氟尿嘧啶联合姜黄素纳米剂型抑制肝癌细胞的研究

Co-delivery of fluorouracil and curcumin by nano-layered double hydroxide for inhibition of hepatocellular carcinoma cells in vitro

摘要:

目的  探究纳米层状双氢氧化物（LDH）共载氟尿嘧啶（Fu）姜黄素（Cur）混合剂型对肝癌细胞的抑制作用。方法  通过共沉淀法合成纳米LDH混合剂型，并对其进行详细表征，如电镜（TEM）、X射线衍射（XRD）及动态光散射技术（DLS）检测，细胞计数试剂盒（CCK8）测定Fu组、Fu+Cur组及纳米剂型组对7721、Hep G2和LM3细胞的增殖抑制作用，同时凋亡试剂盒检测各组对7721细胞的凋亡效应，Western blot检测各组对细胞抗凋亡相关基因Bcl-2蛋白的表达及下游Caspase的活化情况。结果  合成的纳米LDH混合剂型具有较好的均一性，DLS检测提示其粒径约为400nm，XRD提示Fu及Cur通过水平方式插入LDH层间；CCK8结果提示，相对于单纯的Fu组、Fu+Cur组和LDH-Fu组，LDH-Fu-Cur组对LM3、Hep G2和7721细胞具有更强的增殖抑制作用（P <0.05）。相对于单纯的Fu组、Fu+Cur组和LDH-Fu组，LDH-Fu-Cur组对LM3、Hep G2和7721细胞具有更强的促凋亡效应作用（P <0.05），35μg/ml浓度的Fu纳米混合剂型组凋亡率高达（87.0±4.7）%，而单纯Fu仅为（23.0±2.3）%；Western blot检测结果显示，纳米混合剂型能够下调7721细胞Bcl-2水平。结论  纳米LDH混合剂型具有良好的抗肿瘤效应，其机制可能为下调肿瘤抗凋亡基因Bcl-2从而引起下游Caspase活化。

Abstract:

Objective To evaluate the effects of fluorouracil and curcumin co-loaded nano-layered double hydroxide (NLDH) on proliferation and apoptosis of hepatocellular carcinoma cell lines. Methods NLDH was synthesized by co-precipitation method. Detailed characterizations of NLDH such as its morphology, structure and particle size were performed by electron microscopy, X-ray diffraction (XRD) and dynamic light scattering technique (DLS). The function of pure fluorouracil, fluorouracil and curcumin, fluorouracil loaded NLDH (LDH-Fu), fluorouracil and curcumin co-loaded NLDH (LDH-Fu-Cur) on cell viability of 7721, LM3 and Hep G2 cell lines was measured by CCK-8 kit. Meanwhile, apoptosis kit was applied to detect apoptosis of 7721 cells in the aforementioned groups. In order to explore the mechanism of apoptosis induced by fluorouracil and curcumin co-delivered NLDH, Western blot was used to detect the expression levels of apoptotic proteins such as Bcl-2, Caspase-3 and Caspase-9. Results NLDH, with a size of 400 nm, presented a good uniformity under electron microscope. XRD results indicated that fluorouracil and curcumin were horizontally inserted into the interlayer of NLDH. CCK8 results indicated that LDH-Fu-Cur more efficiently inhibited the growth of 7721, LM3 and Hep G2 cell lines compared with pure fluorouracil, fluorouracil plus curcumin, and LDH-Fu (P < 0.05). Similarly, LDH-Fu-Cur was more effective in promoting apoptosis than pure fluorouracil, fluorouracil plus curcumin, and LDH-Fu, as was evidenced by the apoptosis rate of LDH-Fu-Cur which was (87.0 ± 4.7)% with the fluorouracil concentration of 35 μg/ml while the apoptosis rate of pure fluorouracil was only (23.0 ± 2.3)% with that concentration (P < 0.05). Western blot results showed that NLDH could significantly down-regulate the Bcl-2 gene expression of 7721 cells and induce cell apoptosis by activating Caspase-3 and Caspase-9. Conclusions NLDH presents as a good carrier for anti-cancer drugs such as fluorouracil and curcumin and exhibits a potent anti-tumor effect, which make it a promising candicate for cancer therapy.