| Background and Objective：According to the world health organization released data show that regardless oflung cancer in the first place in the world cancer morbidity or mortality, non-smallcell lung cancer accounts for about80%. the main therapeutic measures have surgery,radiotherapy, chemotherapy and biological therapy. Although some patients withearly stage lung cancer can affect a cure, but most patients have to be advanced whendiagnosed, most have lost the chance of operation. Impact on tumor medicineattracted attention at home and abroad in recent years, the pharmaceutical industry,especially in medicine to induce apoptosis in cancer fight has important significance.The method of combining traditional Chinese and western medicine treatment oftumors in both clinical and widely used in scientific research and study. BuShenShuGan Formula is tutor many years of experience in party, clinically can narrow thefocus, so that the disease has been significantly controlled, but also the quality of lifeof patients improved significantly prolong the patient’s life.The present study,serumpharmacological method to study the apoptosis of BuShen ShuGan Formula in humanlung adenocarcinoma A549and explore the mechanism may exist, the possibility oftheoretical and experimental basis for its clinical application. Methods：60male SD rats were randomly divided into6groups(Each group contains10rats,the weight of two groups is no difference),which were Respectively controlgroup, DDP group, low dose group, middle dose group, high dose group, combinedgroup (middle dose BSSGF solution plus DDP group). The concentration oftraditional Chinese medicine (TCM) is set up in accordance with the adult and SDrats weight conversion method and serum pharmacology testing method.Controlgroup was given saline to lavage in4ml every day; DDP group was given2ml everyone by intraperitoneal injection (0.5mg/ml), respectively, on days1,3,5, a total of3days; Low dose group was given traditional Chinese medicine in4ml every day(including crude drugs1.25g/ml, a total of5g); Middle dose group was giventraditional Chinese medicine in4ml everyday (including crude drugs2.5g/ml, a totalof10g); High dose group was given traditional Chinese medicine in4ml everyday(including crude drugs3.75g/ml, a total of15g); combined group was given solution(2.5g/ml) by lavage and l injects DDP(0.5mg/ml) by intraperitoneal on1,3,5days;10%chloral hydrate was used to anesthesia SD rats by intraperitoneal injection afterthe last lavage lasted for2h and blood collection was from abdominal aorticvessel.The samples were centrifuged in3000r/min for20min and the seurms wereinactivated at56°C for30min, which were saved for later use at-20°C thelast.Preparation of rat serum containing MTT assay containing serum on human lungadenocarcinoma A549cell proliferation. PI method flow cytometry containing serumon A549cell cycle; the Annexin V-FITC/PI double staining flow cytometry.Containing serum on apoptosis of A549cells.Western-Blot detection of serumcontaining P53and Bcl-2protein expression. Semi-quantitative RT-PCR DetectNotch signaling pathway.Results：1. Under an inverted microscope, the growth state of A549cells inserum-containing effects A549cells seen in the control group48h good. While themiddle dose group of cells, the cell gap increases slightly, a few fractured cells suspended in culture medium. Most of the high-dose group of cell suspension, a smallnumber of adherent cells become rounded or broken into pieces, the number of cellswas significantly reduced, DDP group and high dose group is substantially the sameunder a microscope. A large number of cells in the combination group off, broken,showing apoptotic state. side effect of the drug-containing serum on apoptosis ofA549cells72h after the most significant.2. MTT assay was applied to determine the cell proliferation of A549cells aftertreated with BSSGF at different concentrations(control, low, middle, high, DDP,combined) for different time durations (24,48and72h). Bushen Shugan Formulacould inhibit growth of A549cells with a dose-and time-dependent manner and havesynergies combined with DDP. The inhibitory rate was26.71%,30.86%and41.74%respectively for24h,48h and72h.3. Flow cytometry instrument detected the change of drug-containing serumon A549cells72h cell cycle and apoptosis rate: with the increasing of thedrug-containing serum concentration of G2/M phase cells percentage increase, withcombined group G2/M phase cells the highest percentage (P <0.05); With Annexin v-FITC/PI double flow cytometry instrument to detect cell apoptosis rate, with theincreasing of the drug-containing serum concentration cell apoptosis rate increased,were higher than the control group, the difference was statistically significant (P <0.05).combined group of apoptosis rate compared with the control group withsignificant difference (P <0.01), the toppest apoptosis rate was almost40%.4. Western Blot detection means different doses of drug-containing serumtreatment of A549cells72h total protein, and its impact on the P53, Bcl-2expression.72h BuShen ShuGan Formula combined group containing serum significantlyinduced activation of P53A549cells to promote apoptosis, while ontained serum canbe reduced apoptosis inhibitory protein Bcl-2.The low, medium, high, combinationgroup, DDP group and control group BCL-2, P53compare gray values were different(P <0.05).5. RT-PCR detected that drug-containing serum of BuShen ShuGan Formulaaffected on the mRNA expression of Notch1, Delta4, the VEGF: drug-containingserum of BSSGF worked on A549cells of72h could inhibit the gene expression of Notch1, Delta4, the VEGF. The mRNA expression Comparison of the Notch1, Delta4,VEGF gene was discovered that the differences of high, DDP group and combinedgroup of Notch1, Delta4, VEGF compared with control group were statisticallysignificant(P <0.05).Conclusion：1. BuShen ShuGan Formula could inhibit the proliferation of A549cells relatedto the time and dose.2. BuShen ShuGan Formula blocked non-small cell lung cancervA549cells inG2/M phase, slowed the speed of cell proliferation and induced apoptosis.3. BuShen ShuGan Formula could induce apoptosis according to reducing theamount of Bcl-2protein, P53protein expression increased.4. BuShen ShuGan Formula which is also according to inhibit the geneexpression of Notch1, Delta4, VEGF induced apoptosis of A549. |
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