灯盏花素对高糖联合低氧诱导人视网膜色素上皮细胞分泌VEGF的影响及其机制

Effect of scutellarin on VEGF secretion induced by high glucose and hypoxia in human retinal pigment epithelium cells and its mechanisms

目的通过观察1μmol/L灯盏花素对高糖、低氧,及高糖联合低氧环境下人视网膜色素上皮(RPE)细胞株D407细胞分泌VEGF的影响,PKCδ的转位激活变化,以及构建PKCδ的小分子干扰载体,探讨灯盏花素对RPE细胞在糖尿病环境下分泌VEGF的调节作用及机制。方法常规培养D407细胞,分为对照组(5.56 mmol/L葡萄糖)、高糖组(25.0mmol/L葡萄糖),低氧组(200.0μmol/L CoCl2),联合组(25.0 mmol/L葡萄糖+200μmol/L CoCl2),灯盏花素干预联合组(25.0 mmol/L葡萄糖加200μmol/L CoCl2加灯盏花素1.0μmol/L),ELISA检测VEGF浓度,Western blot检测PKCδ的转位激活水平;构建PKCδ小分子干扰载体转染D407细胞后,Western blot检测PKCδ表达水平,ELISA检测VEGF分泌水平。结果高糖组、低氧组、联合组分别与对照组比较,细胞分泌VEGF与PKCδ转位激活均增加(均P<0.05),联合组增加最明显(P<0.01);灯盏花素在1μmol/L的浓度下可以显著降低高糖联合低氧诱导的VEGF分泌;灯盏花素可以抑制高糖联合低氧引起的PKCδ转位激活;所构建的sh3-PKCδ干扰载体可以降低PKCδ表达水平,干扰PKCδ后联合组的VEGF分泌水平降低(P<0.05)。结论灯盏花素可以抑制高糖联合低氧诱导RPE细胞分泌VEGF,这可能与灯盏花素抑制了PKCδ转位激活有关,提示PKCδ可能是DR的一个药物靶点,灯盏花素具有作为DR治疗药物的前景。

Objective To investigate the effect of scutellarin on the VEGF secretion in retinal pigment epithelium （RPE） cells treated with high glucose, hypoxia, and high glucose ＋ hypoxia combination, and the mechanisms. Methods D407 cells were cultured under 5.56 mmol/L glucose （control group）, 25.0 mmol/L glucose （high glucose group）, 200.0 μmol/L CoCl2 （hypoxia group）, and 25.0 mmol/L glucose with 200.0 μmol/L CoCl2 （combination group）, with or without scutellarin（ 1.0 μmol/L）. VEGF secretion was measured by ELISA, PKCδ translocation were detected by Western blot. For short hairpin RNA vector construction, three short hairpin RNA vectors predicted to have the interference effect were designed and constructed. Western blot was used to se- lect the strongest interference shRNA after 24 h co-expression with PKCδ expression vector in 293T cells. After the shRNA vector transfection into RPE cells, PKCδ expression and VEGF secretion were measured. Results VEGF secretion of the RPE cells were increased under high glucose, hypoxia, and combination （all P〈0.05）, the highest increase was exhibited in combination group （P〈 0.01）. The scutellarin treatment could block that effect （P〈0.05）. PKCδ was activated and translocated to the membrane under all treatments, which could be inhibited by scutellarin, sh3-PKCδ could knockdown the PKCδ expression, and after the transfection, VEGF secretion was lowered （P〈0.05）. Conclusion Scutellarin could inhibit the increase of VEGF secretion induced by high glu- cose and hypoxia combination in RPE cells, and its inhibitory effect on PKCδ activation is in the mechanisms.