| Benign lesions of the breast are far more frequent than malignant ones. Breast hyperplasia is one of these lesions and is also one of the risk factors for breast cancer.The World Health Organization advocates early prevention and detection in order to improve breast cancer outcome, and these measures remain the cornerstone of breast cancer control. However, patients with breast hyperplasia are reluctant to undergo tissue resection because it is painful, often allows recurrence, and does nothing to treat the underlying, causative endocrine imbalance. Instead, multiple applied estrogen or estrogen receptor antagonist agents, including tamoxifen, have been used. Although these drugs can relieve symptoms in patients, they have many side effects and complications, and their use also can lead to endocrine disorders Mongolian medicine plays an important role in Chinese ethnic medicine. But very little scientific evidence exist that support consumption of Mongolian medicine herbal preparations in traditional medicine. So RuXian-â… is only in clinical use in the Affiliated Hospital of Inner Mongolia University for the Nationalities as a traditionally drug, lacks widespread application. RuXian-â… treatment of breast hyperplasia in 400 patients showed that the cure rate was 98%.But very little scientific evidence exist that support consumption of Mongolian medicine herbal preparations in traditional medicine. Two dimensional difference gel electrophoresis (2D-DIGE) can greatly reduce the inter-gel variation and excessive time/labor costs associated with standard 2-DE and can allow a more accurate qualitative and quantitative analysis. There have been several reports comparing the expression profiles of proteins in breast cancer tissues with those in nontumor breast tissues. However, expression profiles of proteins in breast hyperplasia have not been reported. In order to gain a better insight into the mechanism of RuXian-â… 's efficacy, diagnosis, therapy, outcomes, and prognosis in the breast hyperplasia, and the relation between breast hyperplasia and breast cancer, we used intramuscular estrogen and progesterone to induce rat breast hyperplasia and 2D-DIGE to study the proteins associated with RuXian-â… treatment. Differentially expressed spots were identified by MALDI-TOF/TOF-MS, and changes in the expression of the proteins of interest were further validated using immunohistochemical and biochemical detection methods.In this study, we tested the breast-protective effects of RuXian-â… ,in rats with estradiol/progesterone induced breast hyperplasia. Rats were divided into 5 groups:a control group, a model group, a tamoxifen citrate (positive control) group, a RuXian-â… high-dose group and a RuXian-â… low-dose group.As a first step toward the investigation of biomarkers associated with RuXian-â… treatment, a proteome-wide analysis of rat breast tissue was conducted in this study. First, rat breast hyperplasia was induced by injection with estradiol and progesterone. We used an approach that integrates size-based 2D DIGE, MALDI-TOF/TOF-MS,and bioinformatics to analyze data from the control group, the model group and the RuXian-â… treatment group.After 2D DIGE, the Cy2, Cy3, and Cy5 channels of each gel were individually imaged, and the images were analyzed using DeCyder 5.0 software.The main results of this study are as follows:1.Compared with the model group, the RuXian-â… group experienced markedly limited increases in nipple diameter, reduced histopathological changes, and decreased serum estradiol (E2) and luteinizing hormone (LH) levels.RuXian-â… was increased significantly progesterone (P), testosterone (T) levels and significantly follicle stimulating hormone (FSH),but it did not affect prolactin (PRL) in breast hyperplasia rats at 30 d. RuXian-â… demonstrated the same treatment effect as tamoxifen citrate for nipple diameter size and histopathological change; however, tamoxifen citrate affected the regulation of serum hormones differently than RuXian-â… .2. The RuXian-â… treatment mechanism may involve decreased expression of estrogen receptor(ER), progesterone receptor (PR), androgen receptors(AR),ER mRNA and AR mRNA in breast hyperplasia tissue. The study showed that protein expression levels of ER, PR and AR in the model group increased relative to the control group (P<0.01).ER, PR and AR protein expression levels in RuXian-â… groups all decreased significantly compared with the model group (P<0.01)by immunohistochemistry and western blotting. RuXian-â… regulated mRNA expression of ER, PR and AR of sex hormone receptor transcription. The results demonstrated that the mRNA expression of ER and AR increased (P<0.01) and PR mRNA expression decreased (P<0.01) significantly in the model group compared to the control group. RuXian-â… high and low dose groups remarkably decreased ER and AR mRNA expression and increased PR mRNA expression. In conclusion, RuXian-â… could reduce increases in nipple diameter, decrease histopathological changes and adjust regulation of serum sex hormone. This protective effect may be partially mediated through affecting mRNA and protein expression of ER, PR and AR.3.17 spots corresponding to 16 different gene products were identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). There are two spots (corresponding to Tpil protein and myosin-4) that are significantly down-regulated in the disease model (ratio<1.3, p<0.01) but increased after treatment with RuXian-â… .The other 15 spots were significantly up-regulated (ratio>1.3, p<0.01) in the disease model group, but down-regulated after treatment with RuXian-â… .And two have the opposite change trend.4.Following protein identification, they were categorized using the PANTHER gene ontology database. The PANTHER classification system grouped the 17 proteins from the mammary glands into 10 major subcategories according to "biological processes",including the immune system process, metabolic process, response to stimulus, transport, et al.The conclusions of this study are as follows:1.The pathogenesis of breast hyperplasia is closely related to serum endocrine hormone disorders and hormone receptors gene and protein expression increase in breast tissue;2.RuXian-â… can reduce serum estrogen levels, increased progesterone levels, improve the hormone disorder state in the hyperplasia of mammary glands rats;3. RuXian-â… can adjust mammary glands ER, PR and AR protein expression and their respective mRNA expression, then treatment of rat mammary gland hyperplasia;4. Injection of exogenous estrogen and progesterone-induced hyperplasia of mammary glands in rats, serum lipid metabolism in disturbance;5. RuXian-â… can reduce serum TC,TG and LDL-c levels and regulate lipid metabolism in hyperplasia of mammary glands rats;6.RuXian-â… therapeutic effects of breast hyperplasia related to metabolism, signal transduction, cytoskeleton and cell motility, oxidative stress, immunity, cell cycle and other biological processes;The creative points of the present study are:1.Intramuscular injection to rats with estrogen and progesterone used to establish animal models of hyperplasia of mammary glands studied systematically of the RuXian-â… on the treatment of hyperplasia of mammary glands in rats, from both endocrine hormones and receptors discussed the related Mechanism of action;2.Initial establishment animal models of breast hyperplasia in serum and breast tissue proteomics technology platform by proteomics technology;3.The first use of proteomics technology to study animal models of hyperplasia of mammary glands proteome changes in breast tissue for breast hyperplasia in the pathogenesis of the study provides new ideas and methods;4. Proteomics approach was first introduced to study the Mongolian compound preparation mechanism for the study the problem of Mongolian medicine targets provides a new mode of thinking. |
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