Effect Of Buzhong Yiqi Decoction On PI3K / AKT Signal Transduction Pathway In Human Lung Adenocarcinoma Strain A549 / DDP And Its Transplantation

Purpose： This study explored the mechanism of Buzhong Yiqi Decoction ininterfering phosphatidylinositol3-kinase (PI3K)/protein kinase B (PKB)signaling pathway in pre-resistant strains A549/DDP and lung adenocarcinomaxenografts based on serum pharmacology, cell biology, molecular biologytechniques, through in vitro and in vivo experiments. We aimed to provide a basisfor future molecular biology experiments to find effective target of interferingcisplatin-resistant lung cancer with traditional Chinese medicine, as well asnew lines of thinking and methods in the pathogenesis and treatment theory ofmanaging cisplatin-resistant lung cancer.Materials and methods：Sixty BALB/C mice were randomly divided into controlgroup, tumor-bearing control group, cisplatin group and Buzhong Yiqi Decoctionof high, medium and low dose group+cisplatin group（hereinafter referred toas the high,medium and low combined group）. Adjust A549/DDP to the concentrationof5×106mL-1, Blank control group was given subcutaneous inoculation0.2mL salineeach in groin; other groups of nude mice were inoculated subcutaneously A549/DDPcell suspension0.2mL each in groin. Tumor growth was observed, and when thetumor was visually observed, the control group and tumor-bearing control groupreceived saline0.5mL by gavage; cisplatin group were given the same amount ofsaline as well as intraperitoneal injection of cisplatin2mg kg-1; high, mediumand low combined groups were given a high, medium and low concentrations ofBuzhong Yiqi Decoction (98.5g kg-1,49.3g kg-1,24.6g kg-1) by gavage aswell as intraperitoneal injection of cisplatin2mg kg-1, once a day; observethe general state of nude mice. Fourteen days later, take HE staining of tumortissue for pathological morphology; immunohistochemistry and real time PCRmethods were used to detect the expression levels of PI3K, AKT, Bad, NF-κb, caspase-9, survivin, mTOR protein and mRNA in tumor.Forty SD rats(weight200±20g) were randomly divided into four groups, namely, groups of high, mediumand low dose of Buzhong Yiqi Decoction (respectively given13.13g·kg-1,6.57g·kg-1and3.29g·kg-1by gavage) and the control group given2ml of saline,1time/d for3days. One hour after the last administration, abdominal aorticblood was taken to make the serum samples containing Buzhong Yiqi Decoction.MTT method was used to detect the effect of the drug-containing serum of differentdose in inhibiting the proliferation of A549and A549/DDP cells. A549andA549/DDP cells were cultured and divided into four groups: the high, medium,low and control, cultured in10%drug-containing serum of high, medium and lowdose. The cells were collected respectively after12hours,24hours,48hoursand72hours, and detected with MTT method; the most appropriate concentrationof drug-containing serum and the best time was determined. MTT was used todetermine the inhibition rate of drug-containing serum of most appropriateconcentration combined with cisplatin of different concentrations on A549/DDP,and to observe the resistance reversal effect and the sensitivity of A549/DDPto cisplatin in the most appropriate concentration of drug-containing serum.A549/DDP were divided into control serum group, the best drug-containing serumgroup, LY294002group, LY294002+best drug-containing serum group (hereinafterreferred to as the combined group), given appropriate intervention. We detectedexpression of PI3K and AKT protein and mRNA with methods of immunocytochemistry,Western Blotting, and real time fluorescence quantitative polymerase chainreaction (real time PCR). A549and A549/DDP were divided into the A549controlserum group, A549best drug-containing serum group, A549/DDP control serumgroup and A549/DDP best drug-containing serum group, given the appropriateintervention. We detected expression of Bad, NF-κb and caspase-9protein andmRNA with immunocytochemistry, Western Blotting and real time PCR methods. SPSS16.0statistical software was used for data processing; measurement data wereexpressed as mean±standard deviation; when there was normal distribution ofdata in each group and homogeneity of variance, one-way ANOVA was used to do the analysis; pairwise comparison was done using the SNK method; for those thatdo not meet the parametric test, multiple-dose data rank test was performed.P <0.05was considered statistically significant.Results：1Complex-action of Buzhong Yiqi Decoction and cisplatin can reduce the volumeof transplanted tumor.The comparision of medium and high-combined group withcispiatin group showed there was significant diffrernce(P<0.05).2We observed the