Mechanism of anti-hyperplasia of mammary glands of Xihuang Pills blood-entering component based on UPLC-Q-TOF-MS and network pharmacology / 中国中药杂志

In this study, based on network pharmacology and molecular docking method, the mechanism of anti-hyperplasia of mammary glands of Xihuang Pills blood-entering components was explored, and the efficacy and key targets of Xihuang Pills blood-entering components were experimentally verified by MCF-10A proliferation model of human mammary epithelial cells. In order to clarify the material basis and mechanism of Xihuang Pills in realizing anti-hyperplasia of mammary glands, the blood-entering components of Xihuang Pills were qualitatively analyzed by UPLC-Q-TOF-MS, and 22 blood-entering components were identified. By taking the blood-entering components as the research object, the network pharmacology prediction and molecular docking verification were carried out, and finally, three key targets were screened out, namely JAK1, SRC, and CDK1. In vitro experiments show that Xihuang Pills can inhibit the proliferation of MCF-10A cells, promote the apoptosis of MCF-10A cells, and reduce the expression of JAK1, SRC, and CDK1 targets in cells. To sum up, Xihuang Pills can promote the apoptosis of mammary epithelial cells by regulating the expression of JAK1, SRC, and CDK1 and then play an anti-hyperplasia role, which provides an experimental basis for clarifying the material basis of Xihuang Pills for anti-hyperplasia effect.

Study on the Effects of Calpeptin on Estrogen-induced Transformation and Stemness Markers Expression of Mammary Epithelial Cells MCF- 10A Based on Calpain-ERK Signaling Pathway / 中国药房

OBJECTIVE：To study the effects of Calpeptin inhibitor Calpeptin on the transformation and stemness markers expression induced by estradiol（E2），and to investigate its mechanism. METHODS ：Taking human mammary epithelial cells MCF-10A as research object ，transformed cells were induced by E 2 treatment. Cells were divided into control group （0.1％DMSO）， E2-transformed group （50 nmol/L），E2-transformed+Calpeptin group （50 nmol/L E 2+1 μmol/L Calpeptin），then continuously treated with corresponding drug-containing culture medium for 15 generations. Then ，MTT assay was used to determine the proliferation rate of cells （24，48 h）；plate colony test was used to detect the Clone formation rate of cells ；the number of sphere-forming cells was measured by suspension spheroidization test ；mRNA expressions of stemness marker （CD44，Nanog，OCT4）and extracellular sigal-regulated kinase （ERK）were detected by RT-qPCR ，and protein expressions of CD 44，Nanog，OCT4 ，ERK and p-ERK were detected by Western blotting assay. Another E 2-transformed cells were divided into control group （0.1％DMSO）and U0126 （ERK inhibitor ）group（10 μmol/L）. Clone formation rate ，the number of sphere-forming ，protein expressions of CD 44，Nanog， OCT4，ERK and p-ERK were determined with above methods ，and to validate the relationship of ERK inhibition with transformed cell behavior and the expression of stemness markers. RESULTS ：Compared with control group ，proliferation rate and clone formation rate of E 2 transformed group were increased significantly （P＜0.01），and the number of sphere-forming was increased significantly（P＜0.01）；mRNA expression levels of CD 44，Nanog，OCT4，ERK and protein expression levels of CD 44，Nanog， OCT4 and p-ERK in cells were increased significantly （P＜0.01）. Compared with E 2-transformed group ，proliferation rate （24，48 h）and clone formation rate of E 2-transformed + Calpeptin group were decreased significantly （P＜0.01），and the number of sphere-forming was decreased significantly （P＜0.05）；mRNA expression levels of CD 44，Nanog，OCT4 ，ERK and protein expression levels of CD 44，Nanog，OCT4，p-ERK in cells were decreased significantly （P＜0.05 or P＜0.01）. After treated with ERK inhibitor U 0126，clone formation rate of E 2-transformed cells ，the number of sphere-forming ，protein expression levels of CD44，Nanog，OCT4 and p-ERK were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：Calpeptin can inhibit the transformation and the expression of stemness markers of human mammary epithelial cells MCF- 10A，and the mechanism of it may be associated with inhibiting the activation of Calpain-ERK signaling pathway.

Effect of Xihuang pills on apoptosis and expression of ER and PR in rat mammary epithelial cells / 中国药理学通报

Aim To investigate the effect of Xihuang pills on estradiol (E2) and progesterone (P)-induced apoptosis of rat mammary epithelial cells and the expression of estrogen and progesterone receptors, and to explore its anti-mammary hyperplasia mechanism. Methods Rat mammary epithelial cells were treated with different concentrations of Xihuangpills containing serumafter E2, P induced proliferation. The cell proliferation was detected by CCK-8 method (24, 48, 72 h). The apoptosis was detected by flow cytometry after AnnexinV-FITC/PI double staining. The average optical density of Bax and Bcl-2 was detected by immunohistochemistry; Estradiol receptor alpha (ER-α), estradiol receptor beta (ER-β), progesterone receptor (PR) protein expression were detected by Western blot. Results The serum containing Xihuang pillssignificantly inhibitedthe proliferation of rat mammary epithelial cells after E2and P treatment (P < 0. 05), and induced apoptosis, affecting the expression of apoptosis-related proteins Bcl-2 and Bax (P < 0. 05), and effectively inhibiting the expression of estrogen receptors ERα, ERβ. Conclusions Xihuang pills can induce the apoptosis of mammary epithelial cells by regulating related apoptotic proteins, and regulate the secretion of estrogen and progesterone in breast tissues, affecting the proliferation and rejuvenation of breast, which is of great significance for the treatment of breast hyperplasia.

Innate immune response of bovine mammary epithelial cells to Mycoplasma bovis

Mycoplasma spp. are contagious bacteria, and mycoplasmal mastitis is a serious productivity problem on dairy farms. Bovine mammary epithelial cells (bMECs) have an important role in the elimination of pathogens, but the effect of Mycoplasma bovis on bMECs has not been fully described. To elucidate the immune response against intramammary infection by M. bovis, we undertook microarray analysis to examine and profile mRNA expression in bMECs after stimulation with M. bovis. We also compared the effects of M. bovis, Staphylococcus aureus, and Escherichia coli on immune-related mRNA expression in bMECs. Transcriptome analysis indicated a significant decrease in the level of mRNA-encoding lysine-specific demethylase 4D, suggesting that the immune response is suppressed by a decrease in histone demethylase activity. Interleukin (IL)-1β, IL-6, tumor necrosis factor alpha, toll-like receptor (TLR) 2, and TLR4 mRNA expression levels were significantly increased in bMECs stimulated with heat-killed M. bovis, but the expression levels were lower than those following stimulation by heat-killed S. aureus or E. coli. Our results suggest that M. bovis weakly affects mRNA expression in bMECs compared to the effects of E. coli or S. aureus. Moreover, live M. bovis may induce suppression of the immune response in bMECs.

Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer.

The inhibitory effects of calpain inhibitors on epithelial-mesenchymal transition of MCF-10A mammary epithelial cells induced by fibronectin / 药学学报

The aim of this research is to investigate the inhibitory effects of calpain inhibitors (ALLN and calpain inhibitor IV) on mammary epithelial-mesenchymal transition (EMT) of MCF-10A cells induced by fibronectin (FN). After FN treatment of MCF-10A cells for 48 h, cell migration and invasion were determined by scratch repair assay and matrigel coated transwell assay, respectively. Vimentin, E-cadherin, snail and calpain-2 protein expression were measured by Western blot. The results suggest that FN induced morphological changes in MCF-10A cells, significantly increased the migration and invasion abilities of MCF-10A cells, upregulated the expression of calpain-2, vimentin and snail, and downregulated the expression of E-cadherin. All such changes by FN were reversed with ALLN and calpain inhibitor IV. In conclusion, the upregulation of calpain-2 was involved in FN-induced EMT of MCF-10A mammary epithelial cells, which may be inhibited by ALLN and calpain inhibitor IV.

Invasive potential of biofilm-forming Staphylococci bovine subclinical mastitis isolates

Staphylococcus (S.) aureus is a common infectious agent of bovine chronic mastitis, a disease that is difficult to eradicate. The abilities of Staphylococci to be internalized and form a biofilm can contribute to host immunological defence evasion that subsequently impairs antimicrobial therapy. The invasive capability of six S. aureus field isolates with different biofilm-forming profiles was compared in vitro using a bovine mammary epithelial cell line. This was further confirmed in primary cell cultures using fluorescent rRNA probes against S. aureus. The results suggest that S. aureus invasion levels are not related to biofilm formation.