摘要目的 观察姜黄素对高糖诱导的大鼠视网膜血管内皮细胞(RRVEC)凋亡的影响.方法 取对数生长期原代培养的第4代RRVEC进行实验,采用免疫荧光染色法鉴定培养的细胞,并对内皮细胞的特异性标志物血管性假性血友病因子(vWF)的表达进行检测.将培养的RRVEC分为对照组、高糖组和姜黄素干预组.对照组细胞正常培养.高糖组细胞经30 mmol/L的葡萄糖培养.姜黄素干预组细胞经30 mmol/L的葡萄糖培养24 h后,再加入30μmol/L姜黄素处理24 h.采用流式细胞仪检测各组细胞内活性氧(ROS)相对含量及细胞凋亡的变化情况.采用免疫细胞化学染色法检测各组细胞中核因子(NF)-κB p65的蛋白表达.采用蛋白免疫印迹法检测各组细胞中B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关x蛋白(Bax)的蛋白表达.结果 分离得到的RRVEC在1周后呈铺路石样单层贴壁生长.培养获得的细胞vWF表达呈强阳性.对照组、高糖组及姜黄素干预组RRVEC中ROS相对含量、细胞凋亡率比较,差异有统计学意义(F=40.957、325.137,P=0.000、0.000).与对照组比较,高糖组RRVEC中ROS相对含量、细胞凋亡率明显增加,差异有统计学意义(t=8.677、25.500,P=0.000、0.000).与高糖组比较,姜黄素干预组RRVEC中ROS相对含量、细胞凋亡率明显降低,差异有统计学意义(t=6.568、12.818,P=0.000、0.000).与对照组比较,高糖组RRVEC中NF-κB p65(t=8.322,P=0.000)、Bax(t=3.813,P=0.009)蛋白表达明显增加,Bcl-2蛋白表达(t=4.362,P=0.005)及Bcl-2/Bax比值(t=6.449,P=0.001)明显降低,差异有统计学意义(P<0.05).与高糖组比较,姜黄素干预组RRVEC中NF-κB p65、Bax(t=2.577、3.059,P=0.042、0.022)蛋白表达明显降低,Bcl-2/Bax比值(t=3.831,P=0.009)明显提高,差异有统计学意义(P<0.05).结论 姜黄素能抑制高糖诱导的RRVEC凋亡,其机制可能与增强Bcl-2表达、下调Bax表达从而抑制NF-κB信号通路有关.

abstractsObjective To observe the effects of Curcumin on the cellular apoptosis of rat retinal vascular endothelial cells (RRVEC) induced by high glucose. Methods Generation 4 cultured RRVEC were used in this experiment, and identified with anti-vWF factor antibody by immunochemistry and immunofluorescence. The RRVEC were divided into control group (5.5 mmol/L glucose), high glucose group (30 mmol/L glucose), and treatment group (30 mmol/L glucose+30 μmol/L Curcumin), respectively. Flow cytometry was used to measure the cellular reactive oxygen species (ROS) level and apoptosis. The expression intensity and location of nuclear factor (NF)-κB p65 in the cells of the three groups were detected by immunochemistory. The expression of Bcl-2 and Bax protein was detected by Western blot test. Results Immunostaining showed that RRVEC were positive for vWF factor. The flow cytometry showed that the cellular ROS level in treatment group was higher than that in the control group (t=8.677, P=0.000), but less than that in the high glucose group (t=40.957, P=0.000). Compared with the high glucose group, the cellular ROS level in the treatment group was decreased significantly (t=6.568, P=0.000). The cellular apoptosis were significantly different among the three groups (F=325.137, P=0.000). Compared with the high glucose group, the cellular apoptosis in the treatment group was decreased significantly (t=12.818, P=0.000). Immunochemistry showed that NF-κB p65 was expressed strongly in the cellular nuclei and cytoplasm in the high glucose group than that in the control group and the treatment group with the significant differences (t=8.322, P=0.000). Western blot results demonstrated that compared with the control group, the expression of Bcl-2 of RRVEC and Bcl-2/Bax ratio decreased (t=4.362, 6.449; P=0.005, 0.001) and Bax increased (t=3.813, P=0.009)in the high glucose group, with statistically significant differences. Compared with the high glucose group, the expression of NF-κB and Bax decreased (t=2.577, 3.059; P=0.042, 0.022) and Bcl-2/Bax ratio increased significantly (t=3.831, P=0.009) in the treatment group. Conclusion Curcumin could suppress the cellular apoptosis of RRVEC induced by high glucose. The mechanism of Curcumin protecting RRVEC may be via regulating NF-κB signal pathway.