| 1. A model of breast cancer in rats being replicated with7,12-demethylbenz[a]anthraceneObject:To assure the endpoint for drug administrating in rat with breast cancer from DMBA.Methods:Rats were divided randomly into 2 groups, model ones (ni=40) were intragastrically adeministrated with DMBA (100.0mg/kg) in olive oil (10.0ml/kg), did control ones (nj=10) with olive oil. Tumor volumes in breasts were calculated as formulae (1/6πab2) from measured both of longest (a) and shortest (b) diameters weakly, to indicate total bearing tumors. The median duration (hours) of breast cancers (MT50) was nonlinearly regressed from the increased ratios of total bearing tumors with the use of Graph Pad Prism 6.01 under various sloope, as the endpoint for drug administrating window as the most sensitivity time range that is affected by an intervened factor.Results:The model of bearing breast cancer in rats was replicated successfully, with. The median duration of breast cancers and its 95% confidence interval were 1960h (81.7days) and 1843-2095h (76.8-87.3 days), the equation between the total bearing tumors and the during hours was Y=-1.695+91.985/(1+101374169-4.173X), R2=0.99.Conclusions:The endpoint of drug administrating was 81.7 (IC95:76.8-87.3) days in breast cancer-bearing rats dealt with demethylbenz[a]anthracene.2. Leading infiltrated immunocytes rat breast cancer from Qingdu dose-effectiveObject:To qualify the infiltrated immunocytes in rat breast cancer from Qingdu dose-effective.Methods:Breast cancer was replicated in 90 rats with DMBA at do, did so with olive oil in 10 control rats. Nine dose of Qingdu granules (k=0.316,0.0107-107g/kg, ni=3) were administrated intragastrically and daily from d91 in rats with breast cancer, so did SSa (k=0.316, 0.0153-153mg/kg, ni=2), and 10.0ml/kg CMC in control ones (ni=5). Body weight and total tumor volume were measured weekly until d147, tumor inhibitory ratio was calculated with area under the curve (AUC).The dose effect relationship of Qingdu granules or SSa was regressed from inhibiting ratios with the use of Graph Pad Prism 6.01 under various sloope. Tumor inhibitory ratioes were calculated from (AUC deriving from total tumor volume/Body weight). Four biomarkers of immunocytes infiltrated in breast cancer tissues were demonstrated with immunohistochemical staining (IHC) and qualified with morphometery in situ, as CD4, CD8, F4/80 or CD57 indicating for infiltrated CD4+T lymphocyte, CD8+T lymphocyte, macrophage, or natural killer; so did as Ki-67 indicating for the biomarker of proliferative oncocytes, as cancer background;the increase rate of immune cells was calculated in all dose groups.Results:ID50 of Qingdu granules and SSa were 7.82nmol/kg/d and 179.5nmol/kg/d, respectively, the non-SSa compounds in Qingdu granules enhanced 23 timely the effectiveness from SSa. Qingdu granules had effect for reduceing the activity of tumor cell proliferation, promoting CD4+T, CD8+T, macrophages and natural killers infiltrated in tumor tissue. The positive gradiens of lineaer equations between the dosages of Qingdu granule and the levels of immune biomarkers were KF4/80> KCD4>KCD8> KCD57, respectively, to enhance the infiltration of CD4+ T lymphocyte, macrophage, CD8+ T lymphocyte.Conclusion:Pharmacologic mechanism in Qingdu granule for breast cancer therapy has been demonstrated that non-SSa enhance SSa 23 times, the effectiveness has mainly derived from infiltrated immunocytes, especially, CD4+T lymphocyte and macrophage.3. Both pathways involving in Qingdu granules inhibiting for breast cancer immune evasionObject:To confirm CD47/SIRPa and STAT4 pathways involving in Qingdu granules therapy for breast cancer via inhibiting cancer immune evasion.Methods:D0,135 rats were given DMBA by gavage once,15 rats of control were given by gavage once with equal oil. D91, rats with breast cancer were divided into 9 groups by tumor volume and weight ratio, such as model, tamoxifen (TAM), Saikosaponin a(SSa), Fluoxetine(FXT), Qingdu granules with low(QDL), middle(QDM) and high(QDH) dose group. The rats were irrigated once a day, tumor volumes were measured once a week. D147, the rats were killed, the inhibiting ratio of tumor was calculated, Ki-67, CD4, CD8, F4/80, CD57, SIRPa and CD47 in tumor was tested by immunohistochemical staining and the content of IL-12, IFN-γ, IL-4 and IL-10 in serum was determined by Elisa. Expression of IL-12, IL-12R and pSTAT4 were determined with immunohistochemical staining, real time RT-PCR and Western blot.Results:Anti-tumor effects of Qingdu granules were shown as decreased tumor volume, the inhibitory rate (%) of Qingdu granules with low, middle, high dose and Saikosaponin a is 30.93, 43.84,44.17 and 43.48. Compared with the control group the expression of Ki-67, CD47 and SIRPα was reduced and CD4, CD8, F4/80 and CD57 was increased in Qingdu granules and Saikosaponin a groups. The content of IL-12 and IFN-γ in serum was increased and the level of IL-4 and IL-10 was reduced in these groups. Qingdu granules increased existed levels, gene levels, and protein amounts of IL-12, IL-12R and pSTAT4.Conlusion:Qingdu granules inhibitted the growth of breast cancer in rats by adjusting the immune system to suppress the immune escaping, enhance antitumor immune. |
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