六君子汤乙酸乙酯提取物干预CAFs 条件培养基下EC9706 细胞能量代谢的机制研究
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作者单位:

1.河南中医药大学,郑州 450046;2.河南省中医方证信号传导重点实验室,郑州 450046

中图分类号:

R-33


Mechanism of ethyl acetate extract of Liujunzi Decoction in the energy metabolism of EC9706 cells in CAF-conditioned medium
Author:
Affiliation:

1.Henan University of Traditional Chinese Medicine, Zhengzhou 450046, China. 2. Henan Provincial Key Laboratory of Prescription and Syndrome Signal Transduction, Zhengzhou 450046

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    摘要:

    目的 探索六君子汤乙酸乙酯提取物(EAELD)对癌相关成纤维细胞(CAFs)条件培养基下食管癌EC9706 细胞能量代谢影响的分子机制。 方法 噻唑蓝(MTT)法检测EAELD 对EC9706 增殖活性的影响;比色法检测EAELD 对CAFs 条件培养基(CAFM)下EC9706 细胞上清中乳酸及葡萄糖含量的影响;Seahorse 能量代谢分析系统检测EAELD 对CAFM 下EC9706 细胞能量代谢的影响;实时荧光定量PCR(RT-qPCR)、蛋白免疫印迹(Western blot)法检测EAELD 对能量代谢相关分子mRNA 及蛋白表达的影响。 结果 与DMEM 相比,除10 μg/ mL组外,EAELD 对EC9706 细胞增殖活性均有明显抑制作用(P<0. 05),选取抑制浓度(IC30)25 μg/ mL,半抑制浓度(IC50)40 μg/ mL,作为低、高剂量组进行后续实验。在CAFM 培养的EC9706 细胞各组中,EAELD 低、高剂量组都能显著降低非线粒体耗氧、基础呼吸值、最大呼吸值、合成ATP 耗氧量、备用呼吸能力、基础糖酵解、补偿糖酵解、糖酵解潜能(P<0. 01),减少EC9706 细胞上清乳酸含量(P<0. 01),下调GLUT1 的mRNA 表达(P<0. 05,P<0. 01),下调p-PKM2、HK2、PKM2、MCT1 蛋白表达(P<0. 01);EAELD 高剂量组能够下调EC9706 细胞的线粒体耗氧与基础糖酵解比值(P<0. 05),减少EC9706 细胞葡萄糖摄取(P<0. 05),下调p-PKM2、GLUT1 的蛋白表达(P<0. 01,P<0. 05);EAELD低剂量组能够下调MCT1 的mRNA 表达(P<0. 05)。 结论 六君子汤乙酸乙酯提取物能够干预CAFs 条件培养基下EC9706 细胞的能量代谢,其机制可能与EAELD 调控HK2、PKM2、GLUT1、MCT1、MCT4 的mRNA 和蛋白表达相关。

    Abstract:

    Objective To explore the molecular mechanism of ethyl acetate extract of Liujunzi Decoction (EAELD) on energy metabolism in esophageal cancer EC9706 cells in conditioned medium from cancer-associated fibroblasts (CAFs). Methods Methyl thiazol tetrazolium assays were used to assess the effect of EAELD on EC9706 cell proliferation. The effects of EAELD on lactate and glucose in the culture supernatant of EC9706 cells in CAF-conditioned medium were assessed by colorimetry. A seahorse system for energy metabolism analysis was used to assess the effect of EAELD on energy metabolism of EC9706 cells in CAF-conditioned medium. Real-time quantitative PCR (qPCR) and Western blot were used to measure mRNA and protein expression of energy metabolism-related molecules. Results Compared with DMEM, except for the 10 μg/ mL group, EAELD had a significant inhibitory effect on EC9706 cell proliferation (P<0. 05). The 30% inhibitory concentration (IC30) of 25 μg/ mL and half inhibitory concentration (IC50) of 40 μg/ mL were selected as low and high doses for subsequent experiments. In EC9706 cells cultured in CAFM, both low and high dose EAELD groups had significantly reduced non-mitochondrial oxygen consumption, basal respiration, maximum respiration, oxygen consumption of ATP synthesis, spare respiration capacity, basal glycolysis, compensative glycolysis, and glycolysis potential (P<0. 01). Lactate content of EC9706 cells was decreased (P<0. 01), mRNA expression of GLUT1 (P< 0. 05, P< 0. 01) was downregulated, and protein expression of p-PKM2, HK2, PKM2 and MCT1 was downregulated (P<0. 01). The high dose EAELD group had downregulated mitochondrial oxygen consumption and basal respiration. The glycolytic ratio of (P<0. 05) and glucose uptake of EC9706 cells were reduced (P<0. 05) and protein expression of p-PKM2 and GLUT1 was downregulated ( P< 0. 01, P< 0. 05). The low dose group of EAELD had downregulated mRNA expression of MCT1 (P<0. 05). Conclusions EAELD interferes with the energy metabolism of EC9706 cells in CAF-conditioned medium, and its mechanism may be related to regulation of HK2, PKM2, GLUT1, MCT1 and MCT4 mRNA and protein expression.

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陈星,娄翔宇,尚艺婉,周哲旭,刘洋,刘娅茹,胡啸博,陈玉龙.六君子汤乙酸乙酯提取物干预CAFs 条件培养基下EC9706 细胞能量代谢的机制研究[J].中国比较医学杂志,2023,33(11):17~24.

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  • 收稿日期:2022-11-25
  • 在线发布日期: 2023-12-29
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