Experimental Research On Effect Of AKF On The Expression Of Bad,Caspase-9,Survivin,mTOR Protein And Gene In Human Lung Adenocarcinoma A549 And Mouse Lewis Cells

Objective To observe the effect of Ai Kang-Fang(AKF)on the expression of Bad,Caspase-9,Survivin,m TOR protein and gene in human lung adenocarcinoma A549 and mouse Lewis cells.Based on the previous experiments of our research group,exploring the possible mechanism by which AKF promotes apoptosis of lung cancer cells and synergizes with cyclophosphamide in deeper.Method 40 SPF male rats were selected and weighed between 220±20 grams,and then randomly divided into four groups,including blank control group(normal saline gavage),cyclophosphamide group(cyclophosphamide intraperitoneal injection),AKF group(AKF gavage)and AKF combined with cyclophosphamide group(AKF gavage combined with cyclophosphamide intraperitoneal injection),each group has 10 rats.After 5 days of continuous gavage in rats,blood was taken from the heart,serum was separated and inactivated,and stored in a refrigerator at-80°C.Purchasing A549 and Lewis cells for culture,groups were divided according to the different drug-containing serum:blank control group,cyclophosphamide group,AKF group and AKF combined with cyclophospham-ide group,cultured for 48 h and 72 h respectively,and then collected for detection.The expression levels of Bad,Caspase-9,Survivin and m TOR were detected by immunofluores-cence and Western blotting respectively.The m RNA expression levels of Bad,Caspase-9,Survivin and m TOR in the samples were detected by Real-Time PCR.To clarify the possible mechanism by which AKF promotes apoptosis of lung cancer cells and synergi-zes with cyclophosphamide.Results1.The effects of AKF and AKF on the expression of Bad and Caspase-9 in A549cells: immunofluorescence and Western blotting showed that the expression levels of Bad and Caspase-9 proteins increased with the increase of drug efficacy.And the increase was observed;the expression level of the blank control group was the lowest,an-d the expression level of the AKF combined with the cyclophosphamide group was the highest;compared with the blank control group,the expression of the AKF group,the cyclophosphamide group,the AKF combined with cyclophosphamide.The level of water increased significantly,and the difference was statistically significant(P<0.01).C-ompared with cyclophosphamide group,the expression level of AKF combined with c-yclophosphamide was more obvious,the difference was statistically significant(P<0.05).The results of RT-PCR showed that the m RNA expression levels of Bad and Casp-ase-9 increased with the increase of drug efficacy;the expression trend was the sameas that of Western blotting.2.Effects of AKF and AKF on the expression of Survivin and m TOR in A549 cells by synergistic cyclophosphamide:Immunofluorescence and Western blotting showed that the expression levels of Survivin and m TOR protein decreased with the increasing of drug efficacy.The expression level of the blank control group was the highest,and the expression level of AKF combined with cyclophosphamide group was the lowest.Compared with the blank control group,the expression levels of the AKF group,cyclophosphamide group,AKF and cyclophosphamide group were significantly decreased,the difference was statistically significant(P<0.01);Compared with the cyclophosphamide group the expression level of the group of AKF combined with cyclophosphamide was more obvious,and the difference was statistically significant(P<0.05).The results of RT-PCR showed that the m RNA expression levels of Survivin and m TOR decree-sed with the increase of drug efficacy;the expression trend was the same as that of Westernblot.3.The effect of AKF on the expression of Bad,Caspase-9,Survivin,m TOR protein and gene in Lewis cells was the same as that of A549 cells.Conclusion1.AKF and AKF synergistic cyclophosphamide can up-regulate the expression leve ls of Bad,Caspase-9 protein and gene in human lung adenocarcinoma A549 and mous e Lewis cells,and down-regulate the expression levels of Survivin,m TOR protein andgene.2.AKF can promote the apoptosis of human lung adenocarcinoma A549 and mouse Lewis cells,and synergize with cyclophosphamide.The mechanism may be:(1)Related to the regulation of apoptosis-related proteins Bad,Caspase-9 and Survivin;(2)By downregulating the expression of m TOR protein,inhibiting the abnormal activation of PI3K/AKT-m TOR signaling pathway in lung cancer cells,thereby promoting apoptosis and reducing the resistance level of cyclophosphamide and the degree of apoptosis is relate d to time.