛋白1（SIRT1）／核因子-kβ信号途径异常在内皮细胞炎性反应激活中的作用。方法合成含绿色荧光蛋白报告基因的小鼠SIRT1基因重组质粒，以Lipofectamine2000转染IMVC。之后将IMVC分为4组：正常对照组、高糖高脂组、高糖高脂＋SIRT1基因转染组、高糖高脂＋基因转染对照组。高糖高脂组以33．3mmol／L葡萄糖＋0．5mmol／L棕榈酸处理48h。高糖高脂＋SIRT1基因转染组在高糖高脂处理前以SIRT1基因重组质粒预处理48h；高糖高脂＋基因转染对照组在高糖高脂处理前以空质粒进行预处理48h。应用实时荧光定量PCR法检测SIRT1基因转染效果及各组核因子-kβ的mRNA水平，应用酶联免疫吸附法测定细胞培养上清中TNF—α、IL-1β、E-选择素的水平。结果与正常对照组相比，高糖高脂组SIRT1 mRNA表达明显下降（0．58±0．12，P〈0．01）；而高糖高脂＋SIRT1基因转染组的SIRT1基因表达明显升高，与高糖高脂组相比差异显著[（1．55±0．43）比（0．65±0．37），P〈0．01]。与正常对照组相比，高糖高脂组核因子-kβ mRNA（1．59±0．32，P〈0．01）、E-选择素[（48．46±1．04）比（67．12±0．57）ng／L，P〈0．01]、TNF—α[（467．46±8．98）比（621．14±11．26）ng／L，P〈0．01]、IL-1β[（63．32±1．48）比（118．43±1．40）ng／L，P〈0．01]的水平明显升高（P〈0．01）。与高糖高脂组相比，高糖高脂＋SIRT1基因转染组核因子-kβ mRNA（0．95±0．31，P〈0．01）及TNF-α[（451．38±15．91）比（618．89±7．23）ng／L，P〈0．01]明显下降（P〈0．01），E-选择素、IL-1β无明显变化（P〉0．05）。结论高糖高脂环境下，IMVC存在炎性反应激活，可释放大量炎性反应细胞因子。SIRT1-核因子-KB．TNF—d通路异常与IMVC的炎性反应激活密切相关，早期干预该信号通路可能在预防及治疗2型糖尿病的胰岛炎性反应中具有重要意义。

Objective To explore the alteration of E-selectin, tumor necrosis factor （TNF） -α,inter-leukin （IL） -1 β in high-glucose and lipid cultured islet microvascular endothelial cells （IMVC）, and analysis the effects of sirtuin 1 （SIRT） 1/nuclear factor-kβ （NF-KB） signaling pathway on the inflammatory activa-tion of IMVC. Methods Recombinant mouse SIRT1 plasmid including green fluorescent protein report gene was synthesized and transfected to IMVC by using Lipofectamine 2000. IMVC were divided into 4 groups： normal control group, high glucose/hpid group, SIRT1 plasmid plus high glucose/lipid group, and empty plas-mid plus high glucose/lipid group. Cells in high glucose/lipid group were cultured in 33.3 mmol/L glucose and 0.5 mmol/L palmic acid for 48 hours. Cells in SIRT1 plasmid plus high glucose/lipid group were pre-treated with SIRT1 recombinant plasmid for 48 hours before the treatment with high glucose and lipid. Ceils in empty plasmid plus high glucose/lipid treatment group were pretreated with empty plasmid before the treat- ment with high glucose and lipid. Real time quantitive-PCR was used to test the transfecting efficacy of SIRT1 recombinant plasmid and NF-kβ mRNA expression in each group. ELISA was used to exam the levels of TNF-α, IL-1β ,E-selectin in cell culture supernatant. Results Compared with normal control group, the ex-pression of SIRT1 mRNA in high glucose/lipid group was decreased significantly （0.58 ± O. 12 ,P 〈 0.01 ）. However, in SIRT1 plasmid plus high glucose/lipid group, the expression of SIRT1 mRNA was increased greatly compared with that in high glucose/lipid treatment group [ （ 1.55 ± O. 43 ） vs. （0.65 ± 0.37 ）, P 〈 O. O1 ]. Compared with normal control group, the expressions of NF-kβ mRNA （ 1.59 ± 0.32, P 〈 O. O1 ） , E-selectin [ （48.46 ± 1.04） vs. （67.12 ± 0.57 ） ng/L, P 〈 0.01 ], TNF-α[ （467.46 ± 8.98 ） vs. （ 621. 14 ± 11.26） ng/L,P 〈 0.011, IL-1β [ （63.32-± 1.48 ） vs. （ 118.43 ± 1.40 ） ng/L, P 〈 0.01 ] were increased in high glucose/lipid group. However,in SIRT1 plasmid plus high glucose/lipid group, the expression of NF-kβ mRNA（0.95 ±0.31,P 〈 0.01 ） and TNF-α level[ （451.38 ± 15.91 ）vs. （618.89 ± 7.23 ） ng/L,P 〈 0.01 ] were decreased compared with high glucose/lipid group, but E-selectin and IL-1β level were not changed sig-nificantly（ P 〉 0.05 ）. Conclusions In hyperglycemic and hyperlipid situation, IMVC is activated and can release many inflammatolT factors. Abnormal SIRT1-NF-kβ-TNF-α pathway plays roles in the inflammatory activation of IMVC, and early intervention may have great promise in the prevention and treatment of islet in-flammation in type 2 diabetes.