自噬调节剂对莱菔硫烷诱导Caco-2细胞自噬效应及UGT1A1表达的影响

Effectsof autophagy modulator on autophagy and uridine 5＇-diphospho-glucuronosyltransferase 1A1 induced by sulforaphane

目的观察以人结肠腺癌细胞Caco-2为模型，探讨3-甲基腺嘌呤（3-MA）及雷帕霉素（Rapa）对莱菔硫烷（SFN）诱导细胞自噬及葡萄糖醛酸转移酶1A1（UGT1A1）的影响。方法采用Western印迹法测定微管相关蛋白1轻链3（LC3）和UGT1A1，采用免疫荧光法观察LC3的胞内分布及核因子E2P45相关因子2（Nrf2）的核内转位，实时荧光定量PCR法测定UGT1A1、人孕烷X受体（hPXR）的mRNA表达。结果SFN对LC3-Ⅱ蛋白的诱导呈浓度和时间依赖性，3-MA及Rapa可分别减弱或增强LC3-Ⅱ蛋白的表达；与对照组相比，Rapa、SFN及SFN＋Rapa可诱导UGTlAlmRNA的表达增强（分别为对照组的2．4倍、4．12倍、2．41倍，均P〈0．01），且诱导UGT1A1蛋白的表达增强；对照组未见明显的Nrf2核内荧光标记，而含有SFN的处理组中可见强烈的Nrf2核内荧光标记，且SFN＋Rapa更为明显；与对照组相比，Rapa及SFN＋Rapa的hPXRmRNA表达增强（分别为对照组的1．82倍、1．4倍，均P〈0．01）。结论3-MA和Rapa可分别减弱或增强SFN对Caco-2细胞自噬效应的诱导；Rapa可进一步增强SFN对UGTlAl的诱导；Rapa联合SFN可能通过同时激活Nrf2和hPXR增强了对UGTlAl的诱导。

Objective To explore the effects of3-methyladenine（3-MA）and rapamycin（Rapa）on autophagy and uridine 5 ＇-diphospho-glucuronosyltransferase 1A1 （ UGT1A1 ） induced by sulforaphane （ SFN ） in human colon cancer Caco-2 cells. Methods Western blot was used to detect the expression of microtubule-associated protein 1 light chain 3 （ LC3 ） and UGT1AI proteins. And immunocytochemistry was employed to observe the intracellular distribution of LC3 and nuclear localization of NF-E2-related factor 2 （Nrf2）. Quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）was employed to examine the mRNA expression of UGT1 A1 and human pregnane X receptor （hPXR）. Results After the treatment of SFN, the LC3-Ⅱ protein was induced in a dose and time-dependent manner. SFN-induced LC3- Ⅱ protein could be attenuated and enhanced by 3-MA and Rapa respectively. In comparison with the control group, UGT1A1 mRNA levels increased significantly after the treatment of Rapa, SFN or their combination （2. 4, 4. 12 and 2.41 folds respectively, all P 〈 0. 01 ）. And the combination of SFN and Rapa possessed the highest level. UGT1A1 protein band intensity was also enhanced in three groups. There was no obvious nuclear staining of Nrf2 in control group while intense nuclear fluorescent labeling of Nrf2 could be observed in the SFN-treated groups, especially the combination group of SFN and Rapa. The hPXR mRNA levels increased significantly in the Rapa and combination groups （1.82 and 1.4 folds respectively, both P 〈 0.01）. Conclusion The treatment of 3-MA or Rapa may attenuate or enhance SFN-indueed autophagy respectively. And Rapa also potentiates SFN-indueed UGT1A1 expression. The mechanism for the synergic effect of Rapa and SFN on UGT1 A1 induction may be a simultaneous activation of Nrf2 and hPXR signaling pathway.