补中益气汤含药血清联合顺铂对人肺腺癌A549/DDP细胞Nrf2、PRX1、SOD、GPX4的影响

Effects of Buzhong Yiqi Decoction medicated serum combined with cisplatin on Nrf2,PRX1,SOD and GPX4 protein in human lung adenocarcinoma A549/DDP cells

目的探讨补中益气汤含药血清联合顺铂对人肺腺癌A549/DDP细胞中核因子红系2相关因子2(Nrf2)及其下游抗氧化蛋白的影响。方法将60只大鼠按随机数字表法分为空白组、低剂量组、中剂量组、高剂量组,每组15只。低、中、高剂量组分别灌胃3.29、6.57、13.13 g/kg补中益气汤,1次/d,持续7 d。末次给药后1 h,腹主动脉取血,制备补中益气汤含药血清。将A549/DDP细胞按随机数字表法分为空白组(空白组大鼠血清)、模型组(空白组大鼠血清+顺铂)、低剂量组(低剂量含药血清+顺铂)、中剂量组(中剂量含药血清+顺铂)、高剂量组(高剂量含药血清+顺铂),相应药物干预24 h后,采用CCK-8法检测各组A549/DDP细胞对顺铂的半数抑制浓度(IC50);共聚焦荧光定位法检测p-Nrf2荧光表达强度;RT-qPCR法检测Nrf2 mRNA水平;Western blot法检测各组A549/DDP细胞中Nrf2、p-Nrf2、过氧化物还原酶1(PRX1)、谷胱甘肽过氧化物酶4(GPX4)、SOD蛋白相对表达。结果与模型组比较,低、中、高剂量组A549/DDP细胞对顺铂的IC50降低(P<0.01);p-Nrf2荧光强度降低(P<0.01);Nrf2 mRNA水平降低(P<0.01);Nrf2、p-Nrf2、SOD、PRX1、GPX4蛋白表达下调(P<0.01),尤以中、高剂量组效果更显著(P<0.05)。结论补中益气汤含药血清通过抑制A549/DDP细胞中Nrf2活性表达及下调其靶基因SOD、PRX1、GPX4蛋白表达,改善肺腺癌顺铂耐药。

Objective To discuss the effects of Buzhong Yiqi Decoction medicated serum combined with cisplatin on the expression of Nrf2 and its effects of downstream antioxidant proteins in human pulmonary adenocarcinoma A549/DDP cells.Methods Totally 60 rats were divided into blank group,low-dosage group,medium-dosage group,and high-dosage group using a random number table method,with 15 rats in each group.The low-,medium-,and high-dosage groups were orally administered with 3.29,6.57,and 13.13 g/kg Buzhong Yiqi Decoction for gavage,once a day,for 7 days.1 hour after the last administration,blood was collected from the abdominal aorta to prepare the serum containing Buzhong Yiqi Decoction.A549/DDP cells were divided into 5 groups:blank group(rat serum),model group(rat serum+cisplatin),low-dosage group(low-dosage Buzhong Yiqi Decoction medicated serum+cisplatin),medium-dosage group(medium-dosage Buzhong Yiqi Decoction medicated serum+cisplatin),and high-dosage group(high-dosage Buzhong Yiqi Decoction medicated serum+cisplatin).And 24 h after the Buzhong Yiqi Decoction intervention,CCK-8 method was used to detect the IC50 of cisplatin in each group.The fluorescence expression intensity of p-Nrf2 in A549/DDP cells of each group was determined by confocal fluorescence localization method.The mRNA relative expression of Nrf2 in A549/DDP cells of each group was detected by RT-qPCR.The relative expressions of Nrf2,p-Nrf2,GPX4,PRX1 and SOD in A549/DDP cells of each group were detected by Western blot.Results Compared with the model group,the IC50 of A549/DDP cells to cisplatin decreased in the low-,medium-,and high-dosage groups(P<0.01);the fluorescence intensity of p-Nrf2 decreased(P<0.01);the level of Nrf2 mRNA decreased(P<0.01);the protein expressions of Nrf2,p-Nrf2,SOD,PRX1,and GPX4 were down-regulated(P<0.01),with the effect being more significant in the medium-and high-dosage groups(P<0.05).Conclusion Buzhong Yiqi Decoction medicated serum can improve cisplatin resistance in human pulmonary adenocarcinoma cancer by inhibiting the active expression of Nrf2 in A549/DDP cells and down-regulating the target genes of SOD,PRX1 and GPX4 in vivo.