姜黄素对兔视网膜色素上皮细胞DNA含量、线粒体膜电位及胞质内钙离子的影响

目的 探讨姜黄素对兔视网膜色素上皮细胞(RPE)DNA含量(凋亡率)、线粒体膜电位(Aψm)和胞质内钙离子(Ca2+)的影响.方法 实验研究.选取体外培养的第4代RPE细胞进行试验,分为姜黄素组和空白对照组(10% FBS-DMEM含二甲基业砜0.5‰),姜黄素组设10、15、20 mg/L 3个质量浓度.噻唑蓝(MTT)法检测姜黄素10、15、20 mg/L 3个质量浓度分别作用24、48、72及96 h后对体外培养的RPE细胞增殖72及96 h后对体外培养的RPE细胞增殖的抑制率.相关回归分析计算24、48、72及96 h的半数抑制率(IC50)剂量.流式细胞仪检测姜黄素15 mg/L作用8、24、48及72 h,后RPE细胞DNA含量(凋亡率)、△Ψm和胞质内Ca2+的变化.对抑制率分别在同浓度不同时间组间、同时间不同浓度组间进行方差分析(多个样本问两两比较);采用相关回归分析计算姜黄素各时间段半数抑制率剂最;对凋亡率、线粒体△Ψm、细胞质内Ca2+等对照组与姜黄素组间进行两独市样本t检验;对凋亡率、线粒体△Ψm、细胞质内Ca2+等姜黄素不同时间组间进行方差分析(多个样本间两两比较).结果姜黄素对RPE细胞有明显抑制作用,呈时间和剂量依赖件.姜黄素在24、48、72及96 h对RPE细胞的IC50分别是29.31、17.50、13.24及10.99 mg/L.姜黄素作用8、24、48、72 h后,RPE细胞质内Ca2+浓度明显升高与空白对照组相比,差异有统计学意义(t=7.50,10.61,20.74,21.14;P＜0.01)△Ψm明显下降,与对照组相比均有统计学意义(t=7.50,11.74,14.91,15.29;P＜0.01).姜黄素作用8 h DNA含量未见明显下降,24、48及72 h DNA含量明显下降(即凋亡率升高),与空白对照组相比均有统计学意义(t=10.00,14.68,13.68;P＜0.01).结论姜黄素可以使RPE细胞质内Ca2+浓度升高,△Ψm下降,进而使DNA含量明显下降,诱导RPE细胞凋亡.姜黄素可以有效抑制RPE细胞增殖,有望成为防治增生性玻璃体视网膜病变的理想天然药物.(中华眼科杂志.2009,45:210-215)

The effect of curcumin on DNA content, mitochondrial transmembrane potential and calcium of rabbit cultured retinal pigment epithelial cells

Objective To investigate the Change of DNA content(apoptosis rate),mitochondrial transmembrane potential(△Ψm)and calcium(Ca2+)of rabbit retinal pigment epithelial(RPE)cells cultured with curcumin.Methods It was an experimental study.The RPE cells were dissociated from rabbit eyes and cuhured.The RPE cells in the 4th passage were divided into 2 groups:curcumin group and control group(10% FBS-EMI)M contains 0.05% dimethyl sulfoxide).The curcumin group contained 3 mass concentration:10 mg/L,15 mg/L and 20 mg/L.The MTT assay was used to evaluate the inhibition effect of RPE cells cultured with curcumin after 24 h,48 h,72 h and 96 h respectively.The IC50 value in 24 h,48 h,72 h and 96 h were gotten by Linear Regression.Flow cytometry was performed to detect the change of DNA content(apoptosis rate),△Ψm and Ca2+ of RPE cells cultured with curcomin(15 mg/L)after 8 h,24 h,48 h and 72 h respectively.Results RPE cells were significantly inhibited by curcumin in a dose dependent and time dependent manner.The IC50 value of curcumin at 24 h,48 h,72 h and 96 h was 29.31 mg/L,17.50 mg/L,13.24 mg/L and 10.99 mg/L respectively.Ca22+ was significantly increased at 8 h,24 h,48 h and 72 h after cultured with curcumin(15 mg/L)than that of the control group respectively(t=7.50,10.61,20.74,21.14,P＜0.01),and △Ψm was significantly decreased at 8 h,24 h,48 h and 72 h after cultured with curcumin(15 mg/L)than that of the control group respectively(t=7.50,11.74,14.91,15.29,P＜0.01).There was no change of DNA content in RPE cells at 8h after cultured with curcumin(15 mg/L),but significantly lower than that of the control group at 24 h,48 h and 72 h respectively(t=10.00,14.68,13.68,P＜0.01).Conclusion The apeptosis of RPE cells induced by curcumin is cansed by increase of Ca2+ and decrease of △Ψsm that causes decrease of DNA content.The RPE cells are significantly inhibited by curcumin,which may become a potential drug to prevent and treat proliferative vitreoretinopathy.(Chin J Ophthalmol,2009,45:210-215)