光敏化姜黄素对人肝癌细胞HepG2的增殖影响

Effects of Photosensitized Curcumin on Proliferation in HepG2 Cells

目的:研究姜黄素的光敏化效应并探讨光敏化姜黄素对人肝癌细胞株HepG2细胞的增殖和凋亡的影响。方法:采用人肝癌细胞株HepG2进行研究。细胞用不同浓度的姜黄素(2.5μmol/L,5μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L,160μmol/L)孵育2 h后,用0.4 J/cm2剂量的紫光照射,光照的功率为10 mW。利用MTT法测定不同浓度的姜黄素对HepG2细胞的抑制作用,观察其紫光光敏化效应。在选择的姜黄素剂量下,分别用不同剂量的紫光照射,检测能量密度改变后光敏化姜黄素对细胞的杀伤作用。最终选定在20μmol/L浓度下,姜黄素用0.2 J/cm2紫光照射其对细胞的杀伤作用。利用Hoechst33342染色从形态学上在观察光敏化姜黄素对HepG2细胞的促凋亡效应,采用流式细胞仪定量检测光敏化姜黄素的凋亡率。结果 :姜黄素在紫光下具有光敏化效应,IC50从单独作用的92.49μmol/L降到27.06μmol/L,且这种效应在低浓度姜黄素低剂量的能量密度下更加明显。形态学和流式细胞仪检测到光敏化姜黄素的促凋亡效应,且凋亡率是同剂量姜黄素作用的3倍。结论:紫光照射可明显增强低剂量姜黄素对HepG2的细胞毒性,并可显著诱导细胞凋亡。

Objective： To investigate the photosensitive effect of curcumin,and explore the effect of photosensitized curcumin on proliferation,and apoptosis in human hepatocarcinoma cell line HepG2 cells,so as to provide theoretic evidence for the clinic studies of curcumin.Methods： In this study HepG2 cells were incubated with various curcumin（2μmol/L,5 μmol/L,5 μmol/L,10 μmol/L,20 μmol/L,40 μmol/L,80 μmol/L,160 μmol/L） concentrations and then illuminated with purple light radiations（purple：405 nm） at 0.4 J/cm2 to assay the inhibitional rates.Then the 20 μmol/L curcumin concentration was choosen to investigate the photosensitive effect of purple light radiation with different energy density（0.1 J/cm2,0.2 J/cm2,0.6 J/cm2,0.8 J/cm2）.Finally the group of 20 μmol/L curcumin with 0.2 J/cm2 radiation was selected to explore the pro-apoptosis effect of photosensitized curcumin.The HepG2 cells were stained with Hoechst33342 to detect the apoptosis cells.The cells stained with PI were analyzed by flow cytometry to assure the apoptosis rate.Results： Curcumin could be photosensitized on purple light radiation and the anti-proliferation effect of photosensitized curcumin was mainly associated with light doses and curcumin concentrations.IC50 of 0.4 J/cm2 energy density with purple light radiation was 27 μmol/L which was more lower than that of curcumin lonely（IC50 was 92 μmol/L）.In addition photosensitized curcumin could promote the anti-proliferation effect of low curcumin concentrations significantly and the effect was in dose-density-dependent way.The inhibition rate of HepG2 cells in 20 μmol/L curucmin with 0.2 J/cm2 energy density was almost 60%,but in the group of the same curcumin concentration the inhibition rate just 15%.And this advantage effect was suitable for lower curcumin doses and energy densities.Photosensitized curcumin could promote apoptosis in HepG2 cells which was observed with fluorescence microscopy.Treated with photosensitized curcumin 20 μmol/L and 0.2 J/cm2 energy density purple light radiation apoptosis cells could give bright blue fluorescence emission at 405 nm.Using Hoechst33342 staining we found typical morphologic features of apoptosis,characterized by marked chromatin condensation and fragmentation.The cells treated with purple LED light photosensitized curcumin showed more obvious alterations than that of curcumin-treated group.The damaged DNA stained with Hoechst33342 presented particular blue fluorescence.And the cell density was decreased clearly.An analysis of apoptosis rate by hypotonic PI DNA staining revealed that approximately 25% of photosensitized curcumin-treated cells had undergone apoptosis.Conclusions： Curcumin can be photosensitized on purple light radiation.Photosensitized curcumin can improve the anti-proliferation effect of curcumin at the same concentration especially for low curcumin concentration,and it also can induce apoptosis of HepG2 cells obviously with lower curcumin concentrations and lower energy density obviously.