目的 研究芍药苷对雷西莫特诱导的巨噬细胞有氧糖酵解的影响.方法 用佛波酯(PMA)处理THP-1细胞使其分化成巨噬细胞.将细胞分为对照组、模型组和低、中、高剂量实验组.对照组细胞正常培养;模型组用2μg·mL-1雷西莫特刺激24 h诱导巨噬细胞进行有氧糖酵解反应;低、中、高剂量实验组在模型组的基础上分别加入1、10和100 μmol·L-1芍药苷处理24 h.以细胞计数试剂盒-8(CCK-8)法检测细胞活性;以乳酸及葡萄糖测定试剂盒检测各组细胞乳酸的分泌和葡萄糖的消耗;以蛋白质印迹法和实时荧光定量聚核合酶链反应(q-PCR)法分别检测M2型丙酮酸激酶(PKM2)、乳酸脱氢酶A(LDHA)的蛋白和mRNA表达水平;以免疫荧光法比较各组细胞中PKM2的荧光强度.结果 对照组、模型组和低、中、高剂量实验组细胞上清液中的葡萄糖含量分别为(14.70±0.44)、(9.83±0.43)、(10.68±0.29)、(11.79±0.33)和(13.63±0.74)mmol·L-1;细胞分泌的乳酸分别为(6.17±0.48)、(11.94±0.55)、(9.08±0.55)、(7.79±0.66)和(6.50±0.55)mmol·L-1;细胞中PKM2的蛋白表达水平分别为1.00±0.00、1.33±0.18、1.02±0.17、0.74±0.17和0.73±0.18;LDHA的蛋白表达水平分别为1.00±0.00、1.20±0.09、0.90±0.14、0.76±0.12 和 0.78±0.17;PKM2 mRNA 表达水平分别为 1.00±0.09、2.11±0.23、1.98±0.31、1.38±0.25 和 0.93±0.32;LDHA mRNA 表达水平分别为 1.00±0.13、1.85±0.25、1.44±0.21、0.91±0.24 和0.96±0.14;PKM2 的平均荧光强度分别为 136.41±33.63、217.94±5.33、210.27±1.03、204.14±3.27和186.79±14.03.模型组上述指标与对照组比较,在统计学上差异均有统计学意义(P＜0.05,P＜0.01);中、高剂量实验组上述指标与模型组比较,在统计学上差异均有统计学意义(P＜0.05,P＜0.01).结论 芍药苷可以抑制R848诱导的巨噬细胞的有氧糖酵解.
Objective To investigate the effect of paeoniflorin on aerobic glycolysis of macrophages induced by resiquimod.Methods THP-1 cells were treated with phorbol ester(PM A)to differentiate into macrophages.The cells were divided into control group,model group and low,medium,high dose experimental group.The cells in the control group were cultured normally;in the model group,2 μg·mL-1 resiquimod was used to stimulate macrophages for 24 h to induce aerobic glycolysis.The low,medium and high dose experimental groups were treated with 1,10 and 100 μmol·L-1 paeoniflorin for 24 h on the basis of the model group.Cell activity was detected by cell counting kit-8(CCK-8)method.Lactate and glucose determination kit were used to detect lactate secretion and glucose consumption of cells in each group.The protein and mRNA expression levels of(PKM2)and(LDHA)were detected by Western blot and real-time fluorescence quantitative polynucleotide chain reaction(q-PCR)respectively.Immunofluorescence method was used to compare the fluorescence intensity of PKM2 in each group.Results After 24 h stimulation of THP-1 cells with 2 μg·mL-1 resiquimod,the glucose contents in cell culture supernatants of control group,model group and low,medium and high dose experimental groups were(14.70±0.44),(9.83±0.43),(10.68±0.29),(11.79±0.33)and(13.63±0.74)mmol·L-1;the lactate secreted by cells were(6.17±0.48),(11.94±0.55),(9.08±0.55),(7.79±0.66)and(6.50±0.55)mmol·L-1;the protein expression levels of PKM2 in cells were 1.00±0.00,1.33±0.18,1.02±0.17,0.74±0.17 and 0.73±0.18;the protein expression levels of LDHA were 1.00±0.00,1.20±0.09,0.90±0.14,0.76±0.12 and 0.78±0.17;the PKM2 mRNA levels were 1.00±0.09,2.11±0.23,1.98±0.31,1.38±0.25 and 0.93±0.32;the LDHA mRNA levels were 1.00±0.13,1.85±0.25,1.44±0.21,0.91±0.24 and 0.96±0.14;the average fluorescence intensities of PKM2 were 136.41±33.63,217.94±5.33,210.27±1.03,204.14±3.27 and 186.79±14.03.Compared with control group,the above indicators in model group showed statistically significant differences(P＜0.05,P＜0.01);compared with model group,the differences in the above indicators in medium and high dose experimental group were all statistically significant(P＜0.05,P＜0.01).Conclusion Paeoniflorin can inhibit the aerobic glycolysis of macrophages induced by resiquimod.
金莹莹;时乐;郝永熙;唐璠;蒋玟慧;梁涛
南京中医药大学药学院,江苏南京 210023
中医学
芍药苷;巨噬细胞;M2型丙酮酸激酶;乳酸脱氢酶A;有氧糖酵解
paeoniflorin;macrophages;pyruvate kinase M2;lactate dehydrogenase A;aerobic glycolysis
《中国临床药理学杂志》 2024 (005)
683-687 / 5
10.13699/j.cnki.1001-6821.2024.05.011
评论