H2O2对体外培养视网膜色素上皮细胞中BMP-6及其相关受体的影响

Effect of H2O2 on BMP-6 and its related receptors in cultured retinal pigment epithelial cells

目的观察氧化应激对视网膜色素上皮(retinal pigment epithelium,RPE)细胞的损伤作用以及对骨形态发生蛋白(bone morphogenetic protein,BMP)-6及其相关受体的影响。方法RPE细胞随机分为对照组正常培养的RPE细胞;H2O2组加入200μmol·L-1 H2O2处理的RPE细胞;实验组加入200μmol·L-1 H2O2和0.1 ng·L-1 BMP-6的RPE细胞。采用活性氧(reactive oxygen species,ROS)检测试剂盒运用流式细胞仪检测对照组与H2O2组RPE细胞内ROS的变化,运用PCR及Western blot法检查对照组与H2O2组BMP-6基因及蛋白水平的变化;TUNEL染色法观察三组细胞凋亡的变化,并运用PCR及Western blot法观察三组BMP受体及其共受体尤文素(Hemojuvelin,HJV)的变化。结果H2O2组ROS水平(99.25±0.28)%较对照组(70.55±7.71)%明显升高,差异有统计学意义(P<0.05);H2O2组相对于对照组BMP-6 mRNA水平及BMP-6蛋白水平均降低,差异均有统计学意义(均为P<0.05);H2O2组的TUNEL染色阳性细胞数显著高于对照组(P<0.05),而实验组TUNEL染色阳性细胞数明显减少,与H2O2组相比,差异有统计学意义(P<0.05);与对照组相比,H2O2组BMP三种相关受体的BMPR1A、BMPR1B及HJV的表达水平显著降低,而实验组三种受体表达水平较H2O2组显著升高,差异均有统计学意义(均为P<0.05)。结论H2O2导致RPE细胞ROS增加,氧化应激可在基因及蛋白水平抑制BMP-6的表达;BMP-6可减轻氧化应激引起的细胞凋亡,且可能是通过激活其相关受体起作用的,这为探索年龄相关性黄斑变性的发病机制提供了一定的理论基础。

Objective To observe the effects of oxidative stress on damage of retinal pigment epithelium(RPE)cells and on bone morphogenetic protein 6(BMP-6)and its related receptors.Methods RPE cells were randomly divided into control groups(normal cultured RPE cell group),H2O2 group(RPE cells treated with 200μmol·L-1 H2O2)and experimental group(RPE cells treated with 200μmol·L-1 H2O2 and 0.1 ng·L-1 BMP-6).Reactive oxygen species(ROS)detection kit was used to detect ROS changes of RPE cells in control group and H2O2 group by flow cytometry.The changes of BMP-6 gene and protein levels were detected by PCR and western blot.TUNEL staining was used to observe the changes of apoptosis in three groups.The changes of BMPR and hemojuvelin(HJV)receptors were observed by PCR and western blot.Results The ROS level in H2O2 group was(99.25±0.28)%,which was significantly higher than(70.55±7.71)%in normal control group,and the difference was statistically significant(P<0.05).The level of BMP-6 mRNA and protein in H2O2 group significantly decreased compared with those in normal control group,and the differences were statistically significant(both P<0.05).The number of TUNEL positive cells in H2O2 group was significantly higher than that in control group(P<0.05),while the number of TUNEL positive cells in experimental group significantly reduced,and the difference was statistically significant compared with H2O2 group(P<0.05).Compared with the control group,the expression levels of BMPRIA、BMPRIB and HJV of the three BMP related receptors in H2O2 group were significantly lower,while the expression levels of the three receptors in experimental group were significantly higher than that in H2O2 group,and the differences were statistically significant(both P<0.05).Conclusion H2O2 promotes the increase of ROS in cells,and oxidative stress can inhibit the expression of BMP-6 at gene and protein level.BMP-6 can alleviate apoptosis induced by oxidative stress,and may play a role in anti-oxidative stress injury by activating its related receptors,which provides a certain theoretical basis for exploring the pathogenesis of age-related macular degeneration.