Inhibitory Effect of Obacunone on Corticosterone Synthesis in Adrenocortical Tumor Cells by Affecting Mitochondrial Function
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摘要: 目的 研究黄柏酮对肾上腺皮质激素合成与分泌的影响。方法 体外培养Y1小鼠肾上腺皮质瘤细胞，采用2.5~160 μmol/L黄柏酮干预细胞24 h，运用MTT法检测细胞活性；以80 μmol/L黄柏酮处理细胞24 h后采用流式细胞术检测细胞周期，共聚焦显微镜检测细胞线粒体膜变化；以ELISA法检测细胞分泌液皮质酮含量；以40~160 μmol/L黄柏酮干预细胞24 h，80 μmol/L黄柏酮干预细胞24~48 h，运用qPCR法检测皮质酮合成酶mRNA表达，Western blot检测其蛋白表达。结果 与对照组比较，2.5~40 μmol/L黄柏酮对细胞的抑制率约为35%(P < 0.05)，160 μmol/L黄柏酮对细胞的抑制率为60%以上(P < 0.01)。黄柏酮导致G1期细胞显著增加并促进线粒体膜亮度增强(P < 0.05)，同时显著抑制线粒体膜Mfn1、Mfn2蛋白表达以及皮质酮分泌(P < 0.05)。进而发现，40~160 μmol/L黄柏酮显著抑制Cyp11a1、Cyp11b1、Hsd3b2、SF-1基因表达(P < 0.01)，160 μmol/L黄柏酮显著增强Star、Cyp21a1、Nr4a1、Nr4a2基因表达(P < 0.01)；80 μmol/L黄柏酮持续给药48 h内，均显著抑制Cyp11a1、Cyp21a1、Cyp11b1、Hsd3b2基因表达(P < 0.01)，并抑制StAR、CYP11A1蛋白表达，然而促进Star基因表达(P < 0.01)。结论 黄柏酮抑制肾上腺皮质细胞皮质酮合成过程，可能与其阻滞细胞周期及影响线粒体膜上类固醇激素合成酶表达有关。Abstract: OBJECTIVE To study the effect of obacunone (OBA) on the synthesis and secretion of adrenal cortex hormones.METHODS Y1 mouse adrenocortical tumor cells were cultured in vitro, and 2.5-160 μmol/L OBA was intervened with for 24 h. MTT assay was used to detect cell viability. Cells were treated with 80 μmol/L OBA for 24 h, flow cytometry was used to detect cell cycle, and confocal microscope was used to detect cell mitochondrial membrane changes. The content of corticosterone in cell secretion fluid was detected by ELISA. Cells were intervened with 40-160 μmol/L OBA for 24 h, and 80 μmol/L OBA for 24-48 h. The qPCR was used to detect corticosterone synthase mRNA expression, and Western blot was used to detect its protein expression.RESULTS Compared with the control group, 2.5-40 μmol/L OBA inhibited cell growth with a rate of about 35% (P < 0.05), and 160 μmol/L OBA inhibited cell growth with a rate of more than 60% (P < 0.01). OBA showed a significant increase in G1 cells and promoted the enhancement of mitochondrial membrane brightness (P < 0.05), while significantly inhibited the expression of Mfn1 and Mfn2 proteins in mitochondrial membranes and the corticosterone secretion (P < 0.05). Furthermore, it was found that 40-160 μmol/L OBA significantly inhibited the expressions of Cyp11a1, Cyp11b1, Hsd3b2, SF-1 genes (P < 0.01), and 160 μmol/L OBA significantly enhanced the expressions of Star, Cyp21a1, Nr4a1, Nr4a2 genes (P < 0.01). 80 μmol/L OBA within 48 h of continuous administration, significantly inhibited the expressions of Cyp11a1, Cyp21a1, Cyp11b1, and Hsd3b2 genes (P < 0.01), and StAR and CYP11A1 protein, but promoted the expression of Star gene (P < 0.01).CONCLUSION OBA can inhibit the corticosterone synthesis by corticosterone in adrenal cortex cells, which may be related to cell cycle arrest and the expression of steroid synthase on mitochondrial membrane.
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Key words:
- obacunone /
- corticosterone /
- Y1 cells /
- adrenal cortex /
- steroid hormone synthase
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表 1 引物序列
基因 序列号 引物序列 长度/bp Cyp11a1 NM_019779 上游5'-GCTGGAAGGTGTAGCTCAGG-3' 224 下游5'-CACTGGTGTGGAACATCTGG-3' Cyp11b1 NM_001033229 上游5'-GTATCGAGAGCTGGCAGAGG-3' 140 下游5'-GGGTTGATGTCGTGTCAGTG-3' Star NM_011485 上游5'-TTGGGCATACTCAACAACCA-3' 103 下游5'-GAAACACCTTGCCCACATCT-3' Cyp21a1 NM_009995 上游5'-CTCCGGCTATGACATCCCTA-3' 151 下游5'-ACAGCCAAAGGATGGTGTTC-3' Hsd3b2 NM_153193 上游5'-TCCAGCTCAGTTGATGTTGC-3' 129 下游5'-TGCCTTCTCAGCCATCTTTT-3' Nr4a1 NM_010444 上游5'-CTTGAGTTCGGCAAGCCTAC-3' 117 下游5'-GGTGTCAAACTCTCCGGTGT-3' Nr4a2 NM_001139509 上游5'-CGGTTTCAGAAGTGCCTAGC-3' 214 下游5'-TTGCCTGGAACCTGGAATAG-3' SF-1 NM_139051 上游5'-CATGGTCTTTAAGGAGCTGGAG-3' 275 下游5'TGAGGAACTTGAGACAGACGAA-3' β-actin NM_007393 上游5'-TGTTACCAACTGGGACGACA-3' 165 下游5'-GGGGTGTTGAAGGTCTCAAA-3' 
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