Henan University of traditional Chinese medicine
The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)
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增值活性的影响；比色法检测六君子汤对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10μg/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）25μg/mL，半抑制浓度（IC50） 40μg/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。
Objective: To explore the molecular mechanism of ethyl acetate extract of Liujunzi Decoction(EAELD) on energy metabolism of esophageal cancer EC9706 cells in conditioned medium of cancer-associated fibroblasts (CAFs). Methods: Methyl thiazol tetrazolium (MTT) assay was used to detect the effect of EAELD on the proliferation activity of EC9706. The effects of EAELD on lactate and glucose in the supernatant of EC9706 cells in CAFs conditioned medium were detected by colorimetry. seahorse system energy metabolism analysis system was used to detect the effect of EAELD on energy metabolism of EC9706 cells in CAFs conditioned medium. Real-time fluorescent quantitative PCR (q-PCR) and western blotting were used to detect the mRNA and protein expression of energy metabolism-related molecules.. Results: Compared with DMEM, except for the 10μg/mL group,EAELD had a significant inhibitory effect on the proliferation of EC9706 cells (P < 0.05). The inhibitory concentration (IC30) of 25μg/mL and half inhibitory concentration (IC50) of 40μg/mL were selected as the low and high dose groups for subsequent experiments. Among EC9706 cells cultured by CAFM, both low-dose and high-dose EAELD groups could significantly reduce Non-mitochondrial oxygen consumption, Basal respiration value, Maximum respiration value, Oxygen consumption of ATP synthesis, Spare respiration capacity, Basal glycolysis, Compensative glycolysis and glycolysis potential (P <0.01). Decreased the lactate content of EC9706 cells (P <0.01), down-regulated the mRNA expression of GLUT1 (P <0.05, P <0.01), down-regulated the protein expression of p-PKM2, HK2, PKM2 and MCT1 (P <0.01); The high-dose EAELD group could down-regulate the Mitochondrial oxygen consumption and basal use The glycolytic ratio of EC9706 cells (P <0.05), reduce glucose uptake of EC9706 cells (P <0.05), down-regulate the protein expression of p-PKM2 and GLUT1 (P <0.01, P <0.05); The low dose group of EAELD could down-regulate the mRNA expression of MCT1 (P <0.05). Conclusions:EAELD can interfere with the energy metabolism of EC9706 cells in CAFs conditioned medium, and its mechanism may be related to the regulation of HK2, PKM2, GLUT1, MCT1 and MCT4 mRNA and protein expression.