CAJ | 학술논문

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E2 P45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UGT1A1 mRNA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hPXR mRNA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UGT1A1的诱导；Rapa联合SFN可能通过同时激活Nrf2和hPXR增强了对UGT1A1的诱导.
목적 관찰이인결장선암세포Caco-2위모형,탐토3-갑기선표령(3-MA)급뢰파매소(Rapa)대래복류완(SFN)유도세포자서급포도당철산전이매1A1 (UGT1A1)적영향.방법 채용Western인적법측정미관상관단백1경련3(LC3)화UGT1A1,채용면역형광법관찰LC3적포내분포급핵인자E2 P45상관인자2(Nrf2)적핵내전위,실시형광정량PCR법측정UGT1A1、인잉완X수체(hPXR)적mRNA표체.결과 SFN대LC3-Ⅱ단백적유도정농도화시간의뢰성,3-MA급Rapa가분별감약혹증강LC3-Ⅱ단백적표체；여대조조상비,Rapa、SFN급SFN+Rapa가유도UGT1A1 mRNA적표체증강(분별위대조조적2.4배、4.12배、2.41배,균P＜0.01),차유도UGT1A1단백적표체증강；대조조미견명현적Nrf2핵내형광표기,이함유SFN적처리조중가견강렬적Nrf2핵내형광표기,차SFN+Rapa경위명현；여대조조상비,Rapa급SFN+Rapa적hPXR mRNA표체증강(분별위대조조적1.82배、1.4배,균P＜0.01).결론 3-MA화Rapa가분별감약혹증강SFN대Caco-2세포자서효응적유도；Rapa가진일보증강SFN대UGT1A1적유도；Rapa연합SFN가능통과동시격활Nrf2화hPXR증강료대UGT1A1적유도.
Objective To explore the effects of 3-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 UGT1A1 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 UGT1A1 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 Nrt2 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-induced autophagy respectively.And Rapa also potentiates SFN-induced UGT1Al expression.The mechanism for the synergic effect of Rapa and SFN on UGT1A1 induction may be a simultaneous activation of Nrf2 and hPXR signaling pathway.