姜黄素抑制棕榈酸诱导的巨噬细胞炎症反应的机制研究

Inhibitory role of curcumin in palmitic acid-induced inflammatory response of macrophages

目的探讨姜黄素抑制棕榈酸诱导的巨噬细胞炎症反应的作用机制。方法以0、250、500μmol／L棕榈酸干预小鼠巨噬细胞RAW264．724h，观察细胞形态，逆转录一聚合酶链反应（RT-PCR）检测细胞中受体相互作用蛋白140（RIPl40）、肿瘤坏死因子a（TNF-a）、白细胞介素-6（IL-6）mRNA水平，酶联免疫吸附实验（ELISA）检测上清中TNF-a、IL-6水平；噻唑蓝法（MTr）确定姜黄素作用RAW264．7细胞的最佳时间和浓度；细胞分为姜黄素组和对照组，分别给予20μmol／L姜黄素和二甲基亚砜（DMSO）预处理1h后，均给予500μmol／L棕榈酸作用24h，观察细胞形态并检测细胞中RIPl40、TNF-a、IL-6mRNA水平和上清中TNF-a、IL-6浓度。采用单因素方差分析和t检验对研究数据进行统计检验。结果500μmol／L棕榈酸组RIPl40mRNA表达较0μmol／L组显著升高（3．40±0．51比1．01±0．21，t=7．436，P〈0．01）；0、250、500μmol／L棕榈酸组TNF-amRNA（1．00±0．03、1．79±0．12、2．16±0．13）和蛋白[（197±25）、（371±10）、（485±17）ng／L]、IL-6mRNA（1．00±0．51、2．55±0．15、2．59±0．17）和蛋白[（953±66）、（1081±36）、（1182±18）ng／L]与棕榈酸剂量明显相关（F=99．308、187．049、152．958、26．594，均P〈0．01）；棕榈酸处理后，细胞变圆、皱缩甚至崩解；姜黄素作用RAW264．7细胞的最佳时间为1h，最佳浓度为20ixmol／L；姜黄素组与对照组相比，RIPl40mRNA（1．00±0．05比0．63±0．01，t=一13．79，P〈0．01）、TNF-amRNA（0．64±0．11比1．00±0．07，t=一4．532，P〈0．05）和蛋白[（322±12）比（485±17）ng／L，t=一13．577，P〈0．01]、IL-6mRNA（0．57±0．05比1．00±0．02，t=一14．167，P〈0．01）和蛋白[（241±47）比（1182±18）ng／L，t=一44．810，P〈0．01]均显著降低，而细胞形态正常，细胞问仍有连接融合。结论RIPl40可能参与姜黄素抑制棕榈酸诱导的炎症反应过程。

Objective To investigate the inhibitory role of cureumin in palmitic acid-induced inflammatory response of macrophages. Methods Murine macrophage cell line RAW264. 7 were treated with 0, 250, 500 μmol/L palmitic acid for 24 h, then the morphology of cells were observed, receptor interacting proteinl40 （RIP140） , tumor necrosis factor eL （TNF-cx） and interleukin 6 （ IL-6 ） mRNA levels were determined by reverse transcription-polymerase chain reaction （RT-PCR） and levels of TNF-a and IL- 6 in supernatant were tested by enzyme- linked immunosorbent assay （ ELISA ）. The optimum time and concentration of curcumin on RAW264. 7 cells were determined by MTT assay. Cells were separated into two groups and treated with 20 μmol/L curcumin and the same volume of dimethyl sulfoxide （DMSO） for 1 h, respectively; then both groups were treated with 500 μmo[/L palmitic acid. After 24 h, the morphology of cells were observed, and the mRNA abundance of RIP140, TNF-a and IL-6 and protein levels of TNF-a, IL-6 in supernatant were detected. The research data was assessed by one-way ANOWA and t test. ResultsAfter intervening with 500 μmol/L palmitic acid for 24 h, RIP140 mRNA level was higher than that in 0 μmol/L palmitie acid group （3.40 ± 0. 51 vs 1.01 ±0. 21, t = 7. 436, P 〈 0. 01 ）. After intervening with 0, 250, 500 p.mol/L palmitic acid for 24 h, TNF-a mRNA levels （1.00 ±0. 03, 1.79 ±0. 12, 2. 16 ＋0. 13） and protein levels （ （ 197 ±25）, （371 ±10）, （485 ± 17） ng/L）, IL-6 mRNA levels （ 1.00 ± 0. 51, 2. 55 ± 0. 15, 2. 59± 0. 17 ） and protein levels （ （953 ± 66）, （ 1081 ± 36 ）, （ 1182 ± 18 ） ng/L） were increased significantly in a dose-dependent manner（ F = 99. 308, 187. 049, 152. 958, 26. 594, respectively, all P 〈 0. 01 ） ; After the treatment of palmitic acid, cells shrank, rounded and even disintegrated; The optimum time of curcumin treating on RAW264. 7 cells was 1 h, and the optimum concentration was 20 μmol/L. After treated with 20 p.moL/L cummin, compared to control group, RIP140 mRNA abundance （0. 63 ±0. 01 vs 1.00 ±0. 05, t = - 13.79, P 〈0. 01）, TNF-a mRNA abundance （0. 64±0. 11 vs 1.00±0.07, t = -4.532, P〈0.05） and protein concentration （（322 ± 12） vs （485 ± 17） ng/L, t = - 13. 577, P 〈0. 01）, IL-6 mRNA abundance （0. 57 ±0. 05 vs 1.00 ±0. 02, t = - 14. 167, P 〈0. 01 ） and protein concentration （ （241 ±47） vs （1182 ± 18） ng/L, t = -44. 810, P 〈0. 01 ） were significantly lower, the morphology of cells was not impaired and cell junction still existed. Conclusions Curcumin may redress nalmitic acid-induced inflammation bv decreasing RIP140 exnression.