姜黄素对IL-1β诱导的兔RPE细胞中核因子-κB相关炎性因子表达的抑制作用

Inhibition of curcumin on the expression of IL-1β-induced nuclear factor-κB-dependent inflammatory gene in rabbit RPE cells

背景白细胞介素-1β（IL-1β）是增生性玻璃体视网膜病变（PVR）早期释放的重要炎性因子，研究证实姜黄素能抑制IL-1β诱导的兔视网膜色素上皮（RPE）细胞的增生，但其是否能够对PVR发挥抗炎作用尚不清楚。目的观察姜黄素对IL-1β诱导的兔RPE细胞移行的影响，探讨姜黄素对IL-1β诱导的RPE细胞中炎性因子表达的影响。方法采用对数生长期原代培养的第4代兔RPE细胞进行实验，在无血清DMEM培养基中分别添加0、0．1、1．0和10．0μg／L的IL-1β作用于细胞24h，分别采用Western blot和逆转录PCR法检测细胞中环氧合酶-2（COX-2）蛋白和mRNA的表达以筛选IL-1β最佳质量浓度。将培养的RPE细胞分为IL-1β组和姜黄素＋IL-1β组，分别在无血清培养基中添加1．0μg／L IL-1β和1．0μg／L IL-1β联合10μg／ml姜黄素作用于细胞24、48和72h，仅用无血清培养基培养的细胞作为对照组。培养的细胞行苏木精-伊红染色，于光学显微镜下计算进入损伤区的细胞数，比较各组细胞的移行能力；分别采用Western blot法和逆转录PCR法检测各组RPE细胞中COX-2蛋白及其mRNA的相对表达量，采用Westernblot法检测并比较各组细胞中核因子-κB p65（NF—κB p65）蛋白和核因子κB抑制蛋白-α（IκB—α）的相对表达量；采用免疫化学染色法检测NF—κB p65、IκB-α和COX-2在各组RPE细胞中的表达和定位。结果初分离的兔RPE细胞呈球形，细胞中可见大量黑色素颗粒；第4代细胞色素颗粒明显减少，接近融合的细胞形态为长梭形，呈拉网状分布。免疫细胞化学染色法结果显示，细胞角蛋白（AE1／AE3）在细胞质呈阳性表达。对照组在培养24、48和72h移行的细胞数分别为（31．93±1．21）、（36．27±2．50）和（38．33±2．40）个，IL-1β组分别为（45．73±2．30）、（71．13±1．92）和（80．60±1．71）个，而姜黄素＋IL-1β组分别为（13．13±2．20）、（14．93±1．10）和（12．60±1．51）个，各时间点IL-1β组细胞移行细胞数均明显高于对照组，而姜黄素＋IL-1β组移行细胞数均明显低于对照组和IL-1β组，差异均有统计学意义（均P〈0.05）。IL-1β质量浓度为1．0μg/L时，RPE细胞中COX-2蛋白及其mRNA的相对表达量达峰，以1．0μg／L IL-1β的剂量进行后续实验。细胞培养后24、48和72h，姜黄素＋IL-1β组细胞中COX-2蛋白及其mRNA的相对表达量均明显低于IL-1β组，差异均有统计学意义（均P〈0.05）。IL-1β作用于细胞后48h，细胞中NF—κB p65蛋白的相对表达量最高，与IL-1β组相比，各时间点姜黄素＋IL-1β组细胞中NF—κB p65蛋白相对表达量降低，差异均有统计学意义（均P〈0．05）。IL-1β组药物作用于细胞后48h细胞中IκB—α降至最低，各时间点姜黄素＋IL-1β组细胞中IκB-α值均明显高于IL-1β组，差异均有统计学意义（均P〈0.05）。免疫细胞染色结果显示，IL-1β组RPE细胞的细胞核及细胞质中NF—κB p65呈强阳性表达，对照组表达较弱，姜黄素＋IL-1β组细胞中NF—κB p65的表达强度较IL-1β组明显降低；与对照组相比，IL-1β组细胞的细胞质中IκB-α表达强度明显减弱，而COX-2表达明显增强；与IL-1β组比较，姜黄素＋IL-1β组细胞中IκB-α表达明显增强，而COX-2表达明显减弱。结论姜黄素可抑制IL-1β引起的兔RPE细胞的移行，IL-1β通过激活NF—κB信号通路刺激细胞中COX-2的表达，姜黄素可通过阻断这一途径而抑制炎性因子的表达，从而发挥抗炎作用。

Background Interleukin-1β （IL-1β） is an important inflammation-related factor in the initial stage of proliferative vitreoretinopathy （PVR）. The previous research showed that curcumin can inhibit IL-1β-induced proliferation of rabbit retinal pigment epithelium （RPE） cells, but the anti-inflammatory mechanism and effect of curcumin are still undefined. Objective This study was to observe the migration of IL-1β-induced rabbit RPE cells, and evaluate the function and mechanism of inhibition of curcumin on IL-1β-induced inflammation of RPE cells. Methods Cultured rabbit RPE cells of generation 4 were used in this experiment. The cells were cultured in serum-free DMEM and 0,0. 1,1.0 and 10. 0 /.zg/L IL-1β were separately added in the medium for 24 hours. The expressions of cyelooxygenase-2 （COX-2） protein and mRNA in the cells were detected by Western blot and reverse transcription PCR to determine the optimal concentration of IL-1β. The cells were divided into IL-1β group and cureumin＋IL-1β group,and 1.0 μg/L IL-1 or 1.0 μg/L IL-1β combined with 10 μg/ml cureumin was respectively added into the medium for 24,48 and 72 hours. The cells cultured by only serum-free medium served as the control group. Hematoxylin and eosin staining was conducted for the cells to count the number of cells migrating into the injured area under the optical microscope. The relative expression levels of COX-2 protein and mRNA in the cells were detected by Western blot and reverse transcription PCR, and the relative expression levels of nuclear factor （NF） - κBp65 and inhibitor of NF-κB-α （IκB-α） protein were also detected by Western blot assay. The expression intensity and location of NF-κBp65 ,IκB-α and COX-2 in the cells were detected by immunochemistry. Results RPE cells just isolated from the rabbit eyes were in round shape and abundant in melanin. The melanin significantly decreased in the fourth generations of RPE cells. The shape of cells became long and narrow, and net shaped distribution. Immunoehemistry demonstrated the strong positive response of RPE cells for keratin （AE1/AE3）. There were （31.93±1.21）,（36.27±2.50） and （38.33±2.40） migratory cells in the control group after 24, 48 and 72 hours respectively. The number of migratory cells increased to 45.73 ±2.30,71. 13 ±1.92 and 80.60±1.71 in the IL-1β group,but obviously decreased to 13. 13±2.20,14.93 ±1. 10 and 12.60±1.51 in the eureumin ＋ IL-1β group. A Significant increase in the migrating clee number was found in the IL-1β group compared with the control group and the cureumin＋IL-1β group in various time points （ all at P〈0. 05）. The relative expression levels of COX-2 protein and mRNA peaked in the 1.0 ixg/L IL-1β group, so 1.0 ~g/L of IL-1β was determined as the optimal concentration in the experiment. In 24,48 and 72 hours after culture,the expression levels of COX-2 protein and mRNA in the cells were significantly lower in the cureumin ＋ IL-1β group than those in the control group （ all at P〈0. 05）. The relative expression level reached peak in NF-κBp65 protein and lowed bottom in IκB-α proteins at 48 hours after cultured in the IL-1β group, and the reverse trend was seen in the eurcumin＋IL-1β group,with the significant differences between the two groups （ both at P〈0.05）. Immunochemistry showed that NF-κBp65 was expressed strongly in the cell nuclei and cytoplasm in the IL-1β group and presented the weaker expression in the control group and the eureumin＋IL-1β group. Compared with the control group,the expression was weaker in IκB-α and stronger in COX-2 in the IL-1β group. In addition,the expression of IκB-α was enhanced and that of COX-2 was attenuated in the cureumin＋IL-1β group in comparison with the IL-1β group. Conclusions Cureumin inhibits the movement of rabbit RPE cells induced by IL-1β. IL-1β up-regulates the expression of COX-2 by activating NF-κB signal pathway, and curcumin plays an anti-inflammatory role by blocking this pathway.