毛蕊异黄酮对神经母细胞瘤细胞系SH-SY5Y细胞增殖及瓦博格效应的影响

Effect of calycosin on the proliferation and Warburg effect in human neuroblastoma SH-SY5Y cells

目的观察毛蕊异黄酮(calycosin,CA)对人神经母细胞瘤(neuroblastoma,NB)细胞系SH-SY5Y细胞增殖和瓦博格效应的影响并探讨其可能的机制。方法将SH-SY5Y细胞培养于DMEM培养基,实验分为对照组和CA组、CA+siRNA阴性对照组和CA+siRNA TAp73α组。CA组的细胞用50、100和150μmol/L CA分别处理24、48和72 h。CA+siRNA阴性对照组的细胞转染阴性对照siRNA质粒24 h后,再加100μmol/L CA处理48 h。CA+siRNA TAp73α组的细胞转染TAp73α干扰RNA质粒24 h后,再加100μmol/L CA处理48 h。用CCK-8法检测细胞的增殖水平,用化学检测法检测细胞培养液中乳酸的水平和细胞中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,用液闪法检测细胞对葡萄糖的吸收量。采用细胞外通量分析仪检测细胞外液酸化速率、细胞氧气消耗速率和细胞糖酵解能力。采用蛋白质印迹法检测TAp73蛋白的表达水平。采用实时定量聚合酶链反应(quantificational real-time polymerase chain reaction,qRT-PCR)检测TAp73αmRNA的相对表达水平。采用单因素方差分析比较各组间的差异,采用LSD-t检验进行组间差异的两两比较。结果与对照组比较,经50、100和150μmol/L CA处理的SH-SY5Y细胞,在24、48和72 h细胞的增殖水平显著降低,呈现剂量和时间依赖性。与对照组比较,经50、100和150μmol/L CA处理的SH-SY5Y细胞在48 h以剂量依赖的方式降低了培养液中乳酸的水平,对照组和CA组(50、100和150μmol/L)的乳酸相对水平分别为(100±0)%、(84.31±5.14)%、(52.06±6.37)%和(32.16±4.11)%,对照组和CA组之间的差异具有统计学意义(均P<0.05)。CA显著降低了SH-SY5Y细胞中LDH活力,LDH的活力在对照组和CA组(50、100和150μmol/L)分别为(24.32±3.77)U/mg、(19.39±2.34)U/mg、(12.34±2.64)U/mg和(7.61±1.06)U/mg,对照组和CA组之间的差异具有统计学意义(P<0.05)。CA显著降低了SH-SY5Y细胞对葡萄糖的吸收,葡萄糖吸收的相对水平在对照组和CA组(50、100和150μmol/L)分别为(100±0)%、(78.36±6.22)%、(42.91±5.07)%和(26.18±4.39)%,对照组和CA组之间的差异具有统计学意义(P<0.05)。CA显著降低了SH-SY5Y细胞的细胞外液酸化速率,细胞外液酸化速率在对照组和CA组(50、100和150μmol/L)的相对水平分别为(100±0)%、(75.37±5.37)%、(38.42±4.11)%和(22.31±2.86)%,对照组和CA组之间的差异具有统计学意义(P<0.05)。细胞氧气消耗速率的相对水平在对照组和CA组(50、100和150μmol/L)分别为(100±0)%、(85.76±5.29)%、(64.11±6.07)%和(42.86±3.85)%,对照组和CA组之间的差异具有统计学意义(P<0.05)。细胞糖酵解能力的相对水平在对照组和CA组(50、100和150μmol/L)分别为(100±0)%、(75.87±8.06)%、(54.93±6.33)%和(36.75±5.28)%,对照组和CA组之间的差异具有统计学意义(P<0.05)。与对照组比较,100、150μmol/L的CA显著上调了TAp73蛋白的表达水平,对照组和CA组(100和150μmol/L)的TAp73蛋白相对表达水平分别为12.76±2.64、35.84±5.61和67.51±5.97。RNA干扰沉默TAp73α的表达,影响CA对SH-SY5Y细胞增殖和瓦博格效应的抑制作用。结论CA可抑制人NB细胞株SH-SY5Y细胞的增殖,作用机制可能与通过上调TAp73蛋白的表达水平从而抑制瓦博格效应有关。

Objective To observe the effect of calycosin(CA)on the proliferation and Warburg effect of human neuroblastoma(NB)cell line SH-SY5Y and to explore its possible mechanism.Methods After culturing in DMEM medium,SH-SY5Y cells were divided into four groups of control,CA,CA+siRNA negative control and CA+siRNA TAp73α .Cells in CA group were treated with mullein(50/100/150μmol/L)for 24/48/72 h respectively.And cells in CA+siRNA negative control group were transfected for 24 h with negative control siRNA plasmid and then 48 h with 100μmol/L CA.And cells in CA+siRNA TAp73α group were transfected for 24 h with TAp73α small interfering RNA plasmid and then 48 h with 100μmol/L CA.CCK-8 method was employed for detecting the level of cellular proliferation.Chemical method was employed for detecting the level of lactic acid in supernatant of cell culture medium and the intracellular activity of lactate dehydrogenase(LDH).Liquid scintillation was utilized for detecting the cellular glucose absorption.Extracellular flux analyzer was employed for detecting the parameters of extracellular acidification rate(ECAR),cellular oxygen consumption rate(OCR)and cellular glycolytic capacity.Western blot was utilized for detecting the expression level of TAp73 protein.Quantificational real-time polymerase chain reaction(qRT-PCR)was used for detecting the relative expression level of TAp73α mRNA.Results As compared with control group,the treatment of CA(50/100/150μmol/L)for 24/48/72 h lowered markedly the cellular proliferation level in a dose-dependent manner.As compared with control group,48 h treatment of CA reduced significantly the levels of lactic acid in supernatant of cell culture medium.The relative level of lactate was 100±0%,84.31±5.14%,52.06±6.37%and 32.16±4.11%in control and CA groups respectively.The difference between control and CA groups was statistically significant(P<0.05).CA reduced significantly the activity of LDH.LDH activity was 24.32±3.77,19.39±2.34,12.34±2.64 and 7.61±1.06 U/mg in control and CA groups respectively.The difference between control and CA groups was statistically significant(P<0.05).CA reduced significantly the cellular absorption of glucose.The relative levels of glucose absorption were 100±0%,78.36±6.22%,42.91±5.07%and 26.18±4.39%in control and CA groups respectively.The difference between control and CA groups was statistically significant(P<0.05).The relative levels of extracellular acidification rates were 100±0%,75.37±5.37%,38.42±4.11%and 22.31±2.86%in control and CA groups respectively.The difference was statistically significant between control and CA groups(P<0.05).The relative level of cell oxygen consumption rate were 100±0%,85.76±5.29%,64.11±6.07%and 42.86±3.85%in control and CA groups respectively.The difference was statistically significant between control and CA groups(P<0.05).Glycolysis capability was 100±0%,75.87±8.06%,54.93±6.33%and 36.75±5.28%in control and CA groups respectively.The difference was statistically significant between control and CA groups(P<0.05).As compared with control group,100/150μmol/L CA increased markedly the expression level of TAp73 protein.The relative expression levels of TAp73 protein were 12.76±2.64 and 35.84±5.61 and 67.51±5.97 in control group and CA groups respectively.RNA interference silencing the expression of TAp73α affected the inhibition of CA on cellular proliferation and Waborg effect(all P<0.05).Conclusions Calycosin suppresses the proliferation of human neuroblastoma cell line SH-SY5Y.The mechanism of action may be correlated with an inhibition of Warburg effect by up-regulating the expression of TAp73.