中华皮肤科杂志 ›› 2023, Vol. 56 ›› Issue (10): 934-939.doi: 10.35541/cjd.20230135

• 论著 • 上一篇    下一篇

雷公藤甲素通过肌醇需求酶1/c-Jun氨基端激酶信号通路诱导人黑素瘤A375细胞凋亡

孙冬红1    刘国豪2    侍姝彤1    包军1    穆耕林1    陶玥1   

  1. 1南京鼓楼医院皮肤性病科，南京  210008；2京都大学大学院医学研究科医化学分野，日本京都  606-8501
  • 收稿日期:2023-03-09 修回日期:2023-08-21 发布日期:2023-10-08
  • 通讯作者: 陶玥；穆耕林 E-mail:taoyue18@126.com; glyydwsj@163.com
  • 基金资助:
    江苏省自然科学基金（BK20150111）

Triptolide induces apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1/c-Jun N-terminal kinase signaling pathway

Sun Donghong1, Liu Guohao2, Shi Shutong1, Bao Jun1, Mu Genglin1, Tao Yue1   

  1. 1Department of Dermatology and Venereology, Affiliated Drum Tower Hospital, Medical School of Nanjing University, Nanjing 210008, China; 2Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
  • Received:2023-03-09 Revised:2023-08-21 Published:2023-10-08
  • Contact: Tao Yue; Mu Genglin E-mail:taoyue18@126.com; glyydwsj@163.com
  • Supported by:
    Natural Science Foundation of Jiangsu Province of China（BK20150111）

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摘要： 【摘要】 目的 探讨雷公藤甲素（TP）通过肌醇需求酶1（IRE1）/c-Jun氨基端激酶（JNK）信号通路对人黑素瘤A375细胞凋亡的影响及其可能的作用机制。方法 采用不同浓度［0（实验对照组）、50、100、200 nmol/L］TP处理A375细胞，同时设置空白对照组（仅有DMEM高糖培养基，不含细胞），采用噻唑蓝（MTT）比色法测定处理24、48和72 h时细胞活性，流式细胞仪检测处理24 h时细胞凋亡率，实时荧光定量PCR（RT-qPCR）和Western印迹法检测IRE1、JNK和c-Jun mRNA及蛋白表达水平。经JNK抑制剂SP600125预处理A375细胞72 h后，再用100 nmol/L TP处理24 h，即SP600125 + 100 nmol/L TP组，观察TP对A375细胞中IRE1、JNK、c-Jun mRNA表达水平及细胞凋亡的影响。统计分析采用两因素方差分析、单因素方差分析及Dunnett-t检验。结果 不同浓度TP作用不同时间，各浓度TP组A375细胞存活率均显著低于实验对照组（FTP浓度 = 18.36，P = 0.002），且随时间延长，A375细胞存活率越低（F时间 = 8.54，P = 0.018）。处理A375细胞24 h，50、100、200 nmol/L TP组及实验对照组细胞凋亡率分别为16.99% ± 0.33%、30.78% ± 0.40%、38.91% ± 0.51%、4.33% ± 0.02%，组间差异有统计学意义（F = 5 234.97，P < 0.001），各TP组细胞凋亡率均显著高于实验对照组（均P < 0.05），且凋亡率随TP浓度的增加而逐渐升高。50、100、200 nmol/L TP组IRE1、JNK和c-Jun mRNA表达水平均显著高于实验对照组（均P < 0.05），且随着TP浓度的增加这些基因mRNA表达水平逐渐升高，TP处理组A375细胞中IRE1、JNK、c-Jun、p-JNK和p-c-Jun蛋白表达水平也显示出相同的趋势。经JNK抑制剂SP600125预处理72 h后，SP600125 + 100 nmol/L TP组细胞凋亡率（21.88% ± 0.55%）显著低于未经SP600125预处理的100 nmol/L TP组（t = -22.51，P < 0.001），且IRE1、JNK和c-Jun mRNA的表达水平也显著降低（均P < 0.05）。结论 TP可能通过激活IRE1/JNK信号通路诱导人黑素瘤A375细胞凋亡。

关键词: 黑色素瘤, 雷公藤内酯, 细胞凋亡, A375细胞, IRE1/JNK通路

Abstract: 【Abstract】 Objective To investigate the effect of triptolide on the apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1 （IRE1）/c-Jun N-terminal kinase （JNK） signaling pathway, and to explore its possible mechanisms. Methods Cultured A375 cells were treated with triptolide at different concentrations of 0, 50, 100, 200 nmol/L （experimental control group, 50, 100, 200 nmol/L triptolide groups, respectively）, and a blank control group （DMEM high-glucose medium without cells） was set up. Methyl thiazol tetrazolium （MTT） assay was performed to evaluate the cell viability at 24, 48, and 72 hours after the start of treatment, flow cytometry to detect cell apoptosis at 24 hours after the start of treatment, and real-time fluorescence-based quantitative PCR （RT-qPCR） and Western blot analysis were conducted to determine mRNA and protein expression of IRE1, JNK, and c-Jun, respectively. After pretreatment with the JNK inhibitor SP600125 for 72 hours, some A375 cells were then treated with 100 nmol/L triptolide for 24 hours （SP600125 + 100 nmol/L triptolide group）, and the A375 cells only treated with 100 nmol/L triptolide served as control group （100 nmol/L triptolide group）. Effects of triptolide on the mRNA expression of IRE1, JNK, and c-Jun in A375 cells, as well as on cell apoptosis, were investigated. Statistical analysis was performed using two-way analysis of variance, one-way analysis of variance, and Dunnett′s test. Results After the treatment with different concentrations of triptolide for different durations, the cell viability was significantly lower in all triptolide groups than in the experimental control group （Ftriptolide concentration = 18.36, P = 0.002）, and gradually decreased over time （Ftime = 8.54, P = 0.018）. After 24-hour treatment, the apoptosis rate of A375 cells significantly differed among the 4 groups treated with different concentrations of triptolide （F = 5 234.97, P < 0.001）; additionally, the apoptosis rate was significantly higher in the 50, 100, and 200 nmol/L triptolide groups （16.99% ± 0.33%, 30.78% ± 0.40%, 38.91% ± 0.51%, respectively） than in the experimental control group （4.33% ± 0.02%, all P < 0.05）, and gradually increased with the rising concentrations of triptolide. The mRNA expression levels of IRE1, JNK, and c-Jun were all significantly higher in the 50, 100, and 200 nmol/L triptolide groups than in the experimental control group （all P < 0.05）, and gradually increased with the increase of triptolide concentration. Moreover, the protein expression levels of IRE1, JNK, c-Jun, p-JNK, and p-c-Jun in A375 cells in the triptolide groups also showed the same trend. After pretreatment with the JNK inhibitor SP600125 for 72 hours, the apoptosis rate was significantly lower in the SP600125 + 100 nmol/L triptolide group （21.88% ± 0.55%） than in the 100 nmol/L triptolide group without SP600125 pretreatment （30.78% ± 0.40%, t = -22.51, P < 0.001）, and the mRNA expression levels of IRE1, JNK, and c-Jun were also significantly decreased in the SP600125 + 100 nmol/L triptolide group compared with the 100 nmol/L triptolide group（all P < 0.05）. Conclusion Triptolide may induce apoptosis of human melanoma A375 cells by activating the IRE1/JNK signaling pathway.

Key words: Melanoma, Triptolide, Apoptosis, A375 cells, IRE1/JNK pathway

引用本文

孙冬红 刘国豪 侍姝彤 包军 穆耕林 陶玥. 雷公藤甲素通过肌醇需求酶1/c-Jun氨基端激酶信号通路诱导人黑素瘤A375细胞凋亡[J]. 中华皮肤科杂志, 2023,56(10):934-939. doi:10.35541/cjd.20230135

Sun Donghong, Liu Guohao, Shi Shutong, Bao Jun, Mu Genglin, Tao Yue. Triptolide induces apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1/c-Jun N-terminal kinase signaling pathway[J]. Chinese Journal of Dermatology, 2023, 56(10): 934-939.doi:10.35541/cjd.20230135