Danzhi Xiaoyao Powder Regulates The Inflammatory Microenvironment To Resist Breast Cancer And Its Molecular Mechanism

Purpose:In this study,we established a breast cancer transplant model in Nude mice to explore the anti-breast cancer effect of Danzhi Xiaoyao Powder and its molecular mechanism,and revealed that Danzhi Xiaoyao Powder may prevent breast cancer by regulating inflammatory microenvironment.Material and method:6-week-old SPF grade BALB/c female Nude mice were fed adaptively for 7 days.MDA-MB-231-GFP cells were transformed stably in logarithmic growth phase and seeded subcutaneously into Nude mice.The number of inoculated cells was5×10⁵.The tumor model was established.The inoculated Nude mice were randomly divided into model group,Danzhi Xiaoyao Powder group(11.69g·kg^-1)and Xihuang Pill group(0.78g·kg^-1),with 10 mice in each group and 10 mice in normal control group.One day after the breast cancer cells were inoculated,drug gavage was performed at a fixed time once a day for 22d.Normal control group and model group were given the same amount of normal saline for intragastric administration.Two hours after the last administration,the eyeball was enucleated and the blood was taken,and the Nude mice were killed according to the routine method,the tumor tissue was stripped,the tumor mass was weighed and the tumor mass inhibition rate was calculated,and the follow-up experiment was carried out.Hematoxylin-eosin staining was used to observe the morphological changes of transplanted tumor in Nude mice.Enzyme-linked immunosorbent assay was used to detect the effect of Danzhi Xiaoyao Powder on the contents of HIF-1αand MIF in plasma of breast cancer Nude mice.The effects of Danzhi Xiaoyao Powder on the expression of HIF-1α,MIF,MCT1 and MCT4 in tumor tissues were detected by immunohistochemical method.The effect of Danzhi Xiaoyao Powder on the protein expression of MIF,IL-6,IL-8,MCT1,MCT4,LOX,VEGF-A and VEGFR-2 in breast cancer tissue was detected by Western blot.Results:1.Effect of Danzhi Xiaoyao Powder on transplanted tumor of Breast Cancer in Nude miceThe results showed that compared with the model group,the tumor quality in Danzhi Xiaoyao Powder group decreased significantly（P<0.01）,and the tumor mass inhibition rate was 26.17%,indicating that Danzhi Xiaoyao Powder can effectively inhibit the growth of breast cancer transplanted tumor in Nude mice.2.Effect of Danzhi Xiaoyao Powder on tumor morphologyThe results of HE showed that in the model group,the tumor cells arranged densely and showed irregular clump or cord distribution,the tumor cells were atypical,the nucleus enlarged was round or oval,the color was dark,and a small number of necrotic foci were scattered.Compared with the model group,the tumor cells in Danzhi Xiaoyao Powder group and Xihuang Pill group showed degenerative changes in different degrees,the necrotic foci of tumor tissue increased significantly.3.Effect of Danzhi Xiaoyao Powder on the contents of HIF-1αand MIF in Plasma of Nude mice with Breast CancerThe results of ELISA showed that compared with the normal control group,the contents of HIF-1αand MIF in the model group increased（P<0.01）,while the contents of HIF-1αand MIF in the Danzhi Xiaoyao Powder group decreased（P<0.01）,and the contents of HIF-1αand MIF in the breast cancer plasma decreased in the Xihuang Pill group（P<0.01）.It is suggested that Danzhi Xiaoyao Powder can reduce the contents of HIF-1αand MIF in plasma of breast cancer.4.Effect of Danzhi Xiaoyao Powder on the expression of HIF-1α,MIF,MCT1 and MCT4protein in tumor tissuesImmunohistochemical results showed that compared with the normal control group,the positive expressions of HIF-1α,MIF,MCT1 and MCT4 in the model group were increased（P<0.01）.Compared with the model group,the positive expressions of HIF-1α,MIF,MCT1 and MCT4 in the Danzhi Xiaoyao Powder group were decreased（P<0.01）,and the positive expressions of HIF-1α,MIF,MCT1 and MCT4 in the Xihuang Pill group were decreased（P<0.01）.These results indicated that Danzhi Xiaoyao Powder could decrease the expression levels of HIF-1α,MIF,MCT1 and MCT4 in tumor tissues.5.Effects of Danzhi Xiaoyao Powder on the protein expression of MIF,IL-6,IL-8,MCT1,MCT4,LOX,VEGF-A and VEGFR-2 in tumor tissuesWestern blot results showed that compared with the normal control group,the protein expressions of MIF,IL-6,IL-8,MCT1,MCT4,LOX,VEGF-A and VEGFR-2 in model group were increased（P<0.01）.Compared with the model group,the protein expressions of MIF,IL-6,IL-8,MCT1,MCT4,LOX,VEGF-A and VEGFR-2 in Danzhi Xiaoyao Powder group were decreased（P<0.01）;Compared with model group,the protein expressions of MIF,IL-6,IL-8,MCT1,MCT4,LOX,VEGF-A and VEGFR-2 in Xihuang Pill group were decreased（P<0.01）.Conclusion:1.Danzhi Xiaoyao Powder can effectively inhibit the growth of breast cancer xenografts in Nude mice.2.Danzhi Xiaoyao Powder can down-regulate the expression of HIF-1α,MIF,IL-6,IL-8,MCT1,MCT4,LOX,VEGF-A,VEGFR-2 protein in tumor tissue,suggesting that Danzhi Xiaoyao Powder may inhibit the growth of breast cancer transplanted tumor in Nude mice by regulating tumor microenvironment.It may be mainly related to the regulation of inflammatory microenvironment,metabolic microenvironment,hypoxic microenvironment and angiogenesis of breast cancer.