[1]闫文帝,李珍玲,刘特思,等.PP242诱导多囊肾大鼠胆管上皮细胞自噬和凋亡的机制研究[J].中国药理学通报,2018,(09):1218-1225.[doi:10.3969/j.issn.1001-1978.2018.09.008]
 YAN Wen-di,LI Zhen-ling,LIU Te-si,et al.Mechanism of PP242 inducing autophagy and apoptosis in cholangiocytes of polycystic kidney rats[J].Chinese Pharmacological Bulletin,2018,(09):1218-1225.[doi:10.3969/j.issn.1001-1978.2018.09.008]
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PP242诱导多囊肾大鼠胆管上皮细胞自噬和凋亡的机制研究()
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《中国药理学通报》[ISSN:/CN:]

卷:
期数:
2018年09期
页码:
1218-1225
栏目:
论著
出版日期:
2018-09-26

文章信息/Info

Title:
Mechanism of PP242 inducing autophagy and apoptosis in cholangiocytes of polycystic kidney rats
文章编号:
1001-1978(2018)09-1218-08
作者:
闫文帝1李珍玲1刘特思1李玉姬1原田宪一3任香善12
延边大学1.肿瘤研究中心、2.医学院病理学教研室,吉林 延边 133002; 3.金泽大学医学部病理学教研室,日本 金泽 9208640
Author(s):
YAN Wen-di1 LI Zhen-ling1 LIU Te-si1 LI Yu-ji1 HARADA Kenichi3 REN Xiang-shan12
1.Cancer Research Center; 2.Dept of Pathology, Medical College, Yanbian University, Yanji, Jilin 133002, China; 3.Dept of Pathology, School of Medical Science, Kanazawa University, Knazawa 9208640, Japan
关键词:
自噬 凋亡 PI3K/Akt/mTOR信号通路 多囊肾大鼠 PP242 Caroli病
Keywords:
autophagy apoptosis PI3K/Akt/mTOR axis polycystic kidey rats PP242 Caroli's disease
分类号:
R-332; R322.47; R329.24; R329.25; R575.7
DOI:
10.3969/j.issn.1001-1978.2018.09.008
文献标志码:
A
摘要:
目的 探讨mTORC1/2抑制剂PP242诱导多囊肾(polycystic kidney,PCK)大鼠胆管上皮细胞凋亡和自噬、抑制细胞增殖及胆管囊性扩张的分子机制。方法 免疫组织化学染色法检测PCK大鼠胆管上皮细胞中p-mTOR和p-Akt的表达; WST-1比色法检测雷帕霉素和PP242对胆管上皮细胞增殖活性的抑制作用,以及LC3、Beclin-1基因沉默和自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)对细胞增殖的影响; 蛋白免疫印迹法检测PP242对PI3K/Akt信号通路相关蛋白、自噬标识蛋白LC3和Beclin-1、cleaved caspase-3表达的影响; 流式细胞术和ELISA法检测PP242对细胞凋亡的影响; 免疫荧光染色法检测LC3在细胞质中的表达情况; 3D细胞培养检测Rictor基因沉默和雷帕霉素联用及雷帕霉素单独应用时对大鼠胆管上皮细胞成球能力的影响。结果 p-mTOR和p-Akt在PCK大鼠胆管上皮细胞中呈过表达,PP242比雷帕霉素抑制胆管上皮细胞增殖效果更为明显,且呈浓度和时间依赖性改变(P<0.05); PP242明显抑制PI3K/Akt信号通路相关蛋白的表达; PP242可诱导细胞凋亡和自噬,上调cleaved caspase-3、Beclin-1和LC3-II/LC3-I比值。Rictor基因沉默和雷帕霉素联用比雷帕霉素单独应用对大鼠胆管上皮细胞的抑制作用更为明显; LC3、Beclin-1基因沉默和3-MA均可明显减弱PP242对细胞增殖的抑制作用(P<0.05)。结论 PP242可通过PI3K/Akt/mTOR信号通路抑制PCK大鼠胆管上皮细胞的增殖,其机制与细胞凋亡和自噬密切相关。
Abstract:
Aim To investigate the molecular mechanism of mTORC1/2 inhibitor PP242, which inhibiting cholangiocyte cell preliferation and cystic diliatation via inducing apoptosis and autophagy in the polycystic kidney(PCK)rats. Methods The expression of p-mTOR and p-Akt in the bile duct epithelial cells was examined by immunohistochemistry. The inhibiting effect of rapamycin and PP242 on cell proliferation activity on bile duct epithelial cells, the effect of gene silence on LC3, Beclin-1 and the effect of the authophagy-specific inhibitor 3-methyladenine(3-MA)on cell proliferation were respectively analyzed by WST-1 assay. The expression of PI3K/Akt signaling pathway related proteins, autophagy-related proteins LC3, Beclin-1 and clevead caspase-3, which were treated by PP242 were determined by Western blot. The effect of PP242 on apoptosis was detected by Annexin V/PI double staining and ELISA. The expression of LC3 in cytoplasm was detected by immunofluorescence. The ability of rat bile duct epithelial cells spheroid formation was detected by 3D cell culture method, and the cells were treated by single applied with rapamycin and applied rapamycin combined with Rictor gene silencing respectively. Results The protein levels of p-Akt and p-mTOR markedly increased in the bile duct epithelium of PCK rats. PP242 inhibited the proliferation of bile duct epithelial cells more effectively than rapamycin and showed a dose- and time-dependent manner(P<0.05). PP242 significantly reduced the levels of PI3K/Akt signaling pathway-related proteins in PCK rat cholangiocytes. PP242 induced apoptosis and autophagy, up-regulated the levels of cleaved caspase-3, Beclin-1 and increased the ratio of LC3-II/LC3-I. The combination of Rictor gene silencing and rapamycin was more effective than rapamycin alone in inhibiting cholangiocytes in PCK rats. The inhibitory effect of PP242 on the cell viability was significantly weakened by treatment with 3-MA and knockdown of LC3 and Beclin-1(P<0.05). Conclusions PP242 inhibits the proliferation of PCK rat cholangiocytes through PI3K/Akt/mTOR signaling pathway, and the mechanism is closely related with autophagy and apoptosis.

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备注/Memo

备注/Memo:
收稿日期:2018-05-15,修回日期:2018-06-10
作者简介:闫文帝(1991-),女,硕士生,研究方向:肿瘤分子病理学,E-mail:304783592@qq.com;
任香善(1976-),女,博士,副教授,硕士生导师,研究方向:肿瘤分子病理学,通讯作者,E-mail:renxsh@ybu.edu.cn
更新日期/Last Update: 2018-08-26