Study On The Effect Of Danzhi Xiaoyao Powder On Breast Cancer Based On Metabolomic

Purpose:Based on untargeted metabolomics technology,this study investigated the effect of Danzhi Xiaoyao Powder on the development and progression of breast cancer and its possible molecular mechanism,providing a reference for basic research on the prevention and treatment of breast cancer.Material and method:1.To observe the effect of Danzhi Xiaoyao Powder on MDA-MB-231 cells of breast cancer in nude mice bearing the tumorForty 6-week-old SPF BALB/C-NU female nude mice were fed adaptically for 7 days,The tumor model was established by inoculating m DA-MB-231-GFP cell suspension(density5×10~5/ml)under breast fat pad in nude mice during logarithmic growth period.The nude mice were randomly divided into normal control group,model group,Danzhi Xiaoyao Powder group(11.69g·kg-1)and Xihuang Pill group(0.78g·kg-1),with 10 mice in each group.One day after the breast cancer cells were inoculated,they were given intragastrically once a day at a fixed time for 22d,and the same amount of normal saline was given to the normal control group and the model group.Two hours after the last drug administration,the eyeball was removed for blood collection,and the tumor tissue was collected for subsequent experiments.The serum of human breast cancer nude mice was detected by liquid chromatography/mass spectrometry(LC/MS).The differential metabolites were screened by unsupervised principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA)and univariate statistical analysis(Student’s test).KEGG annotation and metabolic pathway analysis of differential metabolites were performed.Western blot was used to detect adenylate activated protein kinase(AMPK),acetyl-Co A carboxylase 1(ACC1),fatty acid synthase(FASN),fatty acid desaturase 1(FADS1)and stearoyl-co A satiety in tumor tissues The expression of adenylate activated protein kinase(AMPK),acetyl-Co A carboxylase 1(ACC1),fatty acid synthase(FASN),fatty acid desaturase 1(FADS1),stearoyl-co A desaturase 1(SCD1)and fatty acid receptor(CD36)in tumor tissues were determined by blot.2.To observe the effect of Danzhi Xiaoyao Powder containing serum on MDA-MB-231 cells of breast cancerThe apoptosis of breast cancer MDA-MB-231 cells was observed by acridine orange/ethyl bromide(AO/EB)double fluorescence staining method.The effects of Danzhi Xiaoyao Powder on the content of COX-2 and 5-LOx in breast cancer MDA-MB-231 cells were detected by enzyme-linked immunosorbent assay.The expression of connexins 26(Cx26)and Connexins 43(Cx43)in breast cancer MDA-MB-231 cells were investigated by immunofluorescence staining.And using western blotting to observe the effect of Danzhi Xiaoyao Powder serum on expression of SREBP-1,PPARγand HADHA protein in MDA-MB-231 cells of breast cancer.Results:1.Effects of Danzhi Xiaoyao Powder on serum metabolites of nude mice bearing human breast cancerThe analysis results showed that,compared with the normal control group,a total of 15key differential metabolites in the serum of nude mice in the model group had sustained changes.It is suggested that the abnormality of 7 metabolic pathways,such as biosynthesis of unsaturated fatty acids,metabolism of arachidonic acid,the regulation of inflammatory mediators on tryptophan channel,regulation of lipolysis of adipocytes,the prominent amino acids and 5-hydroxytryptamine,synthesis and secretion of aldosterone,are closely related to the occurrence and development of breast cancer.Danzhi Xiaoyao Powder can significantly up-regulate DHA and erucic acid,and down-regulate DPA and adrenoic acid,suggesting that Danzhi Xiaoyao Powder may exert anti-tumor effect by regulating the biosynthesis of unsaturated fatty acids in nude mice bearing human breast cancer.2.Effects of Danzhi Xiaoyao Powder on the expression of AMPK,ACC1,FASN,FADS1,SCD1 and CD36 proteins in tumor tissuesWestern blot results showed that compared with normal control group,the positive expression of ACC1,FASN,FADS1,SCD1 and CD36 protein in model group was increased(P<0.01),while the positive expression of AMPK protein was decreased(P<0.01).Compared with model group,the positive expressions of ACC1,FASN,FADS1,SCD1 and CD36proteins in Danzhi Xiaoxiao Powder group were decreased(P<0.01),while the positive expressions of AMPK protein were increased(P<0.01).These results indicated that Danzhi Xiaoyao Powder could decrease the expression levels of ACC1,FASN,FADS1,SCD1 and CD36 and increase the expression level of AMPK in tumor tissues.3.Effect of Danzhi Xiaoyao Powder containing serum on MDA-MB-231 cells of breast cancerThe results of AO/EB double fluorescence staining showed that,compared with blank control serum group,most of the MDA-MB-231 cells showed green fluorescence and the number of tumor cells marked with red fluorescence increased significantly in Danzhi Xiaoyao Powder containing drug serum after 48h treatment,suggesting that Danzhi Xiaoyao Powder containing drug serum can induce apoptosis of MDA-MB-231 cells.The results of enzyme-linked immunosorbent assay showed that the contents of COX-2 and 5-LOX in Danzhi Xiaoyao Powder serum group were decreased after treated with MDA-MB-231 cells for 48h(P<0.01).Immunofluorescence staining showed that the expression of Cx26 and Cx43in MDA-MB-231 cells was decreased in Danzhi Xiaoyao Powder containing serum group after treated for 48h.The results of western blotting showed that the expressions of SREBP-1and HADHA and PPARγin Danzhi Xiaoyao Powder serum group were down-regulated after treated with MDA-MB-231 cells for 48h.Conclusion:1.Danzhi Xiaoyao Powder may play an anti-tumor role by regulating the biosynthesis of unsaturated fatty acids in nude mice bearing human breast cancer cells.2.Lipid metabolism is one of the pathways for breast cancer cells to obtain energy.Danzhi Xiaoyao Powder can down-regulate the expression of ACC1,FASN,FADS1,SCD1 and CD36 proteins in tumor tissues,and up-regulate the expression of AMPK protein,suggesting that regulation of lipid metabolism may be the molecular mechanism of Danzhi Xiaoyao Powder in the prevention and treatment of breast cancer.3.Danzhi Xiaoyao Powder may promote the apoptosis of breast cancer MDA-MB-231 cells by regulating lipid metabolism-related proteins.