Effect And Mechanism Of Danzhi Xiaoyao Powder On Energy Metabolism Of Breast Cancer Cells

Purpose:Based on the Qi transformation theory of traditional Chinese medicine,this paper discusses the effect and mechanism of Danzhi Xiaoyaosan（DZXYS）on energy metabolism of breast cancer cells,so as to provide experimental basis for DZXYS against breast cancer.Material and method:1.To observe the effect of DZXYS medicated serum on proliferation,cell migration,cell cycle and apoptosis of MDA-MB-231 breast cancer cells.The medicated serum of DZXYS was prepared.Thirty SD rats were divided into blank control group,DZXYS group and Xihuang Pill group,each group 10 rats.DZXYS was8.99g·kg^-1,Xihuang Pill 0.55g·kg^-1,and the blank control group was given normal saline once a day for 7 days.After the last administration for 2 hours,the abdominal aorta took blood and separated the serum.DZXYS medicated serum intervened breast cancer MDA-MB-231 cells,MTT method was used to observe the effect of DZXYS medicated serum on the proliferation of breast cancer MDA-MB-231 cells,Cell scratch test was used to observe the effect of DZXYS medicated serum on the migration ability of breast cancer MDA-MB-231 cells,Flow cytometry was used to observe the effect of DZXYS medicated serum on the proliferation of breast cancer MDA-MB-231 cells,The effect of DZXYS medicated serum on MDA-MB-231cell apoptosis was observed by Annexin V/PI double staining,Hoechst 33342 and Mito-tracker Red fluorescence probe method was used to observe the effects of DZXYS medicated serum on the nuclear and mitochondrial morphology of breast cancer MDA-MB-231 cells,The effects of DZXYS medicated serum on BCL-2 associated X protein（BAX）and B cell lewkmia-2（BCL-2）of breast cancer MDA-MB-231 cells were observed by Western Blot.2.To observe the effect of DZXYS medicated serum on the metabolism of breast cancer MDA-MB-231 cellsDZXYS medicated serum intervened breast cancer MDA-MB-231 cells,Glucose content detection kit was used to observe the effect of DZXYS medicated serum on the glucose content of breast cancer MDA-MB-231 cells,Seahorse XFe96 Analyzer was used to test the extracellular acidification rate（ECAR）and oxygen consumption rate（OCR）,and to observe the effect of DZXYS medicated serum on the glycolysis ability and mitochondrial metabolism ability of breast cancer MDA-MB-231 cells,Immunofluorescence staining was used to observe the effect of DZXYS medicated serum on the expression of Glucose transporter 1（GLUT1）of breast cancer MDA-MB-231 cells,Western blot was used to observe the effect of DZXYS medicated serum on the expression of Hexokinase 2（HK2）,Pyruvate kinase M2（PKM2）and Lactate dehydrogenase A（LDHA）in breast cancer MDA-MB-231 cells.3.To observe the effect of DZXYS on breast cancer MDA-MB-231 cells transplanted subcutaneously in nude miceThe breast cancer MDA-MB-231 cell suspension was inoculated subcutaneously in nude mice.After inoculation,the nude mice were randomly divided into model group,DZXYS group and Xihuang Pill group,10 in each group.The clinical doses of DZXYS and Xihuang Pill were 11.69g·kg^-1 and 0.78g·kg^-1 respectively.The blank control group and model group were given normal saline once a day for 22 days.Blood was taken 2 hours after the last administration,and the tumor tissue was completely stripped.The tumor volume of tumor bearing nude mice was calculated;The effects of DZXYS on the contents of lactic acid and ATP in tumor tissue of tumor bearing nude mice were detected by lactic acid content and ATP content detection kit;Enzyme linked immunosorbent assay was used to detect the effect of DZXYS on the content of LDHA in serum of tumor bearing nude mice;Immunohistochemical staining was used to detect the effect of DZXYS on the expression of GLUT1 and LDHA in tumor tissues of tumor bearing nude mice;Western blot was used to detect the expression of Hypoxia-inducible factor-1α（HIF-1α）,GLUT1,PKM2 and LDHA of tumor tissue of nude mice treated with DZXYS.Results:1.The effect of DZXYS medicated serum on the proliferation,cell migration,cell cycle and apoptosis of breast cancer MDA-MB-231 cellsMTT results showed that DZXYS medicated serum inhibited the proliferation of breast cancer MDA-MB-231 cells,and 15%DZXYS medicated serum had the best effect on treating breast cancer MDA-MB-231 cells for 48 hours（P<0.01）,The results of cell scratch test showed that DZXYS decreased the migration ability of MDA-MB-231 cells,The results of flow cytometry showed that the DZXYS medicated serum blocked the growth of MDA-MB-231 cells in G0/G1 phase,Annexin V/PI double staining showed that DZXYS medicated serum promoted the apoptosis of MDA-MB-231 cells（P<0.01）,Hoechst 33342staining and Mito tracker red mitochondrial fluorescence probe results showed that DZXYS medicated serum induced mitochondrial structural damage,Western blotting showed that DZXYS medicated serum could up regulate the expression of BAX and down regulate the expression of BCL-2 in MDA-MB-231 cells.2.The effect of DZXYS medicated serum on the metabolism of breast cancer MDA-MB-231cellsThe results of glucose content test showed that DZXYS medicated serum decreased the glucose content in breast cancer MDA-MB-231 cells（P<0.01）,ECAR results showed that DZXYS medicated serum decreased the basic glycolysis,glycolysis capacity and glycolysis reserve of breast cancer MDA-MB-231 cells,OCR results showed that DZXYS medicated serum decreased the basic glycolysis capacity of breast cancer MDA-MB-231 cells basal respiration,ATP production and spare respiratory capacity,Immunofluorescence staining showed that DZXYS medicated serum decreased the expression of GLUT1 in MDA-MB-231cells,Western blot results showed that DZXYS medicated serum down regulated the expression of HK2、PKM2 and LDHA in MDA-MB-231 cells.3.The effect of DZXYS on breast cancer MDA-MB-231 cell transplantation in nude miceThe nude mouse model of breast cancer MDA-MB-231 cell transplantation was established.The results of lactic acid content test showed that DZXYS reduced the content of lactic acid in tumor tissue（P<0.01）,The results of ATP content test showed that DZXYS reduced the content of ATP in tumor tissue（P<0.01）,The results of enzyme-linked immunosorbent assay showed that DZXYS decreased the content of LDHA in serum of nude mice（P<0.01）,The results of immunohistochemistry showed that DZXYS decreased the expression of GLUT1 and LDHA in tumor tissue,Western blot showed that DZXYS down regulated the expression of HIF-1α,PKM2 and LDHA in tumor tissue.Conclusion:1.The DZXYS medicated serum can affect the function of breast cancer MDA-MB-231 cells,which may induce apoptosis of breast cancer MDA-MB-231 cells through mitochondria mediated apoptosis pathway.2.Glycolysis is one of the pathways for breast cancer MDA-MB-231 cells to obtain energy.DZXYS medicated serum can reduce the level of glycolysis and mitochondrial metabolism of breast cancer MDA-MB-231 cells.3.Under hypoxia condition,glycolysis and glucose transport of MDA-MB-231 cells were enhanced,it may inhibit glycolysis and glucose transport by down regulating Hypoxia-inducible factor-1α,so as to delay the growth of breast cancer MDA-MB-231 cell transplanted tumor in nude mice.