姜黄素调控炎症对内皮细胞增殖凋亡的影响

目的明确姜黄素对炎症诱导的血管内皮细胞损伤的保护机制。方法将人单核／巨噬细胞系（THP-1）分为空白对照组、高脂高糖组、高脂高糖＋姜黄素预处理组（高脂高糖浓度为25mmol／L葡萄糖＋500μmol／L棕榈酸），分别给予相应处理24h，更换培养基继续培养24h，利用酶联免疫吸附法（ELISA）检测上清及实时定量聚合酶链反应（RT-PCR）分析THP-1细胞内肿瘤坏死因子-α（TNF-α）、白介素-6（IL-6）的表达，同时将上清作用脐静脉血管内皮细胞（HUVECs），HUVECs分为空白对照组、空对照条件培养基组、高脂高糖条件培养基组、姜黄素处理条件培养基组，并行噻唑蓝法（MTT）检测HUVECs增殖、流式细胞仪检测凋亡，Westernblotting分析磷酸化细胞外调节蛋白激酶（p-ERK）及B淋巴细胞瘤-2（Bcl-2）蛋白表达。组间比较采用独立样本t检验，多组间比较采用单因素方差分析。结果高脂高糖组THP-1细胞内受体相互作用蛋白140（RIPl40）、TNF-α和IL-6mRNA表达及上清中炎症因子TNF-α和IL-6浓度明显高于空白对照组（t=8．55、9．44、9．73、16．01、19．22，均P〈0．05）。相对于高脂高糖组，姜黄素预处理组THP-1细胞内RIPl40、TNF-α和IL-6mRNA表达及上清中TNF-α和IL-6浓度明显下降（t=3．59、5．96、5．59、6．95、23．91，均P〈0．05）；高脂高糖条件培养基组HUVECs细胞活力明显低于空对照条件培养基组（24h，1．22±0．07比1．85±0．14，t=6．58，P〈0．05；48h，1．72±0．02比2．49±0．09，t=10．08，P〈0．05），而姜黄素处理条件培养基组能够改善高脂高糖条件培养基对HUVECs增殖的抑制作用（24h，1．22±0．07比1．72±0．11，t=2．13，P〈0．05；48h，1．72±0．02比2．33±0．11，t=6．92，P〈0．05）；与空对照条件培养基组比较，高脂高糖条件培养基组能够明显增加HUVECs凋亡率、p-ERK表达及降低Bcl-2表达（t=9．82、9．69、4．61，均P〈0．05），姜黄素处理条件培养基组与高脂高糖条件培养基组比较，下调RIPl40表达后能明显降低HUVECs凋亡率、p-ERK表达及增加Bcl-2表达（t=6．35、7．17、3．26，均P〈0．05）。结论姜黄素能够通过抑制高脂高糖诱导的炎症来调控HUVECs的增殖凋亡。

Curcumin suppresses the effect of inflammation on proliferation and apoptosis of vascular endothelial cells

Objective To investigate the protective role of curcumin in the dysfunctional vascular endothelial cells induced by inflammation. Methods THP-1 cells were pretreated with 20 μmoL/L curcumin under the condition of 25 mmol/L glucose and 500 μmol/L palmitic acid, while other groups received 25 mmol/L glucose and 500μmoL/L palmitic acid and dimethyl sulfoxide （ DMSO ） , seperately. After 24 h treatment, the real time Polymerase Chain Reaction （PCR） was explored to detect the mRNA expression of receptor interaction protein 140（RIP140） , tumor necrosis factor-α （TNF-α） and interleukin- 6 （ IL-6）, while the concentration of TNF-α and IL-6 in the supernatant was detected by the enzyme-linked immunosorbent assay （ELISA）. Human umbilical vein endothelial cells （HUVECs） were incubated with the supernatant from the THP-1 in each group. The methodology of 3-（4, 5-Dimethylthiazol-2-yl）-2, 5- diphenyhetrazolium bromide （MTT） assay, flow cytometry and western-blot was applied to investigate the proliferation, apoptosis and the protein expression of phosphorylated extracellular regulated protein kinases （ERK） and B-cell lymphoma 2 （Bcl-2）. Results The mRNA expression of RIP140, TNF-α and IL-6 in the THP-1 cells and the concentration of TNF-α and IL-6 in the supernatant were significantly higher when treated with 25 mmol/L glucose and 500μmol/L palmitic acid, than those in the control group （t = 8.55, 9. 44, 9.73, 16.01, 19.22 with all P 〈 0. 05）. In the groups treated with 25 mmol/L glucose and 500 μmol/L palmitic acid, the pretreatment with curcumin significantly decreased the mRNA expression of RIP140, TNF-α and IL-6 in the THP-1 cells and the concentration of TNF-α and IL-6 in the supernatant （t = 3.59, 5.96, 5.59, 6. 95, 23.91 with all P 〈 O. 05）. At 24 h and 48 h treatment, compared with the control group, the supertant from the groups treated with 25 mmol/L glucose and 500 Ixmol/L palmitic acid significantly suppressed the proliferation of HUVECs （24 h ： 1.22 ± 0. 07 vs 1.85 ± 0. 14, t = 6. 58, P 〈 0. 05 ; 48 h ： 1.72 ± 0. 02 vs 2. 49 ± 0. 09, t = 10. 08, P 〈 0. 05 ）, while the proliferation of HUVECs could be improved by the pretreatment with 20 μmol/L curcumin （24 h ： 1.22 ± 0. 07 vs 1.72 ± 0. 11, t = 2. 13, P 〈 0.05 ; 48 h： 1.72 ± 0.02 vs 2. 33 ± 0.11, t = 6.92, P 〈 0.05）. Compared with the control group, the incubation with the supertant from the groups treated with 25 mmol/L glucose and 500 /xmol/L palmitic acid significantly increased the apoptosis rate and the level of phosphated ERK, while decreased the Bcl-2 protein expression （t =9.82, 9.69,4. 61, all P 〈0. 05）. Such effect could be reserved by the pretreatment with 20μmol/L curcumin （ t = 6. 35, 7. 17, 3.26, all P 〈 0. 05 ）. Conclusion Curcumin could protect the vascular endothelial cells from the dysfunction induced by inflammation.