Danzhi Xiaoyao San's Anti-breast Cancer Through Apoptosis Pathway And Its Molecular Mechanism

Purpose:The experiment based on breast cancer liver depression and spleen deficiency,Qiand blood weakness of the basic pathogenesis of Chinese medicine.Select soothing liver a-nd strengthening spleen,regulating qi and blood of the classic prescription Danzhi Xiaoyao Power.From the perspective of apoptosis,explore Danzhi Xiaoao Power on breast cancer in-hibition.Revealed Danzhi Xiaoyao Power may induce apoptosis of breast cancer cells though anti-breast cancer.Material and method:Thirty-two female SPF BALB/c-nu nude mice of 6 weeks old were include.After 7d adaptive feeding,MCF-7 cell suspension with a density of 1×10⁸/mL was inoculated subcutaneously in the axilla of nude mice.10d after inoculation success model,the nude mice were randomly divided into 4 groups:The model group,Danzhi Xiaoyao Po-wer high,medium and low dose(17.98,8.99,4.50g·kg^-1)group,n=8ineachgroup.Mice were gavaged after surgery,drugs by human and mice body surface area equivalent dose ratio calculated gavage.The mice were dosed twice a day for 15 day,the mice in the modle group were given equal volume of normal saline.After the last administration,the nude mice were sacrificed and the subcutaneous tumor tissue in the axilla of nude mice was dissected.W-eigh fresh nude mice tumor mass,calculate the mass inhibition rate.Hematoxylin and eosin（HE）staining was used to observe the morphological changes of xenografted tumor in nude mice.Tunel staining was used to detect tumor cell apoptosis.Western blotting was used to de-tect the expression of Bcl-2,Bax,Caspase-3 and Cyt-c in tumor tissues.Preparation of Dan zhi Xiaoyao Power medium dose group containing serum.Breast cancer cells MCF-7 were d-ivided into two groups,namely blank serum group and Danzhi Xiaoyao Power medium dose serum group.The proliferation rate of MCF-7 cells was determined by MTT（thiazole blue）colorimetry,the volume fraction of drug containing serum was 10%,15%and 20%respec-tively,and the intervention time was 12h,24h,48h and 72h respectively.Flow cytometry analysis of MCF-7 cell cycle and apoptosis.Apoptosis of MCF-7 cells was detected by acri-dine orange/ethidium bromide（AO/EB）double staning.The changes of mitochondrial me-mbrane potential in MCF-7 cells were detected by rhodamine 123（Rh 123）staining.Western blotting was used to detect the expression of Bcl-2,Bax,Caspase-3 and Cyt-c in MCF-7cells.Results:1.Effect of Danzhi Xiaoyao Power on Transplanted Tumor in MCF-7 Nude MiceTumor mass inhibition rate results show that,compared with the model group,the inhibitory rates of Danzhi Xiaoyao Power high and medium dose groups on the xenografts in MCF-7 n-ude mice were 29.89%（P<0.05）,47.89%（P<0.01）.There was no significant difference between low dose group and model group.HE staining results show,compared with the mod-el group,Danzhi Xiaoyao Power high and medium dose groups significantly increased tumor necrosis,cytoplasmic staining,of which the most effective dose group,the low dose group and the model group had no significant difference.Tunel staining showed that,compared with the model group,the tumor cells apoptotic rates of Danzhi Xiaoyao Power high and medium dose groups were 22.45±7.55%（P<0.05）、30.41±10.01%（P<0.01）,the low dose group and the model group had no significant difference.Due to the results of the tumor inhi-bition rate determination,HE staining and Tunel stainting,there was no significant differen-ce between the experimental results of Danzhi Xiaoyao Power low group and the model group,so the Danzhi Xiaoyao Power low group was not included in the follow-up Western blot exp-eriment.Western blot result showed that Dan zhi Xiaoyao Power high dose group upregualted the exp-ression of Bax（P<0.01）and Cyt-c（P<0.01）,and downregulated the expression of Bcl-2（P<0.01）;Danzhi Xiaoyao Power medium dose group upregualted the expression of Bax（P<0.01）,Caspase-3（P<0.01）and Cyt-c（P<0.01）,and downregulated the expression of Bcl-2（P<0.01）,Danzhi Xiaoyao Power medium dose group is better than high dose group.2.Effect of Danzhi Xiaoyao Power Containing Serum on MCF-7 CellsThe results of MTT assay showed that compared with the blank serum group,the cell prolifer-ation rate of the 15%drug-containing serum group decreased significant at 12h,24h,48h and 72h（P<0.05）,15%drug-containing serum group after 48h the most obvious effect,there was no significant difference between other groups and blank serum group（P>0.05）.Therefore,the serum volume fraction was selected as 15%and the intervention time was 48h as the intervention condition for the follow-up experiment.AO/EB staining results showed t-hat compared with the blank serum group,Danzhi Xiaoyao Power medium-containing serum group treated with MCF-7 cells 48h,most of cells were stained green fluorescence,the nu-mber of fluorescent tumor cells labeled with ethidium bromide increased significantly.The r-esults of flow cytometry showed that compared with the blank serum group,the apoptosis rate of MCF-7 cells was increased to 19.51%after treated with Danzhi Xiaoyao Power medium containing serum for 48 hours,the proportion of cells the blank serum group and Dan zhi Xi-aoyao Power-containing serum in G1 phase was 42.83%and51.98%,theproportionofSph-ase cells were 54.23%and 46.79%.Compared with the blank serum group,Danzhi Xiaoyao Power medium-containing serum group G1 phase ratio was significantly increased,Sphase was significantly reduced.Rh 123 fluorescence staining showed that,compared with the bla-nk serum group,the mitochondrial membrane potential of Danzhi Xiaoyao Power medium-containing serum group was significantly reduced.Western blot rsults showed that Danzhi X-iaoyao Power serum-containing group upregualted the expression of Bax（P<0.01）,Caspa se-3（P<0.01）and Cyt-c（P<0.01）,downregulate the expression of Bcl-2（P<0.01）.Conclusion:1.Danzhi Xiaoyao Power may inhibit the growth of tumor by inducing the apoptosis of tumor cells,and the best dosage is medium dosage(8.99g·kg^-1).2.Danzhi Xiaoyao Power induced tumor cell apoptosis may be related to the up-regulation of apoptosis proteins Bax,Caspase-3,Cyt-c expression,down-regulation of apoptosis protein Bcl-2 expression.3.Danzhi Xiaoyao Power medium-dose serum can induce apoptosis of breast cancer cell MC F-7,the best working concentration of 15%serum volume fraction,the best time for 48h.4.The drug-containing serum of Danzhi Xiaoyao Power medium-dose group may induce the apoptosis of breast cancer cell MCF-7 through the mitochondrial-mediated apoptosis pathway.5.Apoptosis factor Bcl-2 and Bax were applied to mitochondria,the abnormal opening of the mitochondrial permeability transformation hole（mPTP）,resulting in decreased mitochondrial membrane potential.The apoptosis factor Cyt-c was released into the cytoplasm,thus activati-ng the caspase-3 in the cell,triggering a series of cascade reactions,which eventually induced apoptosis.This may be one of the basic molecular mechanisms of Danzhi Xiaoyao Power to i-nduce apoptosis.