Pharmacodynamic structure of deer antler base protein and its mammary gland hyperplasia inhibition mechanism by mediating Raf-1/MEK/ERK signaling pathway activation

Mammary gland hyperplasia (MGH) is a common mammary disease whose main pathogenesis is the disruption of estradiol (E2) and progesterone (P) secretion, thereby causing overproliferation of mammary epithelial cells and mammary gland tissue hyperplasia. Deer antler base is a traditional Chinese medicine that has been used for many years to treat MGH. However, its pharmacological mechanism and pharmacodynamic material basis are unclear. In this study, we for the first time used the graded salting method to classify deer antler base protein (CNCP) as CNCP-A, CNCP-B, and CNCP-C and explored the pharmacological mechanism of the anti-MGH properties of CNCP. We found that CNCP could regulate the hormonal levels of E2, P, and follicle stimulating hormone (FSH) and improve the histopathological condition. The potential mechanism might be related to the recombinant C-Raf proto oncogene serine/threonine protein kinase/mitogen-activated protein/extracellular regulated protein kinase (Raf-1/MEK/ERK) signaling pathway. By upregulating the protein expression of the follicle stimulating hormone receptor (FSHR), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) inhibited the activation of the downstream Raf-1/MEK/ERK signaling pathway, which in turn inhibited the proliferation of mammary epithelial cells. We analyzed the physicochemical properties of CNCP-A, CNCP-B, and CNCP-C and obtained CNCP-C-I by column chromatographic purification of the best pharmacophore protein CNCP. Using high-performance liquid gel filtration chromatography (HPGFC), we determined the molecular weight of CNCP-C-I and identified it by high-performance liquid tandem mass spectrometry (LC-MS/MS) to obtain the first match for a high confidence protein KRT1. This study provides a theoretical basis for the development of effective traditional Chinese medicines with low toxicity levels for the prevention and treatment of mammary gland diseases.