蜡菊亭抑制MITF/PGC-1α轴介导的MGC-803细胞线粒体有氧磷酸化

Helichrysetin inhibits MITF/PGC-1αaxis mediated mitochondrial oxidative phosphorylation in MGC-803 cells

目的:考察蜡菊亭对人胃癌MGC-803细胞线粒体有氧磷酸化的影响,并探讨其分子机制。方法:①将细胞分为对照组和蜡菊亭不同浓度(10、20、40μmol/L)组,分别给予相应干预。药物作用24、48 h后,试剂盒检测线粒体有氧磷酸化相关指标。②将细胞分为对照组和蜡菊亭(20μmol/L)组,分别给予相应干预。药物作用12、24 h后,PCR检测过氧化物酶体增殖物活化受体γ共刺激因子-1α(PGC-1α)、黑色素细胞诱导转录因子(MITF)m RNA表达,Western blot检测PGC-1α蛋白表达。③将细胞分为对照组、蜡菊亭(20μmol/L)组、PGC-1α激动剂(10μmol/L)组、蜡菊亭+激动剂联用组,各组分别给予相应干预,处理细胞24 h后,CCK-8法检测各组细胞活力。结果:①与对照组相比,药物干预24 h后,蜡菊亭20μmol/L组细胞基础呼吸和质子渗漏均受到抑制(P<0.05,P<0.01),蜡菊亭40μmol/L组细胞基础呼吸、最大呼吸、质子渗漏、ATP生成和备用呼吸能力均受到抑制(P<0.05,P<0.01);药物干预48 h后,蜡菊亭各浓度组细胞的基础呼吸、最大呼吸、质子渗漏和备用呼吸能力均受到抑制(P<0.05,P<0.01),蜡菊亭20、40μmol/L组细胞ATP生成亦明显降低(P<0.01)。②蜡菊亭(20μmol/L)干预12、24 h后能够显著下调MGC-803细胞PGC-1α的m RNA和蛋白表达(P<0.01)以及MITF m RNA表达(P<0.01)。③蜡菊亭(20μmol/L)能够显著降低MGC-803细胞活力(P<0.01),但该作用可被PGC-1α激动剂拮抗,蜡菊亭+激动剂组的细胞活力明显高于蜡菊亭组(P<0.01)。结论:蜡菊亭可能通过抑制MITF/PGC-1α轴,降低MGC-803细胞的线粒体有氧磷酸化,进而抑制肿瘤细胞活力。

Objective:To investigate the effects of helichrysetin on mitochondrial oxidative phosphorylation(OXPHOS)in human gastric cancer MGC-803 cells and explore its molecular mechanism.Methods:①The cells were divided into the control group and helichrysetin groups with different concentrations(10,20,40μmol/L),which were treated with the corresponding intervention.After treatment for 24 and 48 hours,the related indexes of mitochondrial OXPHOS were detected by kits.②The cells were divided into the control group and helichrysetin(20μmol/L)group,which were treated with the corresponding intervention.After treatment for 12 and 24 hours,the m RNA expressions of peroxisome proliferator-activated receptorγcoactivator-1α(PGC-1α)and melanocyte inducing transcription factor(MITF)were detected by PCR,and the protein expression of PGC-1αwas detected by Western blot.③The cells were divided into the control group,helichrysetin(20μmol/L)group,PGC-1αagonist(10μmol/L)group and helichrysetin plus agonist group,which were treated with the corresponding intervention.After treatment for 24 hours,the cell viability was detected by CCK-8 assay.Results:①After drug intervention for 24 hours,compared with the control group,the basic respiration and proton leakage were inhibited in the helichrysetin 20μmol/L group(P<0.05,P<0.01),and the basic respiration,maximal respiration,proton leakage,ATP production and spare respiration capacity were inhibited in the helichrysetin 40μmol/L group(P<0.05,P<0.01).After drug intervention for 48 hours,compared with the control group,the basal respiration,maximal respiration,proton leakage and spare respiration capacity were inhibited in the helichrysetin groups with different concentrations(P<0.05,P<0.01),and the ATP production was also significantly decreased in the helichrysetin 20 and 40μmol/L groups(P<0.01).②After treatment with helichrysetin at 20μmol/L for 12 and 24 hours,the m RNA and protein expressions of PGC-1αand the m RNA expression of MITF in MGC-803 cells were significantly down-regulated(P<0.01).③Helichrysetin at 20μmol/L could significantly reduce the cell viability of MGC-803 cells(P<0.01),but this effect could be antagonized by PGC-1αagonist,and the cell viability of the helichrysetin plus agonist group was obviously higher than that of the helichrysetin group(P<0.01).Conclusion:Helichrysetin may inhibit MITF/PGC-1αaxis,reduce mitochondrial OXPHOS of MGC-803 cells,and then suppress the viability of tumor cells.