补中益气汤含药血清逆转A549/DDP的顺铂耐药及对mTOR表达的影响

Rat Serum Containing of Buzhong Yiqi Decoction Reverse Chemotherapeutic Resistance in A549/DDP Cells by Down-regulating Expression Levels of mTOR

摘要:

目的: 研究补中益气汤含药血清对肺腺癌顺铂耐药细胞株(A549/DDP)顺铂耐药的逆转作用及对哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)表达的影响。 方法: 动物随机分组,制备补中益气汤高、中、低剂量血清(给药剂量分别为11.34,5.67,2.83 g·kg^-1·d^-1),将A549,A549/DDP细胞体外分为顺铂(DDP)组,空白血清+DDP组,含药血清高剂量+DDP组,含药血清中剂量+DDP组,含药血清低剂量+DDP组,细胞对照组,经过15%各组血清联合DDP不同浓度(10,20,40,80,100,200 μmol·L^-1)作用48 h,采用MTT法检测补中益气汤不同浓度含药血清对A549/DDP顺铂耐药的逆转作用;采用免疫荧光,免疫细胞化学,Western-blot方法检测mTOR的表达。 结果: 与DDP组比较,补中益气汤不同浓度含药血清作用于A549/DDP后IC₅₀和耐药倍数均有显著降低(P<0.05),以补中益气汤中剂量组(等临床剂量组)作用最为显著(P<0.01),A549/DDP中mTOR的染色吸光度及荧光强度均明显减弱(P<0.01),蛋白表达水平明显降低(P<0.01)。 结论: 补中益气汤能增强A549/DDP对顺铂的敏感性,并能通过降低细胞生存通路中mTOR的表达而有效逆转肺腺癌细胞的顺铂耐药。

Abstract:

Objective: To identify the effect of rat serum containing different concentrations of Buzhong Yiqi decoction(BZYQT) on reverse the chemotherapeutic resistance in A549/DDP cells and on the expression of mTOR. Method: Animals were randomized to prepared the rat serum containing varied does levels of BZYQT(does for 11.34,5.67,2.83 g·kg^-1·d^-1),the A549,A549/DDP were equally divided into six groups in random: the DDP group, BZYQT low does+DDP group, BZYQT middle does+DDP group, BZYQT high does+DDP group, NS+DDP group and the cell control group.Followed by 15%serum of every group and the different concentrations of DDP(10,20,40,80,100,200 μmol·L^-1) for 48 h, assayed by MTT to detect the rat serum containing of BZYQT reverse the chemotherapeutic resistance in A549/DDP.The expression of mTOR in cells were evaluated by indirect immunofluorescence, immunocytochemistry technique and Western-blot. Result: The median inhibitory concentration IC₅₀ and resistant multiple of A549/DDP is significant reduction by the serum containing varied does levels of BZYQT(P<0.05),especially the effect of the middle dose group is the most significant(P<0.01),the optical density and fluorescence intensity of mTOR were significantly reduced(P<0.01) and the protein expression levels were significantly lower in the cells(P<0.01). Conclusion: BZYQT appeared to increase the chemotherapeutic sensitivity of A549/DDP cells, showing regression of multiple chemotherapeutic resistance in pulmonary adenocarcinoma by down-regulating expression levels of mTOR.