姜黄素抑制脂多糖诱导视网膜色素上皮细胞炎症反应

Inhibitory effect of curcumin on inflammatory responses induced by LPS in human retinal pigment epithelial cells

摘要:

目的：探讨姜黄素（curcumin）对脂多糖（lipopolysaccharide，LPS）诱导视网膜色素上皮细胞（retinal pigment epithelial cells, RPE）炎症反应的抑制作用。方法：体外培养人RPE细胞株ARPE-19，CCK-8测定不同浓度姜黄素（5、10、20、30、40 ?滋mol/L）或不同浓度姜黄素和脂多糖（5 ?滋g/mL）刺激后细胞的活性，以此确定姜黄素的最适作用浓度。实时荧光定量（real-time poly－merase chain reaction, RT-PCR）检测白细胞介素（interleukin，IL）-8和IL-6的mRNA表达变化，酶联免疫吸附测定法（enzyme-linked immunosorbent assay，ELISA）测定IL-6和IL-8的蛋白表达水平。Western blot检测IκB-α、p-IκB-α蛋白表达变化。结果：CCK-8：不同浓度姜黄素对ARPE-19细胞活性无影响（P5 ?滋mol/L=0.998，P10 ?滋mol/L=1.000，P20 ?滋mol/L=0.124，P30 ?滋mol/L=0.092，P40 ?滋mol/L=0.078），预先作用不同浓度的姜黄素对细胞活性有保护作用，20 ?滋mol/L保护作用明显（P=0.041）。PCR：姜黄素+LPS组IL-8的mRNA相对表达量（4.965±3.863）比LPS组（106.331±37.314）明显降低（P=0.000），姜黄素+LPS组IL-6的mRNA相对表达量（0.992±0.374）比LPS组（2.175±0.560）明显降低（P=0.005）。ELISA：姜黄素+LPS组IL-8的蛋白表达量[（191.600±18.773） pg/mL]比LPS组[（589.025±38.880） pg/mL]明显降低（P=0.000），姜黄素+LPS组IL-6的蛋白表达量[（113.906±27.947） pg/mL]比LPS组[（243.380±38.001） pg/mL]明显降低（P=0.000）。Western blot：与LPS组相比较，姜黄素+LPS组的p-IκB-α蛋白的表达水平降低（P=0.001）。结论：姜黄素通过抑制NF-κB活性抑制了脂多糖诱导视网膜色素上皮细胞的炎症反应，保护视网膜色素上皮细胞。

Abstract:

Objective：To explore the inhibitory effect of curcumin on inflammatory responses induced by lipopolysaccharide （LPS） in retinal pigment epithelial（RPE） cells. Methods：ARPE-19 cells were cultured in vitro. Before being stimulated with or without LPS （5 μg/mL），ARPE-19 cells were pre-incubated with different concentrations of curcumin（5，10，20，30，40 ?滋mol/L）. The optimum con－centration of curcumin was detected by CCK-8 assay. The mRNA level of IL-6 and IL-8 was measured by real-time polymerase chain reaction（RT-PCR） and their protein expression was detected by enzyme-linked immunosorbent assay（ELISA）. Protein expres－sion levels of IκB-α and p-IκB-α were examined by Western blot. Results：The CCK-8 results showed that the activities of ARPE-19 cells were not affected by different concentrations of curcumin（P5 ?滋mol/L=0.998，P10 ?滋mol/L=1.000，P20 ?滋mol/L=0.124，P30 ?滋mol/L=0.092，P40 ?滋mol/L=0.078）. Curcumin was indicated to be able to protect ARPE-19 cells since pre-treatment with different concentrations of curcumin following by LPS administration. This protection resulted with a maximal protective effect at 20 μmol/L（P=0.041）. The RT-PCR results indicated that the relative mRNA level of IL-8 in curcumin+LPS group（4.965±3.863） was significantly lower than that in LPS group（106.331±37.314）（P=0.000） and the relative mRNA level of IL-6 in curcumin+LPS group（0.992±0.374） was significantly lower than that in LPS group（2.175±0.560）（P=0.005）. The ELISA results showed that protein expression of IL-8 in curcumin+LPS group[（191.600±18.773） pg/mL] was significantly lower than that in LPS group[（589.025±38.880） pg/mL]（P=0.000） and protein expression of IL-6 in curcumin+LPS group[（113.906±27.947） pg/mL] was significantly lower than that in LPS group[（243.380±38.001）pg/mL]（P=0.000）. Meanwhile，the Western blot results indicated that pre-treatment with curcumin inhibit－ed the phosphorylation of IκB-α（P=0.001） mediated by LPS stimulation. Conclusion：Curcumin inhibits the inflammatory responses induced by LPS via suppression NF-κB activity and protects retinal pigment epithelial cells.