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1.
China Journal of Chinese Materia Medica ; (24): 6749-6764, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1008873

RESUMO

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.


Assuntos
Humanos , Cromatografia Líquida de Alta Pressão , Simulação de Acoplamento Molecular , Farmacologia em Rede , Apoptose , Hiperplasia , Medicamentos de Ervas Chinesas/farmacologia
2.
China Pharmacy ; (12): 1549-1556, 2020.
Artigo em Chinês | WPRIM | ID: wpr-822618

RESUMO

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.

3.
Chinese Pharmacological Bulletin ; (12): 710-715, 2020.
Artigo em Chinês | WPRIM | ID: wpr-856978

RESUMO

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.

4.
Journal of Veterinary Science ; : 79-87, 2018.
Artigo em Inglês | WPRIM | ID: wpr-758776

RESUMO

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.


Assuntos
Feminino , Agricultura , Bactérias , Citocinas , Eficiência , Células Epiteliais , Escherichia coli , Perfilação da Expressão Gênica , Histonas , Imunidade Inata , Interleucina-6 , Interleucinas , Mastite , Análise em Microsséries , Mycoplasma bovis , Mycoplasma , RNA Mensageiro , Staphylococcus aureus , Receptores Toll-Like , Fator de Necrose Tumoral alfa
5.
Journal of Veterinary Science ; : 149-158, 2017.
Artigo em Inglês | WPRIM | ID: wpr-109784

RESUMO

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.


Assuntos
Humanos , Neoplasias da Mama , Mama , Linhagem Celular , Classificação , Células Epiteliais , Receptor alfa de Estrogênio , Estrogênios , Fenobarbital , Fenótipo , Progesterona , Proto-Oncogenes , Receptores de Progesterona , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro , Análise de Sequência de DNA
6.
Acta Pharmaceutica Sinica ; (12): 569-574, 2017.
Artigo em Chinês | WPRIM | ID: wpr-779630

RESUMO

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.

7.
Journal of Veterinary Science ; : 95-97, 2011.
Artigo em Inglês | WPRIM | ID: wpr-47183

RESUMO

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.


Assuntos
Animais , Bovinos , Feminino , Biofilmes , Linhagem Celular , Contagem de Colônia Microbiana/veterinária , Células Epiteliais/microbiologia , Hibridização in Situ Fluorescente , Mastite Bovina/microbiologia , Portugal , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/classificação , Fatores de Virulência
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