| Objective:Breast hyperplasia is a benign disease with a high incidence in women.The incidence rate in China is as high as about 40%.Its clinical symptoms include breast tenderness,tingling,tenderness,and sometimes nipple discharge,which seriously affect the quality of life of patients.Breast atypical hyperplasia and breast carcinoma in situ belong to the category of precancerous lesions,which will gradually evolve into invasive cancer as the disease progresses,affecting the survival of patients.Xiaozheng Pill is a proprietary Chinese medicine based on the addition and subtraction of Chaihu Shugan Powder in Zhang Jiebin’s "Jingyue Quanshu".Clinical studies have shown that the drug can effectively treat breast hyperplasia with few adverse drug reactions.It has been widely used in clinical practice.This study will be divided into two parts,respectively,to explore the mechanism of Xiaozheng Pill in the treatment of hyperplasia of mammary glands,and the mechanism of Xiaozheng Pill in the treatment of atypical hyperplasia of mammary glands and carcinoma in situ,to provide experimental data support for the clinical application of Xiaozheng Pill and the expansion of clinical indications.Methods:(1)Research on the mechanism of Xiaozheng Pill in treating breast hyperplasia:Analyze the effective chemical components of Chinese medicine contained in Xiaozheng Pill,as well as the key chemical components,key proteins,main biological processes,and key pathways for the treatment of breast hype rplasia through network pharmacology research.A mouse model of mammary gland hyperplasia was established,and the establishment of the pathological morphological verification model was observed by detecting hormone levels and HE staining.After the intervention of each dose of Xiaozheng Pill,the changes in the organ index of mice were measured.ELISA detecting hormone E2 P,LH,FSH levels.Immunohistochemical method to detect ERα,ERβ,PR levels.Western Blotting to detect the expression of proliferation-related proteins Raf1,ERK1/2,ELK,and the expression of angiogenesis-related proteins HIF-1α,VEGFA,bFGF.Analyze the metabolic differences between the model group and the control group,each dose group of Xiaozheng Pill and the model group through metabolomics,screen different metabolites,analyze their changing trends,the relationship between the different metabolites,and the metabolic pathways involved.(2)Research on the pharmacodynamic mechanism of Xiaozheng Pill in the treatment of atypical hyperplasia of mammary glands and carcinoma in situ:network pharmacology analysis of the key chemical components,key proteins,main biological processes,and key pathways of Xiaozheng Pill in the treatment of pre-breast cancer.A mouse model of breast atypical hyperplasia was established,and the model was verified by HE staining;immunohistochemical detection of ERα,ERβ,PR,TNF-α,IL-β,iNOS levels.Western Blotting to detect the expression of inflammation-related proteins FAK,Gas6,Axl expression.In vitro experiments verified the effect of saikosaponin A,the main active ingredient of Xiaozheng Pill,on HCC1937 cells.MTT was used to detect the cell proliferation inhibition rate and calculate the IC50 value.the cell morphology was observed by an inverted microscope,and the nucleus alterations were observed by Hoechst 33258 staining assay.Scratch assay,transwell chamber assay were performed to observe the cell migration and invasion ability changes.Apoptosis was detected by flow cytometry.The expressions of apoptosis-related proteins Bcl-2,Bax,caspase-9,and caspase-3 were detected by Western blotting.Results:(1)Xiaozheng Pill includes 78 active ingredients of drugs,and the therapeutic effects of the active ingredients are 123.After matching 4656 targets of breast hyperplasia,99 possible targets of Xiaozheng Pill for the treatment of breast hyperplasia are obtained.The active ingredients that are most related to the therapeutic effect include quercetin,luteolin,kaempferol,nobiletin,etc.The core targets related to the treatment include IL6,EGFR,VEGFA,CASP3,MAPK8,ER,etc.The key pathways include the HIF-1 signaling pathway,MAPK signaling pathway,etc(2)Hormone modeling method can effectively establish a mouse mammary gland hyperplasia model.Xiaozheng Pill has no abnormal effect on organ index and liver tissue morphology in mice Xiaozheng Pill can reduce E2 and FSH levels,and increase P and LH levels in mice with mammary gland hyperplasia.Xiaozheng Pills can restore the normal structure of breast tissue and reduce the expression of ERα,ERβ,and PR in breast tissue Xiaozheng Pill can inhibit the expression of Raf-ERK-ELK pathway protein,and also inhibit the protein expression of HIF-1 α and bFGF,but it was no significant effect on VEGFA protein.(3)Metabolomics analysis found a total of 654 metabolites.Compared with the blank group,36 metabolites were significantly up-regulated and 5 metabolites were significantly down-regulated in the model group.Comparing the low,medium,and high dose groups of Xiaozheng Pill with the model group,there were 7,30,and 36 metabolites that were significantly up-regulated,19,25,and 34 metabolites were significantly down-regulated.The intersection of the four sets of data metabolites includes Cholesterol ester,19-Nortestosterone,Naringeninchalcone,Taurocholic acid,Tauro-alpha-Muricholic acid sodium salt,etc.KEGG analysis showed that the metabolic pathways of Xiaozheng Pill in the treatment of mammary gland hyperplasia included Cholesterol metabolism(map04979),Metabolic pathway(map01100),HIF-1 signaling pathway(map04066),etc.(4)After matching the 123 targets of Xiaozheng Pill to 8087 targets of atypical hyperplasia of mammary glands and carcinoma in situ,the key proteins of Xiaozheng Pill for the treatment of atypical hyperplasia of mammary glands and carcinoma in situ include GAS6,AXL,IL6,VEGFA,EGFR,etc.,and key pathways include Apoptosis,TNF signaling pathway,etc.(5)DMBA combined with hormone modeling method can effectively establish a mouse mammary gland atypical hyperplasia model.High-dose Xiaozheng Pill and the combined use of FAK inhibitors can normalize the structure of breast tissue in mice with atypical breast lesions,reduce the expression levels of ERα,ERβ,and PR in the breast tissue,as well as the levels of inflammatory factors TNF-α and IL-1β.High-dose Xiaozheng Pill has obvious inhibitory effects on FAK protein phosphorylation and Gas6 protein,and the combined use of FAK inhibitors has more significant inhibitory effects on Gas6 protein.Each group had no significant effect on Axl protein phosphorylation.(6)Saikosaponin A,the main component of Xiaozheng Pill,can inhibit the proliferation of breast carcinoma in situ HCC1937 cells,destroy cell morphology,reduce cell migration and invasion,and promote cell apoptosis in a dose-dependent manner.Saikosaponin A can up-regulate the expression of Bax and down-regulate the expression of Bcl-2,Caspase-3,and Caspase-9.Conclusions:(1)Xiaozheng Pill can improve the hormone levels of mammary gland hyperplasia mice and inhibit the excessive proliferation of mammary epithelial cells.The mechanism of action is related to the FGF-Raf-ERK-ELK pathway.Metabonomics studies suggest that the mechanism of Xiaozheng Pill in treating mouse mammary gland hyperplasia is related to cholesterol metabolism.(2)Xiaozheng Pill can treat atypical hyperplasia of mammary glands in mice and reduce the level of inflammation in mammary gland tissues.Its mechanism of action is related to the FAK-Gas6 signaling pathway.In vitro experiments show that Saikosaponin A can induce apoptosis of HCC1937 cells,and its mechanism is related to endogenous mitochondrial apoptosis. |
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