金丝桃素对体外培养的人视网膜色素上皮细胞蛋白激酶C活性的影响

Effect of hypericin on the activity of protein kinase C in cultured human retinal pigment epithelial cells in vitro

目的 研究金丝桃素 (hypericin)对体外培养的人视网膜色素上皮 (retinal pigment epi-thelium,RPE)细胞的蛋白激酶 C(protein kinase C,PKC)活性的影响。 方法 用含 0 .5～ 5 .0 μmol/L金丝桃素的 10 %血清培养液培养人 RPE细胞 ,在有或没有 PKC激活剂佛波醇 - 12 -豆蔻酰 - 13乙酸 (phorbol12 - myristate 13- acetate,PMA)预处理 RPE细胞的情况下 ,用 PKC检测试剂盒测定金丝桃素对细胞液PKC(cytosolic PKC,c- PKC)和细胞膜 PKC(membranous PKC,m- PKC)活性变化的影响。 结果 未经PMA处理的人 RPE细胞的 c- PKC和 m- PKC的原始活性分别为 (35 .34± 4 .1) pmol· min- 1· mg- 1和(6 2 .5 2± 8.8) pmol· min- 1 · mg- 1 。 PMA处理细胞后 ,c- PKC在 5 min内就迅速下降 ,2 0 min后下降缓慢 ,6 0 min降至处理前的 18% ,以后一直处于很低的水平。 m- PKC在 5 min后逐渐升高 ,至 4 0 min达到高峰以后下降 ,6 0 min后恢复至对照水平 ,10 h后降至对照组的 30 %以下。用 PMA处理 RPE细胞 4 8h后 ,c- PKC和 m- PKC都降低至不能测出。但若将金丝桃素与 PMA同时加入后 ,能抵消大部分 PMA对 PKC的下降调节。 结论 金丝桃素能阻止 RPE细胞的 PKC转位 ,使 PKC的活性发生变化 ,有可能促进 RPE细胞的凋亡 ,

Objective To investigate the effect of hypericin on the activity of protein kinase C (PKC) in cultured human retinal pigment epithelium (RPE) cells in vitro. Methods RPE cells were cultured in standard medium with 10% serum concentrations containing 0.5 to 5.0 μmol/L hypericin with or without preincubation of phorbol 12 myristate 13 acetate (PMA). The activities of cytosolic PKC (c PKC) and membranous PKC (m PKC) were assayed by PKC kit. Results The original activities of c PKC and m PKC of RPE cells were (35.34±4.10) pmol·min 1 ·mg 1 and (62.52±8.80) pmol·min 1 ·mg 1 . The activity of c PKC in RPE cells with PMA preincubation decreased rapidly in 5 minutes, with a subsequent slow decrease after 20 minutes and a decrease to 18% of the activity of c PKC in RPE cells without PMA preinubation after 60 minutes. While the activity of m PKC in RPE cells with PMA preincubation increased gradually after 5 minutes and reduced after reached the peak at 40 minutes, and then returned to baseline after 60 minutes, eventually decreased below 30% of the control group. When RPE cells were cultured with PMA for 48 hours, the activities of c PKC and m PKC were hardly detectable, while RPE cells were cultured with both PMA and hypericin, hypericin could counteract most of down regulation by PMA. Conclusion Hypericin may inhibit the translocation of PKC in RPE cells,change the activity of PKC, promote the apoptosis of RPE cells likely,and then prevent proliferative vitreoretinopathy.