补中益气汤含药血清对A549/DDP细胞PI3K/AKT信号转导通路影响的实验研究

Experimental Research on Effect of Buzhong Yiqi Decoction Medicated Serum on PI3K/AKT Signal Transduction Pathway in Lung Adenocarcinoma Cell Lines of A549/DDP

目的观察补中益气汤含药血清对肺腺癌顺铂耐药细胞A549/DDP的抑制作用,并探讨其对磷脂酰肌醇3激酶(PI3K)磷酸化蛋白激酶B(p-AKT)蛋白和mRNA表达的影响。方法以SD大鼠制备高、中、低剂量的补中益气汤含药血清和空白对照血清。MTT法筛选最佳含药血清组。培养细胞分为空白血清组、最佳含药血清组、LY294002组(阻滞剂组)、最佳含药血清+LY294002组(联合组)和阴性对照组。免疫细胞化学方法和实时荧光定量PCR(RT-PCR)方法检测PI3K、p-AKT蛋白和mRNA的表达情况。结果随着含药血清浓度的升高,对顺铂的逆转倍数不断增加;中剂量含药血清作用48 h为最佳含药血清及最佳作用时间;最佳含药血清可使A549/DDP细胞中PI3K、p-AKT蛋白表达减少,与空白血清组比较差异有统计学意义(P<0.05);补中益气汤诱导A549/DDP细胞PI3K、p-AKT mRNA表达显著减少,与空白血清组比较,差异有统计学意义(P<0.05),与阻滞剂联合作用p-AKT mRNA下降更为明显。结论补中益气汤可通过降低A549/DDP细胞中PI3K、p-AKT mRNA的表达而影响PI3K/AKT信号转导通路,进而发挥对顺铂耐药的增敏作用。

Objective To observe the inhibitory effect of the Buzhong Yiqi Decoction medicated serum on lung adenocareinoma A549/DDP cells and to discuss its influence on PI3K and AKT protein and mRNA expression. Methods SD rats were randomly divided into 4 groups ： the higher dosage group, the moderate dosage group, the lower dosage group and the blank control group. The best medicated serum was determined by the MTr method. A549/DDP cells were divided into the blank serum group, the best medicated serum group, the LY294002 group （blockers group）, the best medicated serum ＋ LY294002 group （combination group） and the negative control group. The immunocytochemistry method and real-time fluorescence quantitative polymerase chain reaction （RT-PCR） were adopted to detect the protein expression and mRNA expression of PI3K and AKT. Results With the increase in medicated serum concentration, ICso of each group was decreased and the RI of cisplatin was increased. By roll- ing a moderate dose of medicated serum for 48 hours, the best medicated serum was produced and 48 hours was the best time. The best medicated se- rum reduced PI3K and p-AKT protein expression in A549/DDP cells. Compared with the blank serum group, the difference is statistically significant （P 〈 0.05 ）. The Buzhong Yiqi Decoction induced mRNA expression of PI3K and p-AKT in A549/DDP cells significantly reduced. Compared with the blank serum group, the difference is statistically significant （P 〈 0.05 ）. Conclusion the Buzhong Yiqi Decoction can reduce the expression lev- els of PI3K and p-AKT mRNA, which in turn influences PI3K/AKT signal transduction pathway and thus plays a role of sensitization of cisplatin re- sistanee.