5-hydroxytryptophan(5-HTP)regulates Proliferation Of Goat Mammary Epithelial Cells Through MAPK/BCL-3

The regulation of proliferation and apoptosis in mammary epithelial cells play an important role in promoting breast development,lactation and lactation initiation.Mammary epithelial cells are the only cells which could synthesize and secrete milk proteins in mammary gland.5-hydroxytryptamine(5-HT)is a monoamine which synthesize in mammary gland and regulate mammary calcium homeostasis,milk composition and milk production by autocrine-paracrine way.The reagent used in this study is5-hydroxytryptophan(5-HTP),which is a precursor of 5-HT synthesis and has a more stable chemical structure.In order to increase 5-HT concentration in goat's body cycle,we injected0.8mg/kg of 5-HTP into the perinatal goats(the control group was injected with the same dose of physiological saline),compared in two groups,after injection of 5-HTP differentially expressed genes were enriched into the MAPK/ERK signaling pathway.Studies have shown that MAPK/ERK signaling pathway has a regulatory effect on meiosis and mitosis,involved in cell growth regulation and differentiation after mitosis.All of those suggested that 5-HTP may affect cell proliferation and apoptosis by the MAPK/ERK signaling pathway.Also,a few of studies had shown that B-cell leukemia/lymphoma 3(BCL-3),a member of the NF?B inhibitory protein(I?B)family,is also regulated by the MAPK/ERK pathway and involved in cell cycle regulation.Therefore,in this study,goat mammary gland epithelial cells(GMECs)were treated by exogenous addition of 5-HTP to explore the regulatory effects of5-HTP/MAPK/BCL-3 on the proliferation and apoptosis of GMECs.The main results obtained in this study are as follows:(1)CCK-8 test found that 175 ?M and 200 ?M 5-HTP treatment for 12 h could promote the proliferation of GMECs(p <0.05);Annexin V-FITC / PI staining combined with flow cytometry test found that 75 ?M and 175 ?M 5-HTP treatments could decrease apoptosis rate of GMECs(p <0.05).(2)5-HTP of 175 ?M could promote the expression of p-ERK in GMECs(p <0.05);MAPK/ERK inhibitor(U0126)inhibited GMECs proliferation(p <0.05),promoted apoptosis(p <0.05);MAPK/ERK agonist(Honokiol)promoted GMECs proliferation(p<0.05)and inhibited apoptosis(p <0.05);meanwhile phosphorylation of MAPK/ERK could promote BCL-3 expression in the m RNA level.(3)5-HTP of 175 ?M and Honokiol treatment could up-regulate the expression of BCL-3 in m RNA level(p <0.05);U0126 treatment could down-regulate BCL-3 expression inm RNA level(p <0.05).The interference of BCL-3 gene by si RNA showed that when the expression of BCL-3 decreased the expression of p-ERK protein decreased together(p<0.05);Cyclin D1 and Bcl-2 decreased both in m RNA and protein levels(p <0.05).In summary,it is concluded that 5-HTP could promote the proliferation and decrease apoptosis of GMECs through MAPK/BCL-3 axis.This study provides a reference for preventing or curing of perinatal hypocalcemia in dairy cattle and a regulating of physiological state of mammary gland by 5-HTP in theory.