pathological changes of tumor-bearing nude mice in each groupby HE method: with the applications of different doses of Buzhong Yiqi Decoctionand cisplatin, tumor necrosis was common, the density of tumor cells reduced,and inflammatory cell infiltrated more.3The protein and mRNA expression of PI3K, AKT, Bad, NF-κb, caspase-9, survivinand mTOR in tumor in each group by immunohistochemistry and real time PCR methodsshowed: the expression of PI3K, AKT, Bad, NF-κb, survivin and mTOR wassignificantly reduced in medium and high-combined group; the expression ofcaspase-9in tumor-bearing control group was the least, but significantlyincreased to the most in medium and high-combined group; cisplatin group andlow-combined group were at a medium level; the comparision of medium and high-combined groups with tumor-bearing control group, cisplatin group andlow-combined group showed that there was a statistically significant difference(P<0.05), except mRNA expression of survivin and mTOR. The comparison of eachpair in low-combined group, tumor-bearing control group and cisplatin groupshowed there was no significant difference (P>0.05), and there was no differencebetween medium and high-combined groups (P>0.05).4Different doses of Buzhong Yiqi Decoction drug-containing serum could suppressthe growth of A549and A549/DDP, with the higher rate in the high concentrationthan in the medium and low concentration. The10%medium dose of drug-containingserum and48h are the best acting drug-containing serum and the best actiontime(P<0.05), which could be used as dose and time in the follow-up experiments. 5When detecting the best level drug-containing serum and cisplatin by MTT assay,A549/DDP cisplatin resistance index dropped to3.76from7.35and reversal indexwas2.46.6The detection of PI3K, AKT protein and mRNA by Immunohistochemistry, WesternBlotting and real time PCR methods showed: expressions of PI3K and AKT proteinin control serum group were of no significant difference (P>0.05) compared withLY294002group; in the best level drug-containing serum group and the combinationgroup they were significantly reduced, which was statistically significantcompared with the control serum group (P<0.05); mRNA results showed: theexpression of PI3K and AKT mRNA were reduced in A549/DDP best leveldrug-containing serum group, the difference being statistically significantcompared with the control serum group (P<0.05);the expression of PI3K andAKTmRNA was of no difference between LY294002group and the control serum group(P>0.05); in the combination group PI3K and AKT mRNA was less expressed thanin the control serum group, the difference being statistically significant(P<0.05).7The detection of protein and mRNA expression of Bad,NF-κb,caspase-9byimmunochemical methods, Western Blotting and real time PCR showed: Bad andNF-κb in A549/DDP control serum group and A549control serum group had asignificant difference (P<0.05); A549/DDP best level drug-containing serumgroup and A549/DDP control serum group also had a significant difference on Badand NF-κb (P<0.05), and the expression was close to the expression level ofA549control serum group, there being no significant difference (P>0.05);A549/DDP best level drug-containing serum group and A549best level drug-containing serum group had no significant difference (P>0.05); caspase-9expression: A549/DDP control serum group and A549control serum group had asignificant difference (P <0.05); A549/DDP best level drug-containing serumgroup and A549/DDP control serum group had a significant difference (P<0.05),A549/DDP best level drug containing serum group and A549best level drugcontaining serum group had no significant difference (P>0.05). Conclusions：1Complex-action of Buzhong Yiqi Decoction and cisplatin can reduce the volumeof transplanted tumor of A549/DDP.2Inhibiting effect of Buzhong Yiqi Decoction to transplanted tumor of A549/DDPis through the way of regulating PI3K/AKT signaling pathway and promotingapoptosis.3The inhibition on A549/DDP growth with Buzhong Yiqi Decoction drug-containingserum increases with the increasing dose. The10%medium level dose of thedrug-containing serum can be used as the best concentration drug-containingserum, and the48h can be the best time of function. Buzhong Yiqi Decoctiondrug-containing serum can reduce the resistance of A549/DDP to cisplatin.4Buzhong Yiqi Decoction drug-containing serum can reduce the mRNA expressionof PI3K,AKT and their downstream factor Bad, NF-κb protein, while increasethe protein and mRNA expression of caspase-9.5Buzhong Yiqi Decoction drug-containing serum reduces A549/DDP resistance tocisplatin by regulating the expression of PI3K, AKT and its downstream factorBad, NF-κb and caspase-9